Snail Family Transcription Factors Are Implicated in Thyroid Carcinogenesis
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.061211
ISSN1525-2191
AutoresRobert G. Hardy, Carolina Vicente‐Dueñas, Inés González‐Herrero, C E Anderson, Teresa Flores, Sharon Hughes, Chris Tselepis, James A. Ross, Isidro Sánchez-Garcı́a,
Tópico(s)Kruppel-like factors research
ResumoE-Cadherin (CDH1) expression is reduced in thyroid carcinomas by primarily unknown mechanisms. In several tissues, SNAIL (SNAI1) and SLUG (SNAI2) induce epithelial-mesenchymal transition by altering target gene transcription, including CDH1 repression, but these transcription factors have not been studied in thyroid carcinoma. Recently, our group has provided direct evidence that ectopic SNAI1 expression induces epithelial and mesenchymal mouse tumors. SNAI1, SNAI2, and CDH1 expression were analyzed in thyroid-derived cell lines and samples of human follicular and papillary thyroid carcinoma by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The effect of SNAI1 expression on CDH1 transcription was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting in ori-3 cells. Thyroid carcinoma development was analyzed in CombitTA-Snail mice, in which SNAI1 levels are up-regulated. SNAI1 and SNAI2 were not expressed in cells derived from normal thyroid tissue, or in normal human thyroid samples, but were highly expressed in cell lines derived from thyroid carcinomas, in human thyroid carcinoma samples, and their metastases. SNAI1 expression in ori-3 cells repressed CDH1 transcription. Combi-TA mice developed papillary thyroid carcinomas, the incidence of which was increased by concomitant radiotherapy. In conclusion, SNAI1 and SNAI2 are ectopically expressed in thyroid carcinomas, and aberrant expression in mice is associated with papillary carcinoma development. E-Cadherin (CDH1) expression is reduced in thyroid carcinomas by primarily unknown mechanisms. In several tissues, SNAIL (SNAI1) and SLUG (SNAI2) induce epithelial-mesenchymal transition by altering target gene transcription, including CDH1 repression, but these transcription factors have not been studied in thyroid carcinoma. Recently, our group has provided direct evidence that ectopic SNAI1 expression induces epithelial and mesenchymal mouse tumors. SNAI1, SNAI2, and CDH1 expression were analyzed in thyroid-derived cell lines and samples of human follicular and papillary thyroid carcinoma by reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The effect of SNAI1 expression on CDH1 transcription was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting in ori-3 cells. Thyroid carcinoma development was analyzed in CombitTA-Snail mice, in which SNAI1 levels are up-regulated. SNAI1 and SNAI2 were not expressed in cells derived from normal thyroid tissue, or in normal human thyroid samples, but were highly expressed in cell lines derived from thyroid carcinomas, in human thyroid carcinoma samples, and their metastases. SNAI1 expression in ori-3 cells repressed CDH1 transcription. Combi-TA mice developed papillary thyroid carcinomas, the incidence of which was increased by concomitant radiotherapy. In conclusion, SNAI1 and SNAI2 are ectopically expressed in thyroid carcinomas, and aberrant expression in mice is associated with papillary carcinoma development. Thyroid cancer consists of several histological subtypes, including follicular and papillary carcinoma. These two histiotypes exhibit both common and individual alterations in gene expression compared with normal thyroid tissue. At the molecular level, alterations in expression of various proteins have been found between normal and tumor tissue including growth factors, eg, transforming growth factor-β1, epidermal growth factor; proteins regulating the cell cycle and apoptosis, eg, retinoblastoma, cyclin D1; cell adhesion molecules, such as E-cadherin (CDH1) and CD44; and tyrosine kinases and their receptors, eg, RET.1Segev DL Umbricht C Zeiger MA Molecular pathogenesis of thyroid cancer.Surg Oncol. 2003; 12: 69-90Abstract Full Text Full Text PDF PubMed Scopus (77) Google ScholarCadherins are transmembrane proteins mediating homotypic cell-cell adhesion. At a cellular level their expression induces differentiation and growth suppression, which translates at an organ level into the formation of polarized, ordered tissues.2Mege RM Matsuzaki F Gallin WJ Goldberg JI Cunningham BA Edelman GM Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules.Proc Natl Acad Sci USA. 1988; 5: 7274-7278Crossref Scopus (244) Google Scholar The prototypical member, CDH1, is highly expressed in the normal thyroid gland and in benign thyroid lesions, including toxic diffuse goiter, multinodular goiter, and follicular adenomas.3Brabant G Hoang-Vu C Cetin Y Dralle H Scheumann G Molne J Hansson G Jansson S Ericson LE Nilsson M E-cadherin: a differentiation marker in thyroid malignancies.Cancer Res. 1993; 53: 4987-4993PubMed Google Scholar CDH1 expression leads to aggregation of thyrocytes in culture,4Yap AS Stevenson BR Keast JR Manley SW Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation.Endocrinology. 1995; 136: 4672-4680Crossref PubMed Scopus (50) Google Scholar and exposure of thyrocytes to thyroid-stimulating hormone increases CDH1 transcription,5Brabant G Hoang-Vu C Behrends J Cetin Y Potter E Dumont JE Maenhaut C Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro.Endocrinology. 1995; 136: 3113-3119Crossref PubMed Scopus (42) Google Scholar demonstrating the intimate relationship between CDH1 expression and thyroid differentiation.CDH1 expression is decreased in classic papillary thyroid carcinoma3Brabant G Hoang-Vu C Cetin Y Dralle H Scheumann G Molne J Hansson G Jansson S Ericson LE Nilsson M E-cadherin: a differentiation marker in thyroid malignancies.Cancer Res. 1993; 53: 4987-4993PubMed Google Scholar, 6Serini G Trusolino L Saggiorato E Cremona O De Rossi M Angeli A Orlandi F Marchisio PC Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue.J Natl Cancer Inst. 1996; 88: 442-449Crossref PubMed Scopus (96) Google Scholar, 7Soares P Berx G van Roy F Sobrinho-Simoes M E-cadherin gene alterations are rare events in thyroid tumors.Int J Cancer. 1997; 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194: 358-366Crossref PubMed Scopus (67) Google Scholar and also in follicular carcinoma.3Brabant G Hoang-Vu C Cetin Y Dralle H Scheumann G Molne J Hansson G Jansson S Ericson LE Nilsson M E-cadherin: a differentiation marker in thyroid malignancies.Cancer Res. 1993; 53: 4987-4993PubMed Google Scholar, 7Soares P Berx G van Roy F Sobrinho-Simoes M E-cadherin gene alterations are rare events in thyroid tumors.Int J Cancer. 1997; 70: 32-38Crossref PubMed Scopus (83) Google Scholar, 8Cerrato A Fulciniti F Avallone A Benincasa G Palombini L Grieco M β- and γ-Catenin expression in thyroid carcinomas.J Pathol. 1998; 185: 267-272Crossref PubMed Scopus (63) Google Scholar, 11von Wasielewski R Rhein A Werner M Scheumann GF Dralle H Potter E Brabant G Georgii A Immunohistochemical detection of E-cadherin in differentiated thyroid carcinomas correlates with clinical outcome.Cancer Res. 1997; 57: 2501-2507PubMed Google Scholar, 12Naito A Iwase H Kuzushima T Nakamura T Kobayashi S Clinical significance of E-cadherin expression in thyroid neoplasms.J Surg Oncol. 2001; 76: 176-180Crossref PubMed Scopus (39) Google Scholar, 13Smyth P Sheils O Finn S Martin C O'Leary J Sweeney EC Real-time quantitative analysis of E-cadherin expression in ret/PTC-1-activated thyroid neoplasms.Int J Surg Pathol. 2001; 9: 265-272Crossref PubMed Scopus (16) Google Scholar Rarely, this decrease in expression is attributable to mutation of the CDH1 gene,7Soares P Berx G van Roy F Sobrinho-Simoes M E-cadherin gene alterations are rare events in thyroid tumors.Int J Cancer. 1997; 70: 32-38Crossref PubMed Scopus (83) Google Scholar, 9Rocha AS Soares P Seruca R Maximo V Matias-Guiu X Cameselle-Teijeiro J Sobrinho-Simoes M Abnormalities of the E-cadherin/catenin adhesion complex in classical papillary thyroid carcinoma and in its diffuse sclerosing variant.J Pathol. 2001; 194: 358-366Crossref PubMed Scopus (67) Google Scholar loss of heterozygosity,7Soares P Berx G van Roy F Sobrinho-Simoes M E-cadherin gene alterations are rare events in thyroid tumors.Int J Cancer. 1997; 70: 32-38Crossref PubMed Scopus (83) Google Scholar or hypermethylation of the CDH1 promoter.9Rocha AS Soares P Seruca R Maximo V Matias-Guiu X Cameselle-Teijeiro J Sobrinho-Simoes M Abnormalities of the E-cadherin/catenin adhesion complex in classical papillary thyroid carcinoma and in its diffuse sclerosing variant.J Pathol. 2001; 194: 358-366Crossref PubMed Scopus (67) Google Scholar In most tumors the mechanism of CDH1 down-regulation is, however, unknown.Several transcriptional repressors have been shown to modulate cadherin expression, including Snail family members SNAIL (SNAI1)14Cano 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: 76-83Crossref PubMed Scopus (2871) Google Scholar, 15Batlle E Sancho E Franci C Dominguez D Monfar M Baulida J Garcia D Herreros A The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.Nat Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2149) Google Scholar and SLUG (SNAI2).16Hajra KM Chen DY Fearon ER The SLUG zinc-finger protein represses E-cadherin in breast cancer.Cancer Res. 2002; 62: 1613-1618PubMed Google Scholar, 17Bolós V Peinado H Perez-Moreno MA Fraga MF Esteller MA 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: 499-511Crossref PubMed Scopus (911) Google Scholar These transcription factors contain zinc finger motifs and bind to E-boxes in the CDH1 promoter, repressing gene transcription14Cano 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: 76-83Crossref PubMed Scopus (2871) Google Scholar, 15Batlle E Sancho E Franci C Dominguez D Monfar M Baulida J Garcia D Herreros A The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.Nat Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2149) Google Scholar and producing changes in cell phenotype consistent with epithelial to mesenchymal transition including migration, invasion, resistance to cell death, and induction of angiogenesis.18Barrallo-Gimeno A Nieto MA The Snail genes as inducers of cell movement and survival: implications in development and cancer.Development. 2005; 132: 3151-3161Crossref PubMed Scopus (1115) Google ScholarSNAI1 and SNAI2 are ectopically expressed in several tumor cell lines.14Cano 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: 76-83Crossref PubMed Scopus (2871) Google Scholar Furthermore, SNAI1 has been found to be expressed in a number of human tumors derived from epithelial tissues, including gastric and hepatic carcinoma,19Rosivatz 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 (508) Google Scholar, 20Sugimachi K Tanaka S Kameyama T Taquchi K Aishima S Shimada M Sugimachi K Tsuneyoshi M Transcriptional repressor snail and progression of human hepatocellular carcinoma.Clin Cancer Res. 2003; 9: 2657-2664PubMed Google Scholar, 21Miyoshi 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: 252-258PubMed Google Scholar with expression in the latter correlating with both tumor invasiveness and prognosis.20Sugimachi K Tanaka S Kameyama T Taquchi K Aishima S Shimada M Sugimachi K Tsuneyoshi M Transcriptional repressor snail and progression of human hepatocellular carcinoma.Clin Cancer Res. 2003; 9: 2657-2664PubMed Google Scholar, 21Miyoshi 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: 252-258PubMed Google Scholar Recently, we provided the first direct experimental evidence that SNAI1 was linked to tumorigenesis by showing that mice in which SNAI1 was overexpressed by 20% developed both epithelial and mesenchymal tumors.22Pérez-Mancera PA Perez-Caro M Gonzalez-Herrero I Flores T Orfao A de Herreros AG Gutierrez-Adan A Pintado B Sagrera A Sanchez-Martin M Sanchez-Garcia I Cancer development induced by graded expression of Snail in mice.Hum Mol Genet. 2005; 14: 3449-3461Crossref PubMed Scopus (53) Google Scholar Embryonic fibroblasts derived from these mice were subsequently shown to induce tumor formation in nude mice.22Pérez-Mancera PA Perez-Caro M Gonzalez-Herrero I Flores T Orfao A de Herreros AG Gutierrez-Adan A Pintado B Sagrera A Sanchez-Martin M Sanchez-Garcia I Cancer development induced by graded expression of Snail in mice.Hum Mol Genet. 2005; 14: 3449-3461Crossref PubMed Scopus (53) Google ScholarWe sought to investigate the pattern of SNAI1 and SNAI2 expression in the normal human thyroid gland, papillary and follicular thyroid cancers, and transformed lines derived from thyroid carcinomas. We subsequently examined the effects of SNAI1 expression in thyroid cells and analyzed thyroid carcinoma development in mice overexpressing SNAI1.Materials and MethodsHuman Tissue SpecimensFormalin-fixed, paraffin-embedded sections of follicular thyroid carcinoma (n = 31) and papillary thyroid carcinoma (n = 32) were obtained from the pathology archives of the Royal Infirmary of Edinburgh, Edinburgh, UK, and University Hospital Birmingham, Birmingham, UK. Sections of formalin-fixed normal human skin and colorectal and prostate carcinomas were used as positive and negative tissue controls for immunohistochemistry. Ethical approval was obtained for the use of all human material (LREC reference 04/S1101/59). Pathological stage of tumors used was as follows: follicular: minimally invasive, n = 17; invasive, n = 14; papillary: T1, n = 6; T2, n = 11; T3, n = 5; T4, n = 10.Antibodies, Tissue Culture, and PlasmidsPrimary antibodies were as follows. CDH1 antibody (clone 36) was obtained from BD Biosciences, Oxford, UK, and used at a concentration of 1:300 for immunohistochemistry, 1:300 for immunofluorescence, and 1:5000 for Western blotting. SNAI1 and SNAI2 antibodies and specific blocking peptides were obtained from Autogen Bioclear, Wiltshire, UK. SNAI1 (clone E18) was used at a dilution of 1:10 for immunohistochemistry, 1:50 for immunofluorescence, and 1:100 for Western blotting. SNAI2 (clone G18) was used at 1:20 for immunohistochemistry, 1:50 for immunofluorescence, and 1:100 for Western blotting. Monoclonal anti-SNAI1 antibody was a gift from Antonio Garcia Herreros (Unitat de Biologia Cellular i Molecular, Barcelona, Spain) and has been previously described23Francí C Takkunen M Dave N Alameda F Gomez S Rodriguez R Escriva M Montserrat-Sentis B Baro T Garrido M Bonilla F Virtanen I Garcia de Herreros A Expression of Snail protein in tumor-stroma interface.Oncogene. 2006; 25: 5134-5144PubMed Google Scholar and was used at 1:1000 for Western blotting. Mouse monoclonal anti-β-actin (clone AC-15) was obtained from Abcam (Cambridge, UK) and used at 1:10,000 in Western blotting. Secondary antibodies were as follows: goat anti-mouse IgG-horseradish peroxidase-conjugated (Upstate, Dundee, Scotland) and rabbit anti-goat IgG-horseradish peroxidase-conjugated (Autogen Bioclear) were used at 1:5000 for Western blotting. Fluorescein isothiocyanate-linked anti-mouse IgG2a (Sigma, Gillingham, UK) was used at a 1:800 dilution and fluorescein isothiocyanate-linked rabbit anti-goat IgG (Sigma) was used at a 1:200 dilution, both for immunofluorescence.K1 and ori-3 cells were a gift from Professor D. Wynford-Thomas, Department of Pathology, University of Cardiff, Cardiff, UK. B-CPAP, CAL-62, FTC-133, and 8305C cells were a gift from Professor M. d'Armiento, Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," Rome, Italy. Lines were derived from the following human tissues: ori-3, SV40-transformed normal thyrocytes; B-CPAP, K1, papillary thyroid carcinoma; FTC-133, follicular thyroid carcinoma; CAL-62, 8305C, anaplastic thyroid carcinoma. Culture media for individual lines was as follows: B-CPAP, K1, ori-3, 8305C, RPMI 1640 medium + 10% fetal calf serum; CAL-62, Dulbecco's modified Eagle's medium + 5% fetal calf serum; FTC-133, Dulbecco's modified Eagle's medium + Ham's F12 (1:1) + 10% fetal calf serum + 2 mmol/L glutamine. All culture media, serum, and glutamine were purchased from Life Technologies, Paisley, UK, and lines were maintained in a 5% CO2 humidified atmosphere at 37°C. The plasmid pCAG-hSnail was a gift from Professor S. Tsukita, Department of Cell Biology, University of Kyoto, Kyoto, Japan, and has been described previously.24Ikenouchi J Matsuda M Furuse M Tsukita S Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail.J Cell Sci. 2003; 116: 1959-1967Crossref PubMed Scopus (530) Google ScholarCell Transfection, RNA Extraction, and cDNA FormationTo prepare for transfection, 2 × 105 cells were cultured in six-well plates 12 hours before transfection. Cells were then transfected with the plasmid pCAG-hSnail using Fugene 6 reagent (Roche Diagnostics, Lewes, UK) at a plasmid/DNA ratio of 3:2, according to the manufacturer's instructions. Cells were separated from the culture dish by scraping 24 hours after transfection, and RNA was harvested using TRIzol reagent (Invitrogen, Paisley, UK) according to the manufacturer's protocol. cDNA was produced using a reverse transcription kit (Promega, Southampton, UK) after prior treatment of RNA with 1 U of DNase (Promega) per reaction with incubation at 37°C for 30 minutes. Synthesized cDNA was used in subsequent polymerase chain reactions. Cell transfection experiments were performed on three separate occasions, after initial transfection efficiencies of 70 to 80% were documented in ori-3 cells.Total RNA from mice was isolated in two steps using TRIzol (Life Technologies, Inc., Grand Island, NY) followed by RNeasy mini-kit (Qiagen Inc., Valencia, CA) purification following the manufacturer's protocols. RNA was cleaned up with the optional on-column DNase treatment. The integrity and quality of RNA were verified by electrophoresis, and RNA concentration was measured. To analyze expression of CombitTA-Snail in mice, reverse transcriptase (RT) was performed according to the manufacturer's protocol in a 20-μl reaction containing 50 ng of random hexamers, 3 μg of total RNA, and 200 U of Superscript II RNase H− reverse transcriptase (Gibco/BRL, Paisley, UK).Polymerase Chain ReactionOligonucleotides (TAGN, Gateshead, UK) used for amplification from cell lines and polymerase chain reaction (PCR) conditions were as follows, with an initial denaturation step of 94°C for 5 minutes in all cases. CDH1: forward 5′-TGCCCAGAAAATGAAAAAGG-3′, reverse 5′-GTGTATGTGGCAATGCGTTC-3′, with 35 cycles of 94°C × 30 seconds, 58°C × 1 minute, and 72°C × 1 minute. SNAI1: forward 5′-AATCGGAAGCCTAACTACAAG-3′, reverse 5′-AGGAAGAGACTGAAGTAGAG-3′ with 35 cycles of 94°C × 1 minute, 53°C × 1 minute, and 72°C × 1 minute. SNAI2: forward 5′-GCCTCCAAAAAGCCAAACTA-3′, reverse 5′-CACAGTGATGGGGCTGATG-3′ with 35 cycles of 94°C × 1 minute, 53°C × 1 minute, and 72°C × 1 minute. PCR reagent (Promega) concentrations per reaction were as follows: oligonucleotides, 25 pmol; MgCl2, 15 mmol/L; and Taq polymerase, 2 U.To analyze expression of CombitTA-Snail in mice, reverse transcription was performed according to the manufacturer's protocol in a 20-μl reaction containing 50 ng of random hexamers, 3 μg of total RNA, and 200 U of Superscript II RNase H− reverse transcriptase (Gibco/BRL). Exogenous SNAI1 gene product expression in mice was analyzed with mSnailF and Combi-polyA-B1 primers: Combi-polyA-B1: 5′-TTGAGTGCATTCTAGTTGTG-3′; mSnailF: 5′-CAGCTGGCCAGGCTCTCGGT-3′. The PCR conditions used to amplify CombitTA-Snail were as follows: 94°C × 1 minute, 56°C × 1 minute, and 72°C × 2 minutes for 40 cycles. Mouse E-cadherin expression was analyzed with the following primers: mEcadherin forward: 5′-GGACGTCCATGTGTGTGA-3′; mEcadherin reverse: 5′-CTTCTACACACTCAGGGA-3′. The PCR conditions used to amplify E-cadherin were as follows: 94°C × 1 minute, 52°C ×1 minute, and 72°C × 2 minutes for 40 cycles. The PCR products were confirmed by hybridization with specific internal probes. Amplification of β-actin RNA served as a control to assess the quality of each RNA sample from both cell lines and mice.Immunohistochemistry for SNAI1, SNAI2, and CDH1Immunohistochemistry for CDH1 was performed as follows. Sections were dewaxed, incubated in methanol:hydrogen peroxide (H2O2) (10:1) for 5 minutes, and antigen retrieval was performed in 0.01 mol/L sodium citrate three times for 5 minutes each in a microwave. Sections were incubated in normal goat serum for 30 minutes and then in primary antibody for 60 minutes. After washing in Tris-buffered saline (TBS) sections were incubated in horseradish peroxidase Envision solution (DAKO, Ely, UK) for 60 minutes. Diaminobenzidine tetrahydrochloride (DAKO) was then used as a substrate for the peroxidase reaction at a concentration of 1 mg/ml + 1 μl/ml H2O2, and slides were dipped in hematoxylin, dehydrated, and mounted.Immunohistochemistry for SNAI1 and SNAI2 was performed as follows. Slides were immersed in W-cap buffer (Bio-Optica, Milan, Italy) and cycled in a Pixel antigen retriever (CellPath, Newtown, UK) for 60 minutes, washed in running water, and placed in methanol/hydrogen peroxide (10:1) for 5 minutes. Sections were then incubated in primary antibody in TBS 7.5 × buffer (Bios Europe Ltd., Skelmersdale, UK) at 4°C overnight, washed with TBS, and reacted with peroxidase-linked rabbit anti-sheep antibody (DAKO) at a 1:100 dilution in TBS for 1 hour. The immunoreactivity was revealed as above using diaminobenzidine tetrahydrochloride. Slides were then dipped in hematoxylin, dehydrated, and mounted. To control for SNAI1 and SNAI2 antibody specificity, primary antibodies were combined with a 10-fold excess of blocking peptide, incubated for 2 hours at room temperature, and then used as previously described for immunohistochemistry.Intensity and subcellular localization of reactivity was scored as previously described.25Hardy RG Tselepis C Hoyland J Wallis Y Pretlow TP Talbot I Sanders DS Matthews G Morton D Jankowski JA Aberrant P-cadherin expression is an early event in hyperplastic and dysplastic transformation in the colon.Gut. 2002; 50: 513-519Crossref PubMed Scopus (58) Google Scholar In brief, intensity of staining (graded 0 to 3) and percentage of positively staining follicles per lesion, subcellular localization of immunoreactivity, and correlation between positive immunoreactivity, tissue phenotype/morphology, and localization within tumor (ie, centrally or at invasive front) were recorded. The presence of morphologically normal follicles within each section served as additional internal negative/positive controls depending on antibody used. Correlation of staining with tumor characteristics was assessed using Fisher's exact test.Immunofluorescence for SNAI1, SNAI2, and CDH1Cells were grown on coverslips, and then immunofluorescence was performed on confluent cells as described. Cells were washed with phosphate-buffered saline (PBS), fixed with ice-cold methanol/acetone (1:1 ratio) for 5 minutes, and then washed with PBS. Cells were treated with a 0.1% solution of Triton-X for 20 minutes then blocked with a 5% solution of normal goat serum for 20 minutes. Primary antibodies were constituted in 5% normal goat serum and applied for 1 hour; secondary antibody was used for 30 minutes, with multiple washes with 1% bovine serum albumin after both primary and secondary antibody stages. 4,6-Diamidino-2-phenylindole (DAPI) (Sigma) was used at a concentration of 1:10,000 for 1 minute to stain nuclei. Coverslips were then mounted onto slides with immunofluorescence mounting media (Sigma). A Leica fluorescence microscope (Leica, Wetzlar, Germany) was used to analyze sections.Protein Lysate Formation and Western BlottingProtein lysates were made from cell lines by washing in PBS then lysing in radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma). Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hemel Hempstead, UK). For Western blotting, equal concentrations of protein lysates were separated on 8% (CDH1) or 15% (SNAI1 and SNAI2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and proteins were transferred onto Hybond polyvinylidene difluoride nitrocellulose membrane (Amersham, High Wycombe, UK). Membranes were blocked in 10% Marvel in TBS for 20 minutes and then incubated with primary antibodies in TBS-Tween at 4°C overnight. Blots were washed extensively with TBST, probed with secondary antibody, washed in TBS-Tween, and reacted with enhanced chemiluminescence reagent (Amersham) according to the manufacturer's instructions.Mice and TreatmentsAnimals were housed under nonsterile conditions in a conventional animal facility. CombitTA-Snail mice have been previously described.22Pérez-Mancera PA Perez-Caro M Gonzalez-Herrero I Flores T Orfao A de Herreros AG Gutierrez-Adan A Pintado B Sagrera A Sanchez-Martin M Sanchez-Garcia I Cancer development induced by graded expression of Snail in mice.Hum Mol Genet. 2005; 14: 3449-3461Crossref PubMed Scopus (53) Google Scholar Similar phenotypic features were seen in all assays for both of the CombitTA-Snail transgenic lines generated. To study the effect of irradiation on thyroid carcinoma development, 30 CombitTA-Snail and 30 control mice 5 to 6 weeks of age were irradiated with a dose of 400 rads and maintained in microisolator cages on sterilized food and acidified sterile water.For the effect of SNAI1 suppression in thyroid carcinomas after radiation treatment, stock solutions of 4 mg/ml doxycycline (Sigma) were prepared fresh in water and sucrose and administered to mice in the drinking water. Mice were started on doxycycline or placebo (the same volume of diluent) beginning on the day after thyroid carcinoma was confirmed (day 0). Mice tolerated the treatment well, and no interruption of therapy was necessary. Mice were followed clinically three times a week. The death endpoint was determined either by spontaneous death of the animal or by elective sacrificing of the animal because of signs of pain or suffering according to established criteria.Histological Analysis of MiceMice included in this study were subjected to standard necropsy. All major organs were closely examined under the dissecting microscope, and samples of each organ were processed into paraffin, sectioned, and examined histologically. All tissue samples were taken from homogenous and viable portions of the resected sample by the pathologist and were fixed within 2 to 5 minutes of excision. Hematoxylin and eosin (H&E)-stained sections of each tissue were reviewed by a single pathologist (T.F.). For comparative studies, age-matched mice were used (wild-type or CombitTA-Snail mice in the continuous presence of tetracycline). Statistical analysis of rates of tumor development in wild type compared with CombiTA-Snail mice was analyzed using Fisher's exact test.Cell Transfers and Tumorigenicity AssayTo test the tumorigenicity of the various CombitTA-Snail thyroid cancers, 4- to 6-week-old athymic (nude) male mice were injected subcutaneously on both flanks with 106 cells suspended in 200 μl of PBS. The animals were examined for tumor formation once a week and were sacrificed for histopathological studies and collection of tissues for DNA analyses when moribund.ResultsSNAI1 and SNAI2 mRNA and Protein Are Expressed in Thyroid Carcinoma Cell LinesTo test the initial hypothesis that Snail family transcription factors would be expressed in thyroid carcinoma, a panel of human thyroid cell lines were screened for SNAI1 and SNAI2 mRNA expression by reverse transcription-polymerase
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