Regulation of p63 Isoforms by Snail and Slug Transcription Factors in Human Squamous Cell Carcinoma
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090804
ISSN1525-2191
AutoresMichaël Herfs, Pascale Hubert, Meggy Suarez‐Carmona, Anca Reschner, Sven Saussez, Geert Berx, Pierre Savagner, Jacques Boniver, Philippe Delvenne,
Tópico(s)Cancer-related gene regulation
ResumoTP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that encode proteins with (TAp63) or without (ΔNp63) 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 ΔN 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 ΔNp63, 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 ΔNp63 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 for the therapeutic modulation of neoplastic cell invasiveness. TP63 is a p53-related gene that contains two alternative promoters, which give rise to transcripts that encode proteins with (TAp63) or without (ΔNp63) 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 ΔN 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 ΔNp63, 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 ΔNp63 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 for the therapeutic modulation of neoplastic cell invasiveness. 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 (ΔNp63) an amino-transactivating domain. Both TA and ΔN transcripts are alternatively spliced at the 3′ end to yield further carboxyl-terminal isotypes (α, β, γ).1Yang 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 dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar 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.2Mills 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: 708-713Crossref PubMed Scopus (1701) Google Scholar, 3Yang 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: 714-718Crossref PubMed Scopus (1908) Google Scholar Subsequently, additional studies demonstrated that p63 proteins are implicated not only in the stratification of squamous epithelia4Koster 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: 126-131Crossref PubMed Scopus (552) Google Scholar but also in the differentiation of mature keratinocytes5Truong AB Kretz M Ridky TW Kimmel R Khavari PA p63 regulates proliferation and differentiation of developmentally mature keratinocytes.Genes Dev. 2006; 20: 3185-3197Crossref PubMed Scopus (363) Google Scholar and in the maintenance of the proliferative potential of epithelial stem cells.6Senoo M Pinto F Crum CP McKeon F p63 is essential for the proliferative potential of stem cells in stratified epithelia.Cell. 2007; 129: 523-536Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar Recently, in vitro studies have also shown that ΔNp63 isoforms inhibit TAp63 isoforms in a dose-dependent manner.7Petitjean 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: 273-281Crossref PubMed Scopus (73) Google Scholar 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.8Di Como CJ Urist MJ Babayan I Drobnjak M Hedvat CV Teruya-Feldstein J Pohar K Hoos A Cordon-Cardo C p63 expression profiles in human normal and tumor tissues.Clin Cancer Res. 2002; 8: 494-501PubMed Google Scholar However, because of the lack of reliable antibodies for ΔN 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,9Barbieri 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: 2314-2320Crossref PubMed Scopus (72) Google Scholar, 10Flores 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: 560-564Crossref PubMed Scopus (723) Google Scholar, 11Gressner O Schilling T Lorenz K Schulze SE Koch A Schulze-Bergkamen 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: 2458-2471Crossref PubMed Scopus (233) Google Scholar, 12Wu 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 up-regulates the Hsp70 gene in human cancer.Cancer Res. 2005; 65: 758-766PubMed Google Scholar 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 Mills13Mills AA p63: oncogene or tumor suppressor?.Curr Opin Genet Dev. 2006; 16: 38-44Crossref PubMed Scopus (86) Google Scholar). First described in Drosophila melanogaster, Snail represents the founding member of a superfamily of zinc-finger 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 Nieto14Nieto MA The snail superfamily of zinc-finger transcription factors.Nat Rev Mol Cell Biol. 2002; 3: 155-166Crossref PubMed Scopus (1415) Google Scholar). 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 ΔNp63 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 ΔNp63 associated with the induction of TAp63 reduces cell–cell adhesion and increases the migration of squamous malignant cells. 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. 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 RPMI-1640 (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. Immunohistochemical analysis of frozen and paraffin-embedded specimens was performed as previously described.15Herfs 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: 603-614Crossref PubMed Scopus (54) Google Scholar 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. 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 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 +9.16Detry 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: 9-14Crossref PubMed Scopus (14) Google Scholar, 17Hubert 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: 346-355Crossref PubMed Scopus (79) Google Scholar 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 95% specificity of capture tumor cells. One μg 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. 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; ΔNp63 forward, 5′-GAAAACAATGCCCAGACTCAA-3′; ΔNp63 reverse, 5′-TGCGCGTGGTCTGTGTTA-3′, 56°C18Signoretti 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: 1769-1775Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar; Snail forward, 5′-AATCGGAAGCCTAACTACAGCGAG-3′; Snail reverse, 5′-CCTTCCCACTGTCCTCATCTGACA-3′, 65°C; Slug forward, 5′-CCTTCCTGGTCAAGAAGCATTTCA-3′; Slug reverse, 5′-AGGCTCACATATTCCTTGTCACAG-3′, 65°C19Herfs 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: 1391-1402Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; 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. 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 μl of 1× SYBR-Green I qPCR master mix plus (Eurogentec, Seraing, Belgium), containing 200 nmol/L of each primer for Pan-p63, TAp63, ΔNp63 (described above) or 300 nmol/L of following primers: Snail reverse, 5′-GTGGGATGGCTGCCAGC-3′; Snail forward, 5′-TGCAGGACTCTAATCCAAGTTTACC-3′20Rosivatz E Becker I Specht K Fricke E Luber B Busch R Hofler H Becker KF Differential expression of the epithelial-mesenchymal transition regulators snail. SIP1, and twist in gastric cancer.Am J Pathol. 2002; 161: 1881-1891Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar; Slug reverse, 5′-TCCGGAAAGAGGAGAGAGG-3′; Slug forward, 5′-TGTGTGGACTACCGCTGC-3′21Castro 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: 507-515Crossref PubMed Scopus (145) Google Scholar; N-cadherin reverse, 5′-CTCCTATGAGTGGAACAGGAACG-3′; N-cadherin forward, 5′-TTGGATCAATGTCATAATCAAGTGCTGTA-3′22Hotz 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: 4769-4776Crossref PubMed Scopus (343) Google Scholar; HPRT reverse, 5′-GGTCCTTTTCACCAGCAAGCT-3′; HPRT forward, 5′-TGACACTGGCAAAACAATGCA-3′.23Kleinewietfeld 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: 2877-2886Crossref PubMed Scopus (245) Google Scholar 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). 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 μg 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–β-actin (Sigma), anti-ΔNp63 (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 anti-vimentin (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). Small interfering RNA (siRNA) targeting human ΔNp63 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 μl 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. 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 hours after plating, transfection was performed as recommended by the manufacturer by adding, in each well, a mixture containing 200 μl of 150 mmol/L NaCl, 9 μl of Exgene and 2 μg 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. The migratory properties of cells transfected with the siΔNp63 were assessed using the Boyden chamber assay. 104 cells were suspended in 55 μl 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 μl 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. This assay was performed as previously described by Vessey et al.24Vessey 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: 151-159Crossref PubMed Scopus (139) Google Scholar 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 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. 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, Te-13) 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 ΔNp63 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). 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 ΔNp63α and TAp63γ are, respectively, the major ΔN 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 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 ΔNp63 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 ΔNp63 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 ΔNp63. 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).Figure 3ΔN- 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 paraffin-embedded 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 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). 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 ΔNp63 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 ΔN 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. To investigate the functional contribution of ΔNp63 down-regulation 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 ΔNp63 siRNA. Gene silencing efficiency was analyzed by Western blot (Figure 6A) and real-time RT-PCR (Figure 6B). Results indicated that ΔNp63 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 ΔNp63-silenced SCC cells have a lower ability to aggregate compared with cells transfected with a control siRNA (Figure 6C). Moreover, we observed that ΔNp63-silenced SCC cells exhibited significantly higher migratory properties in the Boyden Cha
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