Regulation of p63 Isoforms by Snail and Slug Transcription Factors in Human Squamous Cell Carcinoma

Tumorigenesis and Neoplastic Progression

Regulation of p63 Isoforms by Snail and SlugTranscription Factors in Human Squamous CellCarcinoma

Michael Herfs,* Pascale Hubert,*Meggy Suarez-Carmona,* Anca Reschner,*Sven Saussez,† Geert Berx,‡§ Pierre Savagner,¶

Jacques Boniver,* and Philippe Delvenne*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

TP63 is a p53-related gene that contains two alterna-tive promoters, which give rise to transcripts thatencode proteins with (TAp63) or without (�Np63) anamino-transactivating domain. Whereas the expres-sion of p63 is required for proper development ofepithelial structures, the role of p63 in tumorigenesisremains unclear. Here, we investigated the role ofSnail and Slug transcription factors, known to pro-mote epithelial-to-mesenchymal transitions duringdevelopment and cancer, in the regulation of p63isoforms in human squamous cell carcinoma (SCC).In the present study, we observed that the expres-sions of �N and TAp63 isoforms were, respectively,down- and up-regulated by both Snail and Slug. How-ever, the induction of TAp63 was not directly causedby these two transcription factors but resulted fromthe loss of �Np63, which acts as dominant-negativeinhibitor of TAp63. In SCC cell lines and cancer tis-sues, high expression of Snail and Slug was also sig-nificantly associated with altered p63 expression.Finally, we showed that �Np63 silencing reduced cell–cell adhesion and increased the migratory propertiesof cancer cells. These data suggest that the disruptionof p63 expression induced by Snail and Slug plays acrucial role in tumor progression. Therefore, p63 andits regulating factors could constitute novel prognosismarkers in patients with SCC and attractive targets

for the therapeutic modulation of neoplastic cellinvasiveness. (Am J Pathol 2010, 176:000–000; DOI:

10.2353/ajpath.2010.090804)

p63 is a group of six different transcription factors thatexhibit a high sequence and structural homology to thewell-known p53 tumor suppressor protein. Because ofthe use of alternative promoters and transcription startsites, the TP63 gene gives rise to transcripts that encodeproteins with (TAp63) or without (�Np63) an amino-trans-activating domain. Both TA and �N transcripts are alter-natively spliced at the 3� end to yield further carboxyl-terminal isotypes (�, �, �).1 The extensive defect in a highnumber of epithelial structures including skin, breast, andprostate exhibited by p63-null mice previously suggestedan essential role for p63 isoforms in epithelial develop-ment.2,3 Subsequently, additional studies demonstratedthat p63 proteins are implicated not only in the stratifica-tion of squamous epithelia4 but also in the differentiationof mature keratinocytes5 and in the maintenance of theproliferative potential of epithelial stem cells.6 Recently, invitro studies have also shown that �Np63 isoforms inhibitTAp63 isoforms in a dose-dependent manner.7 In addi-tion to their role in normal development, a potential rolefor p63 proteins in tumorigenesis is supported by thefinding that p63 immunoreactivity is observed in morethan 90% of squamous epithelial malignancies.8 How-ever, because of the lack of reliable antibodies for �N

Supported by the Marshall Program of the Walloon Region (Neoangio N°616476), the Belgian Fund for Medical Scientific Research, the CentreAnti-Cancereux pres l’Universite de Liege, and the Faculty of Medicine ofthe University of Liege. P. Delvenne is a Senior Research Associate of theBelgian National Fund for Scientific Research. M.H. is a Research fellowof 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 Exper-imental Pathology, GIGA-Cancer, University of Liege, 4000 Liege, Belgium.E-mail: [email protected].

The American Journal of Pathology, Vol. 176, No. 4, April 2010

Copyright © American Society for Investigative Pathology

DOI: 10.2353/ajpath.2010.090804

1

Uncorrected Version. Published on February 11, 2010 as DOI:10.2353/ajpath.2010.090804

Copyright 2010 by the American Society for Investigative Pathology.

and TAp63, the p63 isoforms expressed in these malig-nant lesions were not determined in most studies. De-spite some data on their implication in apoptotic path-ways,9–12 the role of p63 proteins in cancer is still unclearand accumulating evidence suggests that p63 proteinscould exert both oncogenic and tumor suppressor func-tions (reviewed by Mills13).

First described in Drosophila melanogaster, Snail rep-resents the founding member of a superfamily of zinc-finger transcriptional regulators. Members of this familyare involved in the formation of mesoderm and neuralcrest as well as in the malignant progression of epithelialtumors. In mammals, the best characterized members ofthe Snail superfamily, Snail and Slug, have been eachimplicated in the loss of epithelial features associatedwith the acquisition of a fibroblast-like motile and invasivephenotype by tumors (reviewed by Nieto14).

The purpose of this study was to examine the implica-tion of Snail and Slug transcription factors in the regula-tion of p63 isoform expression. We showed that these twotranscriptional regulators repress �Np63 expression,which leads to an upregulation of TAp63. These in vitrodata were congruent with results obtained in tissue sam-ples from patients with cervical, esophageal, or head andneck squamous cell carcinoma (SCC). We also observedthat the loss of �Np63 associated with the induction ofTAp63 reduces cell–cell adhesion and increases the mi-gration of squamous malignant cells.

Materials and Methods

Patients and Tissue Samples

One hundred sixty specimens of SCC including 53 cer-vical SCC (mean age: 53 � 7 years), 58 head and neckSCC (50 men, 8 women, mean age: 56 � 9 years), and 39esophageal SCC (24 men, 15 women, mean age: 48 � 9years) were obtained from patients who underwent sur-gery at the University Hospital Center of Liege or Brusselsin the period 2002 to 2008. These tissue samples werecollected at the Tumor Bank of the University of Liege.Tissues were either frozen or fixed in 10% formalin andembedded in paraffin. The protocol was approved by theEthics Committee of the University Hospital of Liege.

Cell Cultures

Four genital SCC cell lines (A431, C4II, SiHa, CaSki) weregrown in a 3:1 mixture of Dulbecco’s modified Eagle’smedium (Gibco-Invitrogen, Carlsbad, CA) and Ham’sF12 (Gibco) containing 10% fetal calf serum (FCS) andsupplemented 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 main-tained in minimal essential medium (Gibco) and in RPMI-1640 (Gibco) containing 10% FCS and supplied with 1%L-glutamine (Gibco). All of the cell lines were incubated at37°C in a humidified CO2 atmosphere until a 50% to 60%confluence was reached.

Immunohistochemistry

Immunohistochemical analysis of frozen and paraffin-em-bedded specimens was performed as previously de-scribed.15 Briefly, paraffin sections were deparaffinized,rehydrated in graded alcohols, and antigens were re-trieved in EDTA or in citrate buffer, whereas frozen sec-tions were fixed with 4% paraformaldehyde and nonspe-cific 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 forthe primary reaction. Immunoperoxidase staining wasperformed using the Envision kit (Dako, Glostrup, Den-mark) according to the supplier’s recommendations. Pos-itive cells were visualized using a 3,3�-diaminobenzidine(DAB) substrate, and the sections were counterstainedwith hematoxylin.

Immunostaining Assessment

The immunolabeled tissues were evaluated by using asemiquantitative score of the intensity and extent of thestaining according to an arbitrary scale. For staining in-tensity, 0 represented samples in which the immunore-activity was undetectable, whereas 1, 2, and 3 denotedsamples with, respectively, a low, moderate, and strongstaining. For staining extent, 0, 1, 2, and 3 representedsamples 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 foreach case, the results obtained with the two scales weremultiplied, yielding a single scale of 0, �1, �2, �3, �4,�6, et �9.16,17 The biopsies were classified into fourgroups: high expression (score �3) for either Snail orSlug 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 obtainedusing a Microm HM 500 M cryostat (Microm International,Francheville, France) and mounted on glass slides cov-ered with a thin membrane (Carl Zeiss Microscopy, Mu-nich, Germany). Sections were then stained with Gill IIIhematoxylin (RNase free) (Merck, Darmstadt, Germany)for 1 minute, washed in distilled water, and dried on icefor a minimum of 30 minutes. Microdissection was per-formed using a P.A.L.M. microdissector (Carl Zeiss Mi-croscopy) and was supervised by a histopathologist toensure �95% specificity of capture tumor cells.

RT-PCR Analysis

One �g of total RNA extracted from either cell cultures orfrozen microdissected biopsies (RNeasy mini kit, Qiagen,Valencia, CA) and quantified with a ND-1000 spectropho-tometer (NanoDrop, Wilmington, DE) was reverse tran-scribed using Superscript II reverse transcriptase (In-vitrogen) according to the manufacturer’s instructions.

2 Herfs et alAJP April 2010, Vol. 176, No. 4

The reactions were performed at 42°C for 50 minutes,followed by inactivation of the enzyme at 75°C for 15minutes. The cDNA was stored at �20°C. For PCR reac-tions, primer sequences and annealing temperatureswere as follows: Pan-p63 forward, 5�-TCCTCAGGGAGCT-GTTATCC-3�; Pan-p63 reverse, 5�-ATTCACGGCTCAGCT-CATGG-3�, 56°C; TAp63 forward, 5�-TGTATCCGCATG-CAGGACT-3�; TAp63 reverse, 5�-CTGTGTTATAGG-GACTGGTGGAC-3�, 56°C; �Np63 forward, 5�-GAAAA-CAATGCCCAGACTCAA-3�; �Np63 reverse, 5�-TGC-GCGTGGTCTGTGTTA-3�, 56°C18; Snail forward, 5�-AATCGGAAGCCTAACTACAGCGAG-3�; Snail reverse,5�-CCTTCCCACTGTCCTCATCTGACA-3�, 65°C; Slugforward, 5�-CCTTCCTGGTCAAGAAGCATTTCA-3�; Slugreverse, 5�-AGGCTCACATATTCCTTGTCACAG-3�,65°C19; HPRT forward, 5�-TTGGATATAAGCCAGACTTT-GTTG-3�; HPRT reverse, 5�-AGATGTTTCCAAACT-CAACTTGAA-3�, 60°C. Thirty (36 for TAp63 detection)cycles, including denaturation at 94°C for 30 s, annealingfor 30 seconds and extension at 72°C for 1 minute, wereused for the analysis. Samples were run on 1.8% agarosegels containing ethidium bromide and visualized with anUV transilluminator.

Quantitative Real-Time RT-PCR Analysis

Total RNA was extracted, and cDNA was generated byreverse transcription as described above. For quantita-tive real-time PCR experiments, 25 ng of cDNA wereamplified in 50 �l of 1� SYBR-Green I qPCR master mixplus (Eurogentec, Seraing, Belgium), containing 200nmol/L of each primer for Pan-p63, TAp63, �Np63 (de-scribed above) or 300 nmol/L of following primers: Snailreverse, 5�-GTGGGATGGCTGCCAGC-3�; Snail forward,5�-TGCAGGACTCTAATCCAAGTTTACC-3�20; Slug re-verse, 5�-TCCGGAAAGAGGAGAGAGG-3�; Slug forward,5�-TGTGTGGACTACCGCTGC-3�21; N-cadherin reverse,5�-CTCCTATGAGTGGAACAGGAACG-3�; N-cadherin for-ward, 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°Cfor 10 minutes, 40 cycles of denaturation at 95°C for 15seconds and annealing/extension at 60°C for 1 minute. Allof the experiments were performed in triplicate, using theABI-Prism 7700 Sequence Detection System (Applied Bio-systems, Foster City, CA) and negative controls (master mixwithout any cDNA or RNA) were added in each run. Eachquantitative real-time PCR experiment was normalized tothe amount of HPRT mRNA from the same sample. Theacquired data were analyzed by Sequence Detector soft-ware, Version 1.9 (Applied Biosystems).

Western Blotting Analysis

Cells were lysed in a buffer containing 50 mmol/L Tris, pH7.5, 300 mmol/L NaCl, 1 mmol/L ethylenediaminetet-raacetic acid, 1% Igepal CA-630 (Sigma, St. Louis, MO),1 mmol/L phenylmethyl sulfonyl fluoride (Sigma), andprotease inhibitors (Roche, Bale, Switzerland). After

quantification (BCA protein assay; Pierce, Rockford, IL),20 �g of proteins were separated by electrophoresis on4% to 12% NuPAGE polyacrylamide gels (Invitrogen) andtransferred onto polyvinylidene difluoride membranes.The membranes were subsequently blocked with 5%skim milk for 30 minutes and incubated overnight at 4°Cwith anti–�-actin (Sigma), anti-�Np63 (anti-p40, Calbio-chem, Gibbstown, NJ), anti-TAp63 (Biolegend, San Di-ego, CA), anti-Snail (Abcam), anti-Slug (Clone G-18,Santa Cruz Biotechnology), anti–E-cadherin (BD Trans-duction Laboratories, Franklin Lakes, NJ), anti–N-cad-herin (Zymed Laboratories, San Francisco, CA), and anti-vimentin (Clone V9, Dako) antibodies. The membraneswere then washed with Tris-Buffered Saline Tween-20(TBS-T) and incubated with appropriate secondary anti-bodies. After washing, the protein bands were detectedusing an enhanced chemiluminescence system (ECLPlus; Amersham Biosciences, Piscataway, NJ).

siRNA Transfection and Gene Silencing

Small interfering RNA (siRNA) targeting human �Np63was designed (5�-UGCCCAGACUCAAUUUAGU-3�) andpurchased from Eurogentec. The sense and the anti-sense strands were annealed to obtain duplexes withidentical 3� overhangs. The sequence was submitted to aBLAST search against the human genome to ensure thespecificity of the siRNA. The day before transfection, 105

cells per well of a six-well plate were seeded in 3 ml ofappropriate growth medium. For each transfection, 50 ngof siRNA duplexes and 3 �l of Transfectin (Bio-Rad,Hercules, CA) were diluted in 1 ml of Optimem (Invitro-gen). The mixture was then incubated at room tempera-ture for 20 minutes to allow the formation of siRNA–liposome complexes. Growth medium was aspiratedfrom the cells and the transfecting solution was addeddrop by drop. The cells were incubated with the com-plexes for 4 hours at 37°C in a CO2 incubator. Afterincubation, 1 ml of growth medium (containing 20% ofserum) was added without removing the transfection mix-ture. Twenty-four hours after transfection, the mediumwas replaced with normal growth medium. The transfec-tion of an ATTO 647N-labeled control siRNA (Eurogentec,Seraing, Belgium) was also performed and revealed asiRNA uptake in more than 95% of the cells.

Transient Transfections of Slug, Snail, and p63cDNAs

To study the regulation of the endogenous p63 gene bySnail and Slug transcription factors, 1.5 � 105 cellsplated in six-well plates were transiently transfected witha pcDNA3.1 Zeo expression vector (Invitrogen) contain-ing a full-length human Slug sequence and/or a pEF6/Myc-His version A expression vector (Invitrogen) contain-ing a human Snail sequence using Exgene transfectionreagent (Fermentas, Burlington, Canada). Similar condi-tions were used to transfect expression vectors (pcDNA3)encoding each p63 isoform (provided by Dr. Caron deFromentel, INSERM U590, Lyon, France). Twenty-four

Snail/Slug-Mediated p63 Regulation in SCC 3AJP April 2010, Vol. 176, No. 4

hours after plating, transfection was performed as rec-ommended by the manufacturer by adding, in each well,a mixture containing 200 �l of 150 mmol/L NaCl, 9 �l ofExgene and 2 �g of the Slug and/or Snail expressionvector. As a control, cells were transfected with the cor-responding empty vector. At 24, 48, and 72 hours aftertransfection, cells were collected for RT-PCR or for West-ern blotting analyses. A control transfection conditionusing a plasmid encoding GFP (pEGFP-IRESpuro, Clon-tech, CA) was performed in parallel to determine thetransfection efficiency. All experiments were set up toobtain at least 60% of transfected cells.

Boyden Chamber Migration Assay

The migratory properties of cells transfected with thesi�Np63 were assessed using the Boyden chamber as-say. 104 cells were suspended in 55 �l of serum-freemedium supplemented with 0.1% BSA and placed in theupper compartment of a 48-well Boyden microchamber(Neuroprobe, Cabin John, MD). The lower compartmentwas filled with 27 �l of medium containing 10% FCS and1% BSA. After 18 hours of incubation at 37°C in a CO2

incubator, the cells that had migrated to the underside ofthe filter (Poretics Corp., Livermore, CA) were fixed andstained with Diff Quick Stain set (Baxter Diagnostics AG,Dudingen, Switzerland). The upper side of the filter wasscraped to remove residual nonmigrating cells. One ran-dom field was counted per well using an eyepiece with acalibrated grid to evaluate the number of fully migratedcells. Experiments were performed at least three times insixplicate.

Cell–Cell Adhesion Assay

This assay was performed as previously described byVessey et al.24 A single cell suspension of 2 � 106 cellsin 2 ml polystyrene tubes was magnetically stirred at37°C in a humidified CO2 atmosphere. The number ofsingle cells was determined using a hemocytometer attime 0, 20, 40, and 60 minutes. The degree of aggrega-tion was represented by the aggregation index Nt/N0,where N0 is the total number of single cells before incu-bation and Nt is the total number of single cells afterincubation for t min.

Statistical Analysis

Statistical analysis was performed with Instat 3 software(Graph-Pad Software, San Diego, CA). The statisticalsignificance of the results was calculated by using aStudent t test. Differences were considered as statisti-cally significant when P values were less than 0.05.

Results

TA- and �Np63 Expressions Show,Respectively, a Positive and NegativeCorrelation to Snail and Slug Levels inHuman SCC Cell Lines

To examine the possible relationship between Snail andSlug transcription factors and p63 isoforms, we first an-alyzed 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, Te-13) SCC cell lines. All of the cells expressed both Snailand 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 Snailand/or Slug expression. Interestingly, in contrast to othercell lines, SiHa, CasKi, and RPMI 2650 weakly expressed�Np63 isoforms whereas TAp63 isoforms were up-regu-lated. 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 areference for identifying p63 isoforms, cDNA correspondingto each isoform was transfected in SiHa cells and analyzedby Western blot (data not shown).

Snail and Slug Regulate p63 Isoform Expression

To determine the exact role of Snail and Slug in theregulation of p63 isoform expression, we transfectedSnail and Slug cDNA sequences in the A431 cell line. Asshown in Figures 2B and 3B, Western blot analyses indi-cated that �Np63� and TAp63� are, respectively, themajor �N and TA isotypes expressed in these cancerouscells. By using primers designed to amplify all p63 tran-scripts, we showed that the transient transfection of Snail

Figure 1. Expression of the p63 isoforms, Snailand Slug in human SCC cell lines. RT-PCR (A)and Western blot (B) analysis of Snail, Slug,�Np63, and TAp63 isoform expression wereperformed on nine human SCC cell lines. HPRTand �-actin were used as controls for RNA andprotein loading, respectively. A representativeexperiment is shown of three independent ex-periments performed.


and Slug cDNA for 24, 48, and 72 hours globally reducesp63 expression (Figures 2A and 3A). However, when thedifferent p63 isoforms were specifically analyzed, theexpressions of �Np63 and TAp63 were, respectively,down- and up-regulated by both Snail and Slug transcrip-tion factors (Figures 2 and 3). These results were ob-tained both at the mRNA and protein levels. No synergis-tic effect was detected when cells were transfected withboth Snail and Slug cDNA (data not shown). Similar re-sults were observed with the cervical C4-II cell line (datanot shown). To investigate whether the increase in TAp63expression was caused by Snail and Slug transcriptionfactors or whether it resulted from the loss of �Np63isoforms, which have a dominant-negative function onTAp63 isoforms, SiHa cells were transfected with Snailand/or Slug cDNA. As shown in Figure 1, this cervical

SCC cell line does not express �Np63. No up-regulationof TAp63 isoforms was observed in SiHa cells trans-fected with Snail or Slug cDNA (Supplemental Figure 2at http://ajp.amjpathol.org).

Snail and Slug Immunoreactivity Is Associatedwith a Global Loss of p63 in Cervical, Head andNeck, and Esophageal SCC

We next investigated the expression of Snail, Slug, andp63 (by using an antibody against all p63 isoforms) in 38cervical, 32 head and neck, and 25 esophageal paraffin-embedded SCC specimens. The immunostaining resultsare shown in Figure 4, A and B. Positive staining for Snailand Slug was observed in 88 tissue samples (93%). As

Figure 2. �N- and TAp63 isoform expressionsare, respectively, down- and up-regulated bySnail transfection. RT-PCR (A) and Western blot(B) analysis of Snail and p63 isoform expressionin A431 cells transiently transfected with SnailcDNA for 24, 48, or 72 hours. A representativeexperiment is shown of three independent ex-periments performed. C: Real-time RT-PCR anal-ysis of p63 isoform expression in A431 cellstransiently transfected with Snail cDNA for 24,48, or 72 hours. Each real-time RT-PCR experi-ment was normalized to the amount of HPRTmRNA from the same sample. Results are themeans � SD of four independent transfection ex-periments performed in duplicate. Asterisks indi-cate statistically significant differences (*P � 0.05,**P � 0.01, ***P � 0.001).

Figure 3. �N- and TAp63 isoform expressionsare, respectively, down- and up-regulated bySlug transfection. RT-PCR (A) and Western blot(B) analysis of Slug and p63 isoform expressionin A431 cells transiently transfected with SlugcDNA for 24, 48, or 72 hours. A representativeexperiment is shown of three independent ex-periments performed. C: Real-time RT-PCR anal-ysis of p63 isoform expression in A431 cellstransiently transfected with Slug cDNA for 24, 48,or 72 hours. Each real-time RT-PCR experimentwas normalized to the amount of HPRT mRNAfrom the same sample. Results are the means � SDof four independent transfection experiments per-formed in duplicate. Asterisks indicate statisticallysignificant differences (*P � 0.05, **P � 0.01).


shown in Figure 4A, variable degrees of nuclear Snail andSlug expression were detected. High expression of Snailand 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 Slugtranscription factors. In addition, we analyzed p63 ex-pression in all these SCC specimens and observed thattumors with a strongly positive Snail and/or Slug immu-noreactivity were significantly associated with a globaldown-regulation of p63, as found by the Student t test(Figure 4B). This inverse association was also observed innormal esophageal and exocervical epithelia. Accordingly,Snail and Slug transcription factors were detected in theupper epithelial cell layers whereas p63 was only presentin the (para)basal cells of the squamous epithelia (Sup-plemental Figure 3 at http://ajp.amjpathol.org).

Reactivation of TAp63 and Down-Regulation of�Np63 Occur in Human SCC when Snailand/or Slug Are Overexpressed

To test whether the expressions of p63 isoforms arealtered during tumorigenesis when Snail and/or Slug tran-scription factors are up-regulated, we microdissectedfrozen biopsies of human SCC (25 cervical, 26 head andneck, and 14 esophageal SCC), extracted total RNA, anddetermined the TA- and �Np63 expression levels byperforming quantitative real-time RT-PCR analysis. Snailand Slug abundance was determined by immunohisto-chemistry. After normalizing gene expression levels to

HPRT, we found that �N and TAp63 expressions were,respectively, significantly down- and up-regulated in cer-vical and head and neck SCC, which display high Snailand/or Slug immunoreactivity (Figure 5). Because of thelimited number of specimens, the differences were notstatistically significant in the esophageal SCC group.

�Np63 Silencing Alters Cell–Cell Adhesion andIncreases the Migration of Cancer Cells

To investigate the functional contribution of �Np63 down-regulation and TAp63 up-regulation in tumor invasionindependently of the well-known epithelial-mesenchymaltransition features induced by Snail and Slug, SCC celllines were transiently transfected with a �Np63 siRNA.Gene silencing efficiency was analyzed by Western blot(Figure 6A) and real-time RT-PCR (Figure 6B). Resultsindicated that �Np63 silencing significantly increasesTAp63 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 aggregatecompared with cells transfected with a control siRNA (Fig-ure 6C). Moreover, we observed that �Np63-silenced SCCcells exhibited significantly higher migratory properties inthe Boyden Chamber assay (Figure 6D).

Discussion

Events frequently observed in the malignant transforma-tion of epithelial cells include the loss of epithelial differ-entiation, a decrease in cell–cell contact, and the acqui-

Figure 4. A loss of p63 immunoreactivity is ob-served in human SCC overexpressing Snailand/or Slug. A: Snail, Slug, and p63 expressionsin paraffin-embedded sections of human SCCsurgical specimens were assessed by immuno-histochemistry. Variable degrees of Snail, Slug,and p63 expression were detected. B: Semi-quantitative evaluation of p63 expression in 38cervical, 32 head and neck, and 35 esophagealparaffin-embedded SCC specimens. The tissuesamples were classified into four groups accord-ing to Snail and Slug immunoreactivity (high:��, low: �/�). Asterisks indicate statisticallysignificant differences (*P � 0.05, **P � 0.01,***P � 0.001). Original magnifications: �200.


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 inthis phenomenon referred to as epithelial-to-mesenchy-

mal transition (reviewed by Nieto14). Accordingly, numer-ous studies have shown that ectopic expression of Snailand/or Slug in epithelial cells induces dramatic pheno-typic changes accompanied by increased cellular motilityand invasiveness.25–28 In addition, these two transcrip-tion factors have been reported to correlate negatively intransformed cell lines with epithelial marker expressionlevels such as E-cadherin, desmoplakin, cytokeratin 18,and MUC-1.26,29–31

Numerous studies have demonstrated that the p63proteins play a crucial role both in the initiation of epithe-lial stratification and in squamous differentiation. Al-though p63 functions in normal development are welldefined, the role for p63 in tumorigenesis still remainscontroversial. Several studies have highlighted the onco-genic potential of �Np63�.32–34 In contrast, other datasuggest that the p63 gene could act as a tumor suppres-sor,10,35 although p63 is rarely mutated in human cancerlike classic tumor suppressor genes.

In the present study, we demonstrated that �N andTAp63 isoform expressions are, respectively, repressedand increased by both Snail and Slug transcription fac-tors. However, as �Np63 isoforms are more expressedthan TAp63 isoforms, we observed a global reduction inp63 expression. Our results are in agreement with thoseof Higashikawa et al,36 who recently reported that Snail-induced epithelial-to-mesenchymal transition was ac-companied by a down-regulation of �Np63�. Interest-ingly, we found no up-regulation of TAp63 when Snailand/or Slug cDNA was transfected in cells lacking �Np63expression (SiHa cells), suggesting that TAp63 inductionis not directly caused by Snail and Slug transcriptionfactors but results from the loss of �Np63 isoforms, whichact as dominant-negative inhibitors of TAp63 isoforms.1,7

This hypothesis was strengthened by the increasedTAp63 expression observed after �Np63 silencing.

To evaluate the association between Snail and Slugtranscription factors and p63 isoform expression in hu-

Figure 5. �N- and TAp63 isoform expressions are, respectively, reducedand increased in human SCC displaying a high immunoreactivity for Snailand/or Slug. The expressions of �N- and TAp63 were determined in 25cervical, 26 head and neck, and 14 esophageal frozen SCC specimens. Thetissue samples were classified in four groups according to Snail and Slugimmunoreactivity (high: ��, low: �/�). Each real-time RT-PCR experimentwas normalized to the amount of HPRT mRNA from the same sample.Asterisks indicate statistically significant differences (*P � 0.05, **P � 0.01).

Figure 6. �Np63 silencing reduces cell aggre-gation and increases migratory abilities of cancercells. A: Western blot analysis of �N- and TAp63isoform expression in the �Np63 siRNA-express-ing A431 cells compared with the control cells. Arepresentative experiment is shown of three in-dependent experiments performed. B: �N- andTAp63 expression levels were also determinedby real-time RT-PCR. Each experiment was nor-malized to the amount of HPRT mRNA from thesame sample. Results are the means � SD offour independent transfection experiments per-formed in duplicate. C: Cell–cell adhesion of�Np63 siRNA-expressing cells compared withcontrol cells. The degree of aggregation wasrepresented by the aggregation index Nt/N0,where N0 is the total number of single cellsbefore incubation and Nt is the total number ofsingle cells after incubation for t min. D: Analysisof the migratory abilities of �Np63 siRNA-ex-pressing cells compared with control cells in theBoyden chamber assay. Data are expressed asfold induction for �Np63 siRNA-expressing cellsrelative to the control cells. Asterisks indicatestatistically significant differences (**P � 0.01,***P � 0.001).


man SCC specimens, we performed immunohistochem-ical and real-time RT-PCR analysis. Statistical analysisrevealed a significant reduction in p63 immunoreactivityin SCC when Snail and/or Slug were overexpressed. Inaddition, high expression of Snail and/or Slug was alsostatistically associated with an up- and down-regulationof TA- and �Np63, respectively, as already demonstratedby the in vitro experiments. Previous studies have shownthat global impaired p63 expression is associated withtumor progression and poor prognosis in bladder can-cer.37,38 Furthermore, aberrant overexpression of TAp63isoforms has been recently reported in head and neckSCC and associated with tumor development and metas-tasis.39 Compelling evidence has also accumulated onthe role of �Np63 proteins in squamous differentia-tion.5,40–42 Thus, a reduced expression of �Np63 iso-forms could be correlated with epithelial dedifferentiation,usually related to an aggressive growth pattern. Interest-ingly, unpublished data have demonstrated a correlationbetween p63 and differentiation markers such as keratin14 in head and neck and esophageal SCC. All these datasuggest a potential prognostic value of p63 and its reg-ulating factors in patient with SCC and may be related tothe effects of these molecules on cell adhesion and mi-gration. Accordingly, we demonstrated that �Np63-si-lenced SCC cells have a lower ability to aggregate andexhibit higher migratory properties compared with controlcells in cell–cell adhesion assay and Boyden chamberassay. In addition, we showed that the silencing of�Np63 in SCC cells results in the acquisition of mesen-chymal phenotypic traits associated with a marked up-regulation of N-cadherin expression (Supplemental Fig-ure 4 at http://ajp.amjpathol.org). In agreement with ourresults, recent studies have reported that the disruptionof p63 in mammary cells and in keratinocytes causedboth a down-regulation of cell-adhesion associatedgenes and an up-regulation of genes involved in invasionand metastasis.43,44 However, in contrast to our results,siRNAs targeting all p63 transcripts were used in thesestudies.

In conclusion, we demonstrated that Snail and Slugtranscription factors affect p63 isoform expression in hu-man SCC, which leads to increased cell migration andreduced cell–cell adhesion. Although further studies areneeded to identify both the upstream transcriptional reg-ulators and the downstream target genes for each p63isoform and to understand the complex role of p63 intumorigenesis, p63 and its regulating factors could con-stitute novel prognosis markers in patients with SCC andattractive targets for the therapeutic modulation of neo-plastic cell invasiveness.

Acknowledgments

We thank Patrick Roncarati for his excellent technicalassistance and Dr. Caron de Fromentel (INSERM U590,Lyon, France) for the generous gift of p63 plasmids.

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Regulation of p63 Isoforms by Snail and Slug Transcription Factors in Human Squamous Cell Carcinoma

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