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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]. 1941 1942 Herfs et al 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 1943 AJP 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 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 1944 Herfs et al AJP April 2010, Vol. 176, No. 4 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 Snail/Slug-Mediated p63 Regulation in SCC 1945 AJP April 2010, Vol. 176, No. 4 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). 1946 Herfs et al 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- Snail/Slug-Mediated p63 Regulation in SCC 1947 AJP April 2010, Vol. 176, No. 4 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. References 1. Yang A, Kaghad M, Wang Y, Gillett E, Fleming MD, Dotsch V, Andrews NC, Caput D, McKeon F: p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominantnegative activities. Mol Cell 1998, 2:305–316 2. Mills AA, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A: p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999, 398:708 –713 3. Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F: p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999, 398:714 –718 4. Koster MI, Kim S, Mills AA, DeMayo FJ, Roop DR: p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 2004, 18:126 –131 5. Truong AB, Kretz M, Ridky TW, Kimmel R, Khavari PA: p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev 2006, 20:3185–3197 6. Senoo M, Pinto F, Crum CP, McKeon F: p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell 2007, 129:523–536 7. Petitjean A, Ruptier C, Tribollet V, Hautefeuille A, Chardon F, Cavard C, Puisieux A, Hainaut P, Caron de FC: Properties of the six isoforms of p63: p53-like regulation in response to genotoxic stress and cross talk with DeltaNp73. Carcinogenesis 2008, 29:273–281 8. Di Como CJ, Urist MJ, Babayan I, Drobnjak M, Hedvat CV, TeruyaFeldstein J, Pohar K, Hoos A, Cordon-Cardo C: p63 expression profiles in human normal and tumor tissues. Clin Cancer Res 2002, 8:494 –501 9. Barbieri CE, Perez CA, Johnson KN, Ely KA, Billheimer D, Pietenpol JA: IGFBP-3 is a direct target of transcriptional regulation by DeltaNp63alpha in squamous epithelium. Cancer Res 2005, 65:2314 –2320 10. Flores ER, Tsai KY, Crowley D, Sengupta S, Yang A, McKeon F, Jacks T: p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 2002, 416:560 –564 11. Gressner O, Schilling T, Lorenz K, Schulze SE, Koch A, SchulzeBergkamen H, Lena AM, Candi E, Terrinoni A, Catani MV, Oren M, Melino G, Krammer PH, Stremmel W, Muller M: TAp63alpha induces apoptosis by activating signaling via death receptors and mitochondria. EMBO J 2005, 24:2458 –2471 12. Wu G, Osada M, Guo Z, Fomenkov A, Begum S, Zhao M, Upadhyay S, Xing M, Wu F, Moon C, Westra WH, Koch WM, Mantovani R, Califano JA, Ratovitski E, Sidransky D, Trink B: DeltaNp63alpha upregulates the Hsp70 gene in human cancer. Cancer Res 2005, 65:758 –766 13. Mills AA: p63: oncogene or tumor suppressor? Curr Opin Genet Dev, 2006, 16:38 – 44 14. Nieto MA: The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002, 3:155–166 15. Herfs M, Herman L, Hubert P, Minner F, Arafa M, Roncarati P, Henrotin Y, Boniver J, Delvenne P: High expression of PGE(2) enzymatic pathways in cervical (pre)neoplastic lesions and functional consequences for antigen-presenting cells. Cancer Immunol Immunother 2009, 58:603– 614 16. Detry C, Waltregny D, Quatresooz P, Chaplet M, Kedzia W, Castronovo V, Delvenne P, Bellahcene A: Detection of bone sialoprotein in human (pre)neoplastic lesions of the uterine cervix. Calcif Tissue Int 2003, 73:9 –14 17. Hubert P, Caberg JH, Gilles C, Bousarghin L, Franzen-Detrooz E, Boniver J, Delvenne P: E-cadherin-dependent adhesion of dendritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions. J Pathol 2005, 206:346 –355 18. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A, Montironi R, McKeon F, Loda M: p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 2000, 157:1769 –1775 19. Herfs M, Hubert P, Kholod N, Caberg JH, Gilles C, Berx G, Savagner P, Boniver J, Delvenne P: Transforming growth factor-beta1-mediated slug and snail transcription factor up-regulation reduces the density of Langerhans cells in epithelial metaplasia by affecting E-cadherin expression. Am J Pathol 2008, 172:1391–1402 20. Rosivatz E, Becker I, Specht K, Fricke E, Luber B, Busch R, Hofler H, Snail/Slug-Mediated p63 Regulation in SCC 1949 AJP April 2010, Vol. 176, No. 4 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Becker KF: Differential expression of the epithelial-mesenchymal transition regulators snail. SIP1, and twist in gastric cancer. Am J Pathol 2002, 161:1881–1891 Castro Alves C, Rosivatz E, Schott C, Hollweck R, Becker I, Sarbia M, Carneiro F, Becker KF: Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin. J Pathol 2007, 211:507–515 Hotz B, Arndt M, Dullat S, Bhargava S, Buhr HJ, Hotz HG: Epithelial to mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer. Clin Cancer Res 2007, 13:4769 – 4776 Kleinewietfeld M, Puentes F, Borsellino G, Battistini L, Rotzschke O, Falk K: CCR6 expression defines regulatory effector/memory-like cells within the CD25(⫹)CD4⫹ T-cell subset. Blood 2005, 105:2877–2886 Vessey CJ, Wilding J, Folarin N, Hirano S, Takeichi M, Soutter P, Stamp GW, Pignatelli M: Altered expression and function of E-cadherin in cervical intraepithelial neoplasia and invasive squamous cell carcinoma. J Pathol 1995, 176:151–159 Bolos V, Peinado H, Perez-Moreno MA, Fraga MF, Esteller M, Cano A: The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci 2003, 116:499 –511 Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA: The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2000, 2:76 – 83 Miyoshi A, Kitajima Y, Kido S, Shimonishi T, Matsuyama S, Kitahara K, Miyazaki K: Snail accelerates cancer invasion by upregulating MMP expression and is associated with poor prognosis of hepatocellular carcinoma. Br J Cancer 2005, 92:252–258 Usami Y, Satake S, Nakayama F, Matsumoto M, Ohnuma K, Komori T, Semba S, Ito A, Yokozaki H: Snail-associated epithelial-mesenchymal transition promotes oesophageal squamous cell carcinoma motility and progression. J Pathol 2008, 215:330 –339 De CB, Gilbert B, Stove C, Bruyneel E, van RF, Berx G: The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res 2005, 65:6237– 6244 Guaita S, Puig I, Franci C, Garrido M, Dominguez D, Batlle E, Sancho E, Dedhar S, de Herreros AG, Baulida J: Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem 2002, 277:39209 –39216 Savagner P, Yamada KM, Thiery JP: The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition. J Cell Biol 1997, 137:1403–1419 Hibi K, Trink B, Patturajan M, Westra WH, Caballero OL, Hill DE, 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. Ratovitski EA, Jen J, Sidransky, D: AIS is an oncogene amplified in squamous cell carcinoma. Proc Natl Acad Sci USA 2000, 97:5462–5467 Hu H, Xia SH, Li AD, Xu X, Cai Y, Han YL, Wei F, Chen BS, Huang XP, Han YS, Zhang JW, Zhang X, Wu M, Wang MR: Elevated expression of p63 protein in human esophageal squamous cell carcinomas. Int J Cancer 2002, 102:580 –583 Patturajan M, Nomoto S, Sommer M, Fomenkov A, Hibi K, Zangen R, Poliak N, Califano J, Trink B, Ratovitski E, Sidransky D: DeltaNp63 induces beta-catenin nuclear accumulation and signaling. Cancer Cell 2002, 1:369 –379 Flores ER, Sengupta S, Miller JB, Newman JJ, Bronson R, Crowley D, Yang A, McKeon F, Jacks T: Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell 2005, 7:363–373 Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N: Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007, 67:9207–9213 Koga F, Kawakami S, Fujii Y, Saito K, Ohtsuka Y, Iwai A, Ando N, Takizawa T, Kageyama Y, Kihara K: Impaired p63 expression associates with poor prognosis and uroplakin III expression in invasive urothelial carcinoma of the bladder. Clin Cancer Res 2003, 9:5501–5507 Urist MJ, Di Como CJ, Lu ML, Charytonowicz E, Verbel D, Crum CP, Ince TA, McKeon FD, Cordon-Cardo C: Loss of p63 expression is associated with tumor progression in bladder cancer. Am J Pathol 2002, 161:1199 –1206 Koster MI, Lu SL, White LD, Wang XJ, Roop DR: Reactivation of developmentally expressed p63 isoforms predisposes to tumor development and progression. Cancer Res 2006, 66:3981–3986 Boldrup L, Coates PJ, Gu X, Nylander K: DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck. J Pathol 2007, 213:384 –391 Romano RA, Birkaya B, Sinha S: A functional enhancer of keratin14 is a direct transcriptional target of deltaNp63. J Invest Dermatol 2007, 127:1175–1186 Romano RA, Ortt K, Birkaya B, Smalley K, Sinha S: An active role of the DeltaN isoform of p63 in regulating basal keratin genes K5 and K14 and directing epidermal cell fate. PLoS ONE 2009, 4:e5623 Barbieri CE, Tang LJ, Brown KA, Pietenpol JA: Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis. Cancer Res 2006, 66:7589 –7597 Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, Brugge JS, Ellisen LW: p63 regulates an adhesion programme and cell survival in epithelial cells. Nat Cell Biol 2006, 8:551–561