Follicular Thyroid Tumors with the PAX8-PPARγ1 Rearrangement Display Characteristic Genetic Alterations
2005; Elsevier BV; Volume: 167; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62967-7
ISSN1525-2191
AutoresLudovic Lacroix, Vladimir Lazar, Stefan Michiels, Hugues Ripoche, Philippe Dessen, Monique Talbot, Bernard Caillou, Jean-Pierre Levillain, Martin Schlumberger, Jean‐Michel Bidart,
Tópico(s)Cancer-related gene regulation
ResumoFollicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) γ1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARγ1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARγ target genes were up-regulated in PAX8-PPARγ-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARγ expression profile presented a putative PPARγ-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARγ and as members of novel molecular pathways involved in the development of thyroid carcinomas. Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) γ1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARγ1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARγ target genes were up-regulated in PAX8-PPARγ-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARγ expression profile presented a putative PPARγ-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARγ and as members of novel molecular pathways involved in the development of thyroid carcinomas. Follicular cell-derived thyroid tumors include benign adenomas, papillary and follicular carcinomas, two entities considered as differentiated carcinomas, and anaplastic carcinomas. Although clinically benign thyroid nodules are common in the general population, thyroid carcinomas are infrequent tumors, and both the putative relationships between adenomas and carcinomas and the mechanisms of thyroid oncogenesis are not clearly understood.1Fagin JA Molecular genetics of tumors of thyroid follicular cells.in: Braverman LE Utiger RD In Werner & Ingbar's The Thyroid: A Fundamental and Clinical Text. Lippincott Williams & Wilkins, Philadelphia2000: 886-898Google Scholar In thyroid tumors, genetic abnormalities have been largely substantiated. Fusion of the tyrosine-kinase domain of the RET gene and the 5′ domain of various genes (RET/PTC) and activating mutation of the B type Raf kinase (BRAF) gene constitute the two major independent and nonoverlapping genetic alterations detected in papillary carcinomas.2Bongarzone I Butti MG Coronelli S Borrello MG Santoro M Mondellini P Pilotti S Fusco A Della Porta G Pierotti MA Frequent activation of ret protooncogene by fusion with a new activating gene in papillary thyroid carcinomas.Cancer Res. 1994; 54: 2979-2985PubMed Google Scholar, 3Xing M Vasko V Tallini G Larin A Wu G Udelsman R Ringel MD Ladenson PW Sidransky D BRAF T1796A transversion mutation in various thyroid neoplasms.J Clin Endocrinol Metab. 2004; 89: 1365-1368Crossref PubMed Scopus (128) Google Scholar A high prevalence of activating mutations of all three RAS genes has been reported in follicular neoplasms.4Fagin JA Minireview: branded from the start-distinct oncogenic initiating events may determine tumor fate in the thyroid.Mol Endocrinol. 2002; 16: 903-911Crossref PubMed Scopus (122) Google Scholar Chromosomal imbalances are also frequent in FTCs;5Roque L Rodrigues R Pinto A Moura-Nunes V Soares J Chromosome imbalances in thyroid follicular neoplasms: a comparison between follicular adenomas and carcinomas.Genes Chromosomes Cancer. 2003; 36: 292-302Crossref PubMed Scopus (55) Google Scholar and the t(2;3)(q13;p25) rearrangement, yielding a PAX8-PPARγ1 fusion gene, is found in 10 to 63% FTCs,6Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000; 289: 1357-1360Crossref PubMed Scopus (729) Google Scholar but also in some follicular adenomas.7Nikiforova MN Biddinger PW Caudill CM Kroll TG Nikiforov YE PAX8-PPARgamma rearrangement in thyroid tumors: RT-PCR and immunohistochemical analyses.Am J Surg Pathol. 2002; 26: 1016-1023Crossref PubMed Scopus (286) Google Scholar, 8Marques AR Espadinha C Catarino AL Moniz S Pereira T Sobrinho LG Leite V Expression of PAX8-PPAR gamma 1 rearrangements in both follicular thyroid carcinomas and adenomas.J Clin Endocrinol Metab. 2002; 87: 3947-3952Crossref PubMed Scopus (292) Google Scholar, 9Cheung L Messina M Gill A Clarkson A Learoyd D Delbridge L Wentworth J Philips J Clifton-Bligh R Robinson BG Detection of the PAX8-PPAR gamma fusion oncogene in both follicular thyroid carcinomas and adenomas.J Clin Endocrinol Metab. 2003; 88: 354-357Crossref PubMed Scopus (169) Google Scholar These observations suggest that FTC proceeds from at least two distinct oncogenic events, namely RAS mutations and PAX8-PPARγ1 rearrangement.10Nikiforova MN Lynch RA Biddinger PW Alexander EK Dorn II, GW Tallini G Kroll TG Nikiforov YE RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma.J Clin Endocrinol Metab. 2003; 88: 2318-2326Crossref PubMed Scopus (568) Google Scholar Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear hormone receptor superfamily and plays a critical role in the differentiation of adipocytes and in the regulation of fat metabolism.11Desvergne B Wahli W Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr Rev. 1999; 20: 649-688Crossref PubMed Scopus (2698) Google Scholar The involvement of PPARγ in the development of tumors, including thyroid carcinomas, is still debated.12Koeffler HP Peroxisome proliferator-activated receptor gamma and cancers.Clin Cancer Res. 2003; 9: 1-9PubMed Google Scholar, 13Michalik L Desvergne B Wahli W Peroxisome-proliferator-activated receptors and cancers: complex stories.Nat Rev Cancer. 2004; 4: 61-70Crossref PubMed Scopus (502) Google Scholar The anti-proliferative effects of PPARγ agonists have been demonstrated in thyroid carcinoma cell lines14Ohta K Endo T Haraguchi K Hershman JM Onaya T Ligands for peroxisome proliferator-activated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells.J Clin Endocrinol Metab. 2001; 86: 2170-2177Crossref PubMed Scopus (201) Google Scholar, 15Martelli ML Iuliano R Le Pera I Sama I Monaco C Cammarota S Kroll T Chiariotti L Santoro M Fusco A Inhibitory effects of peroxisome poliferator-activated receptor gamma on thyroid carcinoma cell growth.J Clin Endocrinol Metab. 2002; 87: 4728-4735Crossref PubMed Scopus (121) Google Scholar and either induce cell-cycle arrest or promote cell-death. Furthermore, thyroid tumors not harboring PAX8-PPARγ translocation displayed decreased PPARγ gene expression,16Aldred MA Morrison C Gimm O Hoang-Vu C Krause U Dralle H Jhiang S Eng C Peroxisome proliferator-activated receptor gamma is frequently downregulated in a diversity of sporadic nonmedullary thyroid carcinomas.Oncogene. 2003; 22: 3412-3416Crossref PubMed Scopus (53) Google Scholar, 17Marques AR Espadinha C Frias MJ Roque L Catarino AL Sobrinho LG Leite V Underexpression of peroxisome proliferator-activated receptor (PPAR)gamma in PAX8/PPARgamma-negative thyroid tumours.Br J Cancer. 2004; 91: 732-738PubMed Google Scholar, 18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar and this suggests that decreased activity of PPARγ might contribute to carcinogenesis. In FTCs, the PAX8-PPARγ1 fusion oncogene appears to act through a dominant-negative effect on the transcriptional activity of wild-type PPARγ1, inhibiting agonist-induced transactivation.6Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000; 289: 1357-1360Crossref PubMed Scopus (729) Google Scholar Recently, in vitro experiments demonstrated that the fusion oncoprotein contributes to the malignant transformation by acting on several pathways, some of which are normally regulated by PPARγ.19Gregory Powell J Wang X Allard BL Sahin M Wang XL Hay ID Hiddinga HJ Deshpande SS Kroll TG Grebe SK Eberhardt NL McIver B The PAX8/PPARgamma fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPARgamma inhibition.Oncogene. 2004; 23: 3634-3641Crossref PubMed Scopus (83) Google Scholar To explore the physiopathological mechanisms associated to the presence of the PAX8-PPARγ1 rearrangement, we analyzed gene profiles of normal thyroid tissues, follicular adenomas and follicular thyroid carcinomas using custom-designed 60-mer oligonucleotides microarrays of 22,000 features, representing about 17,000 distinct genes. Transcriptional changes associated with the presence or the absence of the PAX8-PPARγ1 rearrangement revealed a 93-gene discriminating pattern. Changes in the expression of several genes of this molecular signature were confirmed by real-time quantitative real-time polymerase chain reaction (Q RT-PCR) in a large series of thyroid tissues and at the protein level, by immunohistochemistry on a tissue array. Sixty-five thyroid tissue samples were selected after histological analysis and classified according to World Health Organization recommendations (Table 1).20Hedinger C Williams E Sobin L Histological typing of thyroid tumours. Springer-Verlag, Berlin1988Crossref Google Scholar Normal contralateral thyroid tissues were obtained from patients with a unifocal tumor. All specimens were frozen at −80°C in isopentane and stored in liquid nitrogen directly after surgical resection and until RNA extraction. Informed consent was obtained from all patients. Twenty-three of these 65 samples were used for microarray experiments. All thyroid samples were obtained in euthyroid subjects, as assessed by serum thyroid stimulating hormone (TSH) concentrations in the normal range at the time of surgery. Complete clinical and histological information concerning samples used in this study is available in supplementary Table S1 (http://ajp.amjpathol.org).Table 1Characteristics of Thyroid TumorsQ RT-PCRMicroarrayHistological featuresNormal thyroid1711tissueFollicular164Typical7adenomaAtypical9Follicular278MIF7carcinomaAnaplastic50WIF16carcinomaHürthle4Q RT-PCR and microarray columns represent the number of samples included in the experiments. All tumors used in microarray experiments were included in the Q RT-PCR experiments. Staging is based on the 2002 TNM Classification (American Joint Committee on Cancer, 2002). MIF, minimally invasive; WIF, widely invasive. Open table in a new tab Q RT-PCR and microarray columns represent the number of samples included in the experiments. All tumors used in microarray experiments were included in the Q RT-PCR experiments. Staging is based on the 2002 TNM Classification (American Joint Committee on Cancer, 2002). MIF, minimally invasive; WIF, widely invasive. Total RNA was isolated from frozen tissue samples using Trireagent (Sigma-Aldrich, Saint Louis, MO) and purified on Rneasy columns (Qiagen, Hilden, Germany) according to manufacturer's protocols. Quality of RNA preparation, based on the 28S/18S ribosomal RNAs ratio, was assessed using the RNA 6000 Nano Lab-On-chip as developed on the Agilent 2100 Bioanalyzer device (Agilent Technologies, Palo Alto, CA). All specimens included in this study displayed a ratio of 28S to 18S higher than 1.5 (average 1.8). RNA samples were frozen in nuclease-free water (Promega Corporation, Madison, WI). One microgram of total RNA was reverse-transcribed by Moloney murine leukemia virus reverse transcriptase in the presence of random primers (Applied Biosystems, Foster City, CA). For each sample, the PAX8-PPARγ1 translocation detection assay was performed as previously described.18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar Briefly, the PCR reaction was performed with several primers previously described.6Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA PAX8-PPARgamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000; 289: 1357-1360Crossref PubMed Scopus (729) Google Scholar, 7Nikiforova MN Biddinger PW Caudill CM Kroll TG Nikiforov YE PAX8-PPARgamma rearrangement in thyroid tumors: RT-PCR and immunohistochemical analyses.Am J Surg Pathol. 2002; 26: 1016-1023Crossref PubMed Scopus (286) Google Scholar PCR products were visualized using 2% agarose gel. For each sample with detectable PCR product, all visible bands were purified and directly sequenced to confirm the presence of PAX8-PPARγ1 translocation. The efficiency of reverse transcription was controlled with amplification of several genes with the same cDNA (with quantitative real-time PCR). A pool composed by equal amount of total RNA from each tissue sample was used as the RNA reference. Five-microgram aliquots of total RNA from each sample and from the reference pool were used to generate labeled antisense cRNA with T7 RNA polymerase. Labeling of cRNAs was performed with cyanine 3 (Cy3)-CTP for all samples and cyanine 5 (Cy5)-CTP for the RNA reference (Perkin Elmer NEN, Boston, MA). Reverse transcription, linear amplification, cRNA labeling, and purification were performed using the Agilent Linear amplification kit. The cRNA concentration and Cy3-CTP or Cy5-CTP incorporation were assessed using an UV-visible spectrophotometer. We used custom-designed 60-mer oligonucleotide microarrays of 22,000 features, representing 16,840 known unique genes developed by Agilent Technologies. Hybridization was performed during 17 hours at 60°C, with 1 μg of Cy3-labeled cRNA of each sample mixed to the same amount of Cy5-labeled cRNA reference. The Feature Extraction software (Agilent Technologies) was used to quantify intensity of fluorescent images and to normalize results using the local background subtraction option as recommended for oligo-microarray procedures. Files used for statistical analysis contained, for each tumor sample, the list of 22,000 features associated to a set of values including log ratio compared with reference, P value of log ratio, and intensities. To evaluate the reliability of data, linearity and intrassay reproducibility were checked as follows. First, RNA reference labeled with either Cy3 or Cy5 was hybridized to compare the efficiencies of incorporation for each cyanine and to evaluate the intensity noise. Second, one sample processed in duplicate assessed the reproducibility. All data obtained from microarray analysis have been submitted to Array Express at the European Bioinformatics Institute (http://www.ebi.ac.uk/arrayexpress/). ArrayExpress (at the European Bioinformatics Institute) is a public repository for microarray data, which is aimed at storing well-annotated data in accordance with Microarray Gene Expression Data recommendations (http://www.mged.org). Gene functions were determined using gene ontology database FatiGO (http://fatigo.bioinfo.cnio.es), Panther Ontology (http://myscience.appliedbiosystems.com), and On Line Mendelian Inheritance In Man description (http://www.ncbi.nlm.nih.gov/). Microarray data analysis was performed using the Resolver software (Rosetta Inpharmatics, Kirkland, WA). All data were filtered to eliminate low-intensity value under 200 arbitrary units for both colors, a threshold determined on the basis of the linearity test. Each selected gene had at least a twofold change, with a P value less than 1% in a minimum of four independent samples. Using this procedure, 1859 genes passed the filter. For the unsupervised clustering, a hierarchical agglomerative algorithm that pairs samples according to their similarity was used. Analysis of variance (ANOVA) was performed on the microarray data and on the Q RT-PCR data. For each individual gene, one-way ANOVA tests were performed to compare results obtained from normal tissues and FTCs and from FTCs presenting or not presenting the translocation. Two-way ANOVA tests were applied for the Q RT-PCR data to compare average gene expression values between FTCs with and without the translocation while adjusting for the histological type. To search potential peroxisome proliferator response element (PPRE) sequences within the upstream 2-kb sequence located before the coding determining sequence of the selected genes, a homology matrix was designed on the basis of nine well-characterized PPRE sequences of the human PPARγ promoter, including those regulating acyl-coenzyme A oxidase, apolipoprotein C-III, carnitine palmitoyltransferase 1B, ATP-binding cassette D2 (2 PPRE), aquaporin (AQ7), nuclear receptor LXR, and acyl-coA binding protein genes. The homology matrix associates a value to each nucleotide at each position of the consensus PPRE (AGGTCA N AGGTCA) sequence. This value is related to the frequency of each nucleotide in the nine PPRE analyzed, and when the frequency is null, a negative value is associated. A score between −7 and 98 was attributed to each sequence analyzed. The threshold value for this score was determined as the minimum value of the 95% confidence interval of average of score obtained by the nine well-characterized PPRE. The number of putative promoters found in our gene set was compared with the number of putative PPRE expected by chance and determined in 500 randomly selected gene sets. More information is available in supplementary Table S3 (http://ajp.amjpathol.org). Oligonucleotide primers and Taqman probes specific for PPARγ, PAX8A, FGD3, decorin (DCN), RPLP0, PPIA, or 28S were designed to be intron spanning using the PrimerExpress computer software (Applied Biosystems). Sequences were from the GenBank database, and the oligonucleotides were purchased from MWG Biotech (Courtaboeuf, France). Primers and probes for thyroid-specific genes SLC5A5, TPO, and TG were already published;21Lazar V Bidart JM Caillou B Mahe C Lacroix L Filetti S Schlumberger M Expression of the Na+/I- symporter gene in human thyroid tumors: a comparison study with other thyroid-specific genes.J Clin Endocrinol Metab. 1999; 84: 3228-3234PubMed Google Scholar and 18S, ANGPTL4, acetyl-Coenzyme A acyltransferase 1 (ACAA1), PDE8, and HBP17 were obtained from Assays-On-Demand (Applied Biosystems). Q-PCRs were performed on equivalent of 10 ng per total RNA per tube in a final volume of 18 μl and developed as previously described.18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar The reference pool, corresponding to the microarray RNA reference, was used as a calibrator (1× sample). Normalization was assessed by a combination of four housekeeping genes (18S, 28S, RPL0, and PPIA).22Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol. 2002; 3: 1-11Crossref Google Scholar A tissue array, including all of the samples analyzed in the microarray experiment, was constructed using a tissue-arrayer device (Alphelys, Plaisir, France) with a 1-mm needle. Quadruplicate samples were prepared both from the tumor part and from the normal thyroid tissue at distance from the tumor. A total of 128 spots by antibody were analyzed. Immunohistochemistry was performed on formalin-fixed paraffin-embedded 5-μm sections as described previously.18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar Two independent observers carried out the immunohistochemical analysis, considering both the percentage of positive cells (noted as percentage of stained cells) and the intensity of staining (noted from 0 to 4 +). Antibody against PPARγ was a mouse monoclonal antibody γ, which recognizes the COOH terminus of the protein (Santa Cruz, CA). A 26-amino acid peptide and a 22-amino acid peptide, spanning the COOH-terminal portions of ACAA1 and FGD3, respectively, were synthesized by a conventional solid-phase method using a model 432A peptide synthesizer (Applied Biosystems). The identity and purity of each peptide were verified by 1) amino acid analysis on an α LKB analyzer (LKB, Rockville, MD); 2) HPLC; and 3) sequence analysis using ES-TOF mass spectrometry (Micromass Quattro LCT, Villeurbanne, France). The peptides were then conjugated to keyhole limpet hemocyanin using benzidine as the coupling agent, and two rabbits were immunized by intradermal injections of each synthetic peptide-carrier conjugate. After three subsequent boosts at 3-week intervals, animals were bled, and their sera were tested in an enzyme-linked immunosorbent assay. The PAX8-PPARγ1 rearrangement was detected in 4 of 23 (17%) FTCs and 1 of 16 (6%) benign hypofunctioning follicular adenomas (FTAs), as previously described.18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar In the five positive samples, RT-PCR and sequencing revealed four isoforms generated by the fusion of different exons of PAX8 (exon 7; exons 7 and 8; exons 7, 8, and 9; and exons 7 and 9) with exon 1 of PPARγ1. None of the normal thyroid tissues (n = 17), Hürthle-cell carcinomas (n = 4), and anaplastic carcinomas (n = 5) displayed any evidence of the translocation. Microarray experiments, based on the dual-color technology were performed on 23 thyroid samples, including 8 FTCs, 4 FTAs, and 11 normal contralateral tissues. Three FTCs and one FTA harbored PAX8-PPARγ1 rearrangement. Using the Resolver software, an intensity- and fold changed-based filtering approach selected 1859 features from the 22,000 present on the 60-mer oligonucleotides microarray for further analysis. The nonsupervised hierarchical clustering performed on these features is presented as a hyperbolic view in Figure 1. One cluster included 8 of 11 normal thyroid tissue samples. Two normal contralateral tissues (NT6 and NT9), presenting lymphocytic infiltration at histological examination, segregated separately and were associated with the presence of serum anti-thyroid antibodies. Most FTAs and FTCs clustered together. One carcinoma sample, namely FTC17, displayed a quite different expression profile compared with those of the FTCs and corresponded to a particularly aggressive tumor. Interestingly, a peculiar cluster contained all of the four PAX8-PPARγ-positive tumors. This distinct cluster underlines the existence of a specific gene expression profile associated with the translocation. To explore gene expression profiles related to tumors, an ANOVA test was performed for each of the 1859 filtered genes. Normal thyroid tissues displayed 61 genes differentially expressed at P < 0.01 (N/T− set) when compared with PAX8-PPARγ-negative FTCs and 116 genes when compared with PAX8-PPARγ-positive FTCs at P < 0.01 (N/T+ set). These sets of genes are described in supplementary Table S2 (http://ajp.amjpathol.org). Only 27 genes were differently regulated in both sets of FTCs versus normal tissues (Figure 2). Some of these genes were already found to be up-regulated in human FTCs, such as transforming growth factor α (TGFA), or down-regulated, such as gelsolin, tumor necrosis factor receptor superfamily, member 11b, or trefoil factor 3.23Barden CB Shister KW Zhu B Guiter G Greenblatt DY Zeiger MA Fahey III, TJ Classification of follicular thyroid tumors by molecular signature: results of gene profiling.Clin Cancer Res. 2003; 9: 1792-1800PubMed Google Scholar Interestingly, increased creatine kinase, mitochondrial 1 and decreased DCN gene expressions were also reported in a murine model of follicular carcinoma.24Ying H Suzuki H Zhao L Willingham MC Meltzer P Cheng SY Mutant thyroid hormone receptor beta represses the expression and transcriptional activity of peroxisome proliferator-activated receptor gamma during thyroid carcinogenesis.Cancer Res. 2003; 63: 5274-5280PubMed Google Scholar The low number of genes in common between those two sets might indicate separate oncogenesis pathways. When genes were ordered by ontology, N/T− and N/T+ gene sets displayed important differences (Figure 2). Microarray analysis revealed that the N/T− set mainly included down-regulated genes (84%) as previously described.25Aldred MA Ginn-Pease ME Morrison CD Popkie AP Gimm O Hoang-Vu C Krause U Dralle H Jhiang SM Plass C Eng C Caveolin-1 and caveolin-2,together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis.Cancer Res. 2003; 63: 2864-2871PubMed Google Scholar This included genes involved in cell signaling (CRABP1, DDR2, and DPP6), immunity (CCL21, CXCL12, and DF), cellular metabolism, and enzymes (FGL2 and PGM5). Interestingly, the N/T+ set mainly included up-regulated genes (76%) such as genes involved in cellular energetic metabolism (ACAA1, AK3L1, fructose(1,6) bisphosphatase (FBP1), DGAT2, and AQ7), cell signaling (GDF5, RDC1, DPP4, and FGD3), cellular growth (GADD45G, MYCL1, and TNFRSF21), and transcription (NFE2L3 and PPARγ). Surprisingly, the statistical analysis did not select the thyroid-specific genes present on the microarray, including NIS, TPO, TG, TSH-R, and DUOX2, as differently expressed between FTCs and normal tissue samples. To further investigate the expression profile associated with the PAX8-PPARγ-rearrangement, we compared these two groups of follicular tumors by the ANOVA test (P < 0.01). A set of 93 genes (T−/T+ set) was identified (Figure 3; supplementary Table S2 (http://ajp.amjpathol.org)). Interestingly, a 26-fold up-regulated PPARγ gene expression was detected in the positive follicular tumors. Because the entire PPARγ1 open reading frame is present in the rearranged product, wild-type PPARγ and PAX8-PPARγ transcripts were undistinguishable by the corresponding 60-mer oligonucleotide. In accordance with previous results, PPARγ expression level appears to be the most specific difference between those two tumor groups.8Marques AR Espadinha C Catarino AL Moniz S Pereira T Sobrinho LG Leite V Expression of PAX8-PPAR gamma 1 rearrangements in both follicular thyroid carcinomas and adenomas.J Clin Endocrinol Metab. 2002; 87: 3947-3952Crossref PubMed Scopus (292) Google Scholar, 16Aldred MA Morrison C Gimm O Hoang-Vu C Krause U Dralle H Jhiang S Eng C Peroxisome proliferator-activated receptor gamma is frequently downregulated in a diversity of sporadic nonmedullary thyroid carcinomas.Oncogene. 2003; 22: 3412-3416Crossref PubMed Scopus (53) Google Scholar, 18Lacroix L Mian C Barrier T Talbot M Caillou B Schlumberger M Bidart JM PAX8 and peroxisome proliferator-activated receptor gamma 1 gene expression status in benign and malignant thyroid tissues.Eur J Endocrinol. 2004; 151: 367-374Crossref PubMed Scopus (45) Google Scholar Several genes, known as PPARγ positively regulated targets, were up-regulated in PAX8-PPARγ-positive tumors, including angiopoietin-like 4 and aquaporin 7, which displayed a 23-fold and 4-fold gene expression increase, respectively. Expression of genes involved in cellular metabolism, particularly those associated to lipid, glucose, and amino acid metabolisms, was modified: ACAA1, a gene involved in the β-oxidation process
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