Oncogene (2005) 24, 4155–4161 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc SHORT REPORT profiling reveals specific oncogenic mechanisms and signaling pathways in oncocytic and papillary thyroid carcinoma

Olivier Baris*,1, Delphine Mirebeau-Prunier1, Fre´ de´ rique Savagner1, Patrice Rodien1,2, Benoit Ballester3,Be´ atrice Loriod3, Samuel Granjeaud3, Serge Guyetant4, Brigitte Franc5, Re´ mi Houlgatte3, Pascal Reynier1 and Yves Malthiery1

1INSERM EMI-U 0018, Laboratoire de Biochimie et Biologie Mole´culaire, CHU, 4 rue Larrey, Angers F-49033, France; 2Service d’Endocrinologie, Nutrition et Me´decine Interne, CHU, Angers F-49033, France; 3Laboratoire TAGC/INSERM ERM 206, Marseille F-13009, France; 4Laboratoire d’Anatomie Pathologique, CHRU, Tours F-37044 Cedex 1, France; 5Laboratoire d’Anatomie Pathologique, Hoˆpital A Pare´, Boulogne F-92104, France

The oncogenic pathways in mitochondrial-rich thyroid more frequently in the thyroid gland. Oncocytic thyroid carcinomas are not clearly understood. To investigate the carcinomas are defined as malignant epithelial tumors of possible implication ofmitochondrial abundance in the the thyroid, composed exclusively or predominantly genesis ofthyroid tumors, we have explored the gene (75% or more) of oncocytic cells presenting cytoplasmic expression profile ofsix oncocytic carcinomas and six accumulations of dilated mitochondria. Several studies mitochondrial-rich papillary carcinomas using cDNA- have confirmed the increased activity of the mitochon- microarray technology. A supervised approach allowed drial respiratory chain in these tumors (Ebner us to identify 83 differentially expressed in the two et al., 1991; Savagner et al., 2001a). These lesions are types ofcarcinoma. These genes were classified according mainly classified into oncocytic follicular carcinomas to their ontologic profiles. Three genes, NOS3, alpha- (also known as Hu¨ rthle cell carcinomas). The identifica- actinin-2 and alpha-, suspected ofplaying a role in tion of a Hu¨ rthle cell papillary carcinoma subgroup tumor genesis, were explored by quantitative RT–PCR suggests a close relationship between ordinary PTCs and analysis and immunohistochemistry. Ofthe 59 genes oncocytic carcinomas (Cheung et al, 2000). Also, overexpressed in papillary carcinomas, 51% were involved ordinary PTCs present frequently an increase in in cell communication. Ofthe 24 genes overexpressed in mitochondrial content that may be involved in the oncocytic carcinomas, 84% were involved in mitochon- tumor genesis (Haugen et al., 2003). drial and cellular metabolism. Our results suggest that There is no evidence to suggest that the etiology of mitochondrial respiratory chain complexes III and IV oncocytic malignancies of thyroid is distinct from that play a significant role in the regulation ofreactive oxygen of nononcocytic lesions. The somatic in species production by oncocytic tumors. oncocytic malignancies of thyroid follicular cells are Oncogene (2005) 24, 4155–4161. doi:10.1038/sj.onc.1208578 similar to those of the ordinary papillary and follicular Published online 4 April 2005 carcinomas. Rearrangements involving the proto-onco- gene RET as well as anomalies in the B-RAF, NTRK1, Keywords: carcinoma; thyroid; mitochondria RAS, TC-1 and APC genes have been associated with the development of PTCs (Camiel et al., 1968; Fusco et al., 1987; Chua et al., 2000; Kimura et al., 2003). Mutations in the RAS and PTEN genes, and a fusion between Pax8 and PPARg, have been implicated in the Introduction pathogenesis of follicular thyroid carcinomas (Kroll et al., 2000; Moretti et al., 2000). Nevertheless, the basic ONCOGENOMICS The WHO classification distinguishes between two types mechanisms and signaling pathways involved in the of differentiated thyroid cancer originating from folli- genesis of oncocytic thyroid carcinomas are still poorly cular cells. Papillary thyroid carcinoma (PTC) is known. diagnosed on the basis of architectural and nuclear The role of mitochondrial proliferation in cancer features, whereas follicular thyroid carcinoma is defined development has been evoked (Augenlicht et al., 1999). by capsular or vascular invasion in follicular epithelial A recent cDNA-microarray analysis of oncocytic cell neoplasms. Oncocytic tumors are epithelial tumors thyroid tumors has demonstrated the profound mod- that affect a wide variety of tissues but occur ification of metabolism in oncocytoma (Baris et al., 2004). In this study, the upregulation of genes *Correspondence: O Baris; coding for enzymes playing a role in the glycolysis, E-mail: [email protected] Received 1 June 2004; revised 27 October 2004; accepted 31 January the tricarboxylic acid cycle and oxidative phosphory- 2005; published online 4 April 2005 lation suggested the existence of an aerobic glycolytic Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4156 mechanism in oncocytic adenomas and, unexpectedly, in oxidative phosphorylation, mitochondrial proliferation oncocytic carcinomas as well. In another study, the and cell proliferation in these tumors (Savagner et al., upregulation of the PGC-1-related coactivator (PRC) 2003). indicated a close relationship between the regulation of In the present study, we compare the expression profiles of six oncocytic follicular carcinomas and six ordinary mitochondrial-rich PTCs selected from a

Figure 1 (a) Hierarchical average linkage clustering of the 83 most discriminating genes between follicular oncocytic and PTCs. Samples were obtained from 12patients presenting sporadic thyroid tumors, six oncocytic follicular carcinomas and six ordinary mitochondrial-rich papillary carcinomas, diagnosed according to the WHO classification. All the samples were rendered anonymous before the study. We confirmed the rich mitochondrial content of the tumor cells by using a monoclonal antibody against a complex IV mitochondrial subunit of the respiratory chain (clone 113-1, Biogenex laboratories Inc., San Ramon, CA, USA). According to immunostaining scores, taking into account the signal localization and intensity and the percentage of positive cells, oncocytic carcinomas scored at 3 (more than 75% of staining), papillary carcinomas scored at 2(between 25and 75% of staining) and 3. Patients included three men and nine women, with a mean age of 48 years (range 19–75 years). The average tumor size for oncocytic carcinomas was 29715 mm (mean7s.d.; range 15– 55 mm) and 23710 mm (mean7s.d.; range 10–40 mm) for PTCs. was analysed with several other thyroid tumors by hybridization of nylon Hybond N þ membrane (Amersham Pharmacia Biotech, Little Chalfont, UK) cDNA arrays with radioactive probes, according to the protocols described elsewhere (Thieblemont et al., 2004). The arrays displayed PCR products spotted from 6720 selected IMAGE human cDNA and control clones. The cDNA clones were selected because of their proven or putative implication in carcinogenesis (list available at http:// tagc.univ-mrs.fr/pub/). Total RNA was isolated from frozen thyroid samples using a standard guanidium isothiocyanate protocol (Trizol Reagent, Life Technologies, Gaithersburg, MD, USA). RNA integrity was determined on an RNA 6000 nano labchip (Agilent technologies, Waldbronn, Germany). cDNA from each thyroid sample was obtained from 5 mg of total RNA by simultaneous reverse transcription and [a-33P]dCTP labeling as described elsewhere (http://tagc.univ-mrs.fr/pub/). Each cDNA was hybridized on an individual array prepared as described below. After washing, hybridization images were obtained by scanning with an imaging device (Fuji BAS 5000, Raytest, Paris, France). Signal intensities were quantified using ArrayGauge software (Fujifilm, Japan). Supervised analysis was used to highlight the most important genes discriminating oncocytic carcinomas from papillary carcinomas. The discriminating score (DS) was calculated as follows, DS ¼ (m1Àm2)/(s1 þ s2), where m1 and s1, respectively, represent the mean and the s.d. of the expression levels of a given gene in sample subgroup 1, and m2 and s2, respectively, represent the mean and the s.d. of the expression levels of the same gene in sample subgroup 2(Golub et al., 1999). Owing to the small number of samples in each group, we had to increase the error risk to 1% to select discriminant genes (Magrangeas et al., 2003). This higher risk gives 3.2 false-positive genes meaning that, among 25 genes of our list (83/3.2), one gene could have been randomly selected. Clustering analysis was performed using the Pearson correlation and the average linkage method (Eisen et al., 1998). (b) Gene function distribution (in %) according to the ontologic analysis. Black: papillary carcinomas; Gray: oncocytic carcinomas. Only genes with a known function were considered (N ¼ 77 genes). We searched for the biological processes in which the 77 genes with a known function were involved, according to the GO database. We used the statistical methods available on the FATIGO web server (http://fatigo.bioin- fo.cnio.es, Al-Shahrour et al., 2004) to find over- and under- represented GO terms in the two types of carcinomas. The statistical contrast was performed at the GO level 3, which constitutes the best compromise between information quality and the number of gene annoted

Oncogene Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4157 previous cDNA-microarray analysis on thyroid tumors gene profiling and immunostaining studies, thus validat- (Baris et al., 2004). Applying a supervised strategy, we ing the accuracy of our present data (Huang et al., 2001; have identified a set of genes that clearly differentiates Maeta et al., 2001; Kim et al., 2002). between oncocytic follicular carcinomas and PTCs. According to the (GO) database, we Identification of these genes should improve our under- search for the biological activity for 77 of the 83 genes standing of functional pathways involved in the genesis with a known function, using statistics on FATIGO web of the two types of mitochondrial-rich carcinomas. server (Al-Shahrour et al., 2004). The results are shown in Figure 1b. The 59 genes overexpressed in PTCs are mainly involved in cellular physiological process (52.94%) and cell communication (43.14%) such as Results and discussion cell-to-extracellular matrix and cell-to-cell adhesion. The expression of extracellular matrix components such as Complex targets prepared from several thyroid tumors collagen and biglycan, as well as that of genes involved were hybridized on microarrays containing PCR pro- in adherent junctions (VCL, CDH3, CTNNA1) and ducts from 6720 cDNA clones. After image quantifica- tight junctions (TJP2), was higher in PTCs than in tion and normalization of microarray data, we searched oncocytic carcinomas. Interestingly, we found that for genes with consistent differential expression levels alpha-catenin and are overexpressed in PTCs, between the six follicular oncocytic carcinomas and the whereas alpha-actinin (ACTN2), a main component of six ordinary PTCs. The 83 genes identified at 1% risk the , is underexpressed. Alpha-catenin plays (24 of which were overexpressed in oncocytic carcino- an important role in mediating the binding between mas, and 59 overexpressed in PTCs) were hierarchically beta-catenin and the vinculin–alpha-actinin complex clustered as represented in Figure 1a. This set of genes (Imamura et al., 1999). Cytoplasmic and nuclear allowed a clear distinction between the two classes of accumulation of alpha- and beta-catenin, related to carcinomas, illustrating the efficiency of our supervised high cyclin D1 expression, has been previously reported method. The genes overexpressed in oncocytic carcino- in PTCs (Ishigaki et al., 2002). We used quantitative mas are listed in Table 1 and those overexpressed in RT–PCR analysis and immunohistochemistry to ana- PTCs in Table 2. Certain genes (COX7B, GLG1 and lyse alpha-actinin and alpha-catenin expression in order MMP2) were represented several times in the clusters, to validate the strong difference in expression levels indicating the good reproducibility of the method. For between the two types of carcinoma. We did not test the PTCs, the overexpression of several genes (DUSP6, most differentially expressed gene named TIMP1 as it CCND1, TIMP1, CD44, MMP2) was in agreement with had already been validated as a PTC marker on the basis

Table 1 Genes overexpressed in oncocytic vs papillary carcinomas using a supervised strategy (list available on web site: //lbbma.univ-angers.fr.) Gene symbol O/P ratio Description Function

ADA 5.99 Adenosine deaminase EIF2S2 4.58 Eukaryotic translation initiation factor 2 Translation

STIP1 3.06 Stress-induced phosphoprotein 1 Response to stress PTPRN 2.94 tyrosine phosphatase, receptor type Phosphotyrosine dephosphorylation CYCS 2.86 Cytochrome c, somatic Respiratory chain, apoptosis COX7B 2.84 Cytochrome c oxidase subunit VIIb Respiratory chain ACTN22.59Actinin, alpha 2 Cytoskeleton RBBP5 2.16 Retinoblastoma binding protein 5 Rb binding GCHFR 2.12 GTP cyclohydrolase I regulatory protein NO regulator

CYC1 2.04 Cytochrome c-1 Respiratory chain GSTM3 2.02 Glutathione S M3 Response to oxidative stress KIAA0205 1.91 KIAA0205 gene Unknown U5-200KD 1.91 U5 snRNP-specific protein, 200 kDa Splicing NRF1 1.89 Nuclear respiratory factor 1 Mitochondrial biogenesis ATP6V1D 1.88 ATPase, H+ transporting, V1 subunit D Organelle acidification COPB 1.86 Coatomer protein complex, subunit beta Vesicular transport COX6A1 1.80 Cytochrome c oxidase subunit VIa Respiratory chain ATF4 1.73 Activating transcription factor 4 Transcriptional regulator RPN1 1.64 Ribophorin I Ribosome binding CRKNL1 1.62Crn, crooked-neck-like 1 Splicing COX7A21.56 Cytochrome c oxidase subunit VIIa Respiratory chain GLUL 1.42Glutamate– (glutamine synthase) Glutamine biosynthesis

ERCC5 1.39 Excision repair crosscomplementing repair DNA repair MCP 1.36 Membrane protein Complement activation

The 24 genes overexpressed in oncocytic vs papillary carcinomas are referenced by their HUGO abbreviations as used in Link. The O/P ratio is the ratio between the median gene expression value in oncocytic carcinoma and that in papillary carcinoma (Pp0.01%)

Oncogene Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4158 Table 2 Genes overexpressed in papillary vs oncocytic carcinomas using a supervised strategy (list available on web site: //lbbma.univ-angers.fr.) Gene symbol O/P ratio Description Function

TIMP1 24.55 Tissue inhibitor of metalloproteinase 1 Metalloendopeptidase inhibitor COL8A1 10.94 Collagen, type VIII, alpha 1 Cell adhesion, ECM CCND1 4.60 Cyclin D1 Cell cycle CDH3 4.34 Cadherin 3, type 1, P-cadherin Cell adhesion CCND23.88 Cyclin D2 Cell cycle ABCC3 3.82ATP-binding cassette, subfamily C ABC transporter

GATA3 3.73 GATA binding protein 3 Transcription factor DUSP6 3.69 Dual specificity phosphatase 6 Cell signaling MMP23.10 Matrix metalloproteinase Matrix metalloproteinase TGFB1 3.07 Transforming growth factor, beta 1 Cell proliferation, apoptosis KLF5 2.94 Kruppel-like factor 5 RNA polymerase II transcription LMO7 2.94 LIM domain only 7 Protein–protein interactions

CD44 2.89 CD44 antigen Cell–matrix adhesion RAF1 2.86 v-Raf-1 murine leukemia viral oncogene Cell proliferation, apoptosis CD22.85CD2antigen Cell adhesion, T cells

TNFSF5 2.74 Tumor necrosis factor superfamily B-cell proliferation

ERBB22.59v-Erb-b2erythroblastic leukemia Cell proliferation, oncogenesis viral oncogene homolog 2, TNFSF13 2.59 Tumor necrosis factor superfamily, TNF signaling member 13 ANXA4 2.45 Annexin A4 Calcium-dependent phospholipid binding NEDD8 2.38 Neural precursor cell expressed, Protein modification developmentally downregulated 8 GRB7 2.36 Growth factor receptor-bound protein 7 EGF receptor signaling pathway CALM22.31Calmodulin 2 G-protein-coupled receptor protein signaling pathway

OSBPL1A 2.25 Oxysterol binding protein-like 1A Phospholipid binding, cholesterol metabolism

TNFSF10 2.21 Tumor necrosis factor superfamily, TNF-related apoptosis-inducing ligand TRAIL member 10 RB1 2.20 Retinoblastoma Cell cycle, tumor suppressor IRF1 2.17 Interferon regulatory factor 1 Immune response, transcription factor activity PSCD1 2.17 Pleckstrin homology, Sec7 Protein sorting and membrane trafficking LLGL1 2.16 Lethal giant larvae homolog 1 Cytoskeleton GLG1 2.14 Golgi apparatus protein 1 Golgi apparatus IGF1R 2.13 Insulin-like growth factor 1 receptor Insulin receptor signaling

VCL 2.12 Vinculin Cell adhesion, acting binding ATF1 2.11 Activating transcription factor 1 Transcription factor activity SMARCA5 2.09 SWI/SNF-related, matrix associated, Regulator of transcription, chromatin modeling -dependent regulator of chromatin

HDGF 2.09 Hepatoma-derived growth Cell proliferation NEDD5 2.07 Neural precursor cell expressed, Cytokinesis developmentally downregulated 5 KIAA1354 2.03 KIAA1354 protein Unknown TJP22.01Tight junction protein 2 Cell adhesion CTNNA1 2.00 Catenin alpha 1, 102 kDa Cell adhesion, signaling TOP2B 1.98 Topoisomerase (DNA) II beta 180 kDa DNA topological change FNTA 1.97 Farnesyltransferase, CAAX box, alpha Protein farnesyltransferase

SP110 1.96 SP110 nuclear body protein Regulation of transcription

FGFR4 1.95 Fibroblast growth factor receptor 4 FGF receptor signaling pathway EVPL 1.90 Envoplakin Cytoskeleton IL13RA1 1.84 Interleukin 13 receptor, alpha 1 Cell surface receptor linked signal transduction UBE2C 1.83 Ubiquitin-conjugating E2C Ubiquitin cycle MEL 1.82Mel-transforming oncogene, Intracellular protein transport RAB8 homolog

BGN 1.76 Biglycan Extracellular matrix structural constituent ACY1 1.74 Aminoacylase 1 Amino-acid metabolism RECQL 1.70 RecQ protein like DNA repair

SNRPF 1.68 Small nuclear ribonucleoprotein Splicing

Oncogene Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4159 Table 2 (Continued ) Gene symbol O/P ratio Description Function

CSNK2B 1.66 Casein kinase 2, beta polypeptide Signal transduction SRPR 1.65 Signal recognition particle receptor Signal recognition particle binding UBE1 1.60 Ubiquitin-activating enzyme E1 Ubiquitin cycle FGB 1.60 Fibrinogen, B beta polypeptide Blood coagulation RAD50 1.56 RAD50 homolog DNA repair INPP5D 1.52Inositol polyphosphate-5-phosphatase Signal transduction (PI3) RPL13A 1.49 Ribosomal protein L13a Protein biosynthesis USP1 1.47 Ubiquitin-specific protease 1 Ubiquitin-specific protease PKNBeta 1.41 Protein kinase PKNbeta Signal transduction

The 59 genes overexpressed in papillary vs oncocytic carcinomas are referenced by their HUGO abbreviations as used in Locus Link. The P/O ratio is the ratio between the median gene expression value in papillary carcinoma and that in oncocytic carcinoma (Pp0.01%)

Table 3 Statistical analysis of three nuclear gene expressions in follicular oncocytic and ordinary mitochondrial-rich papillary thyroid carcinomas Oncocytic carcinomaa (n ¼ 5) Papillary carcinomaa (n ¼ 7) Mann–Whitney test

NO synthase IIIb (NOS3) 12717 79257 797872799 Pp0.05 Alpha-cateninb (CTNNA1) 7187250 13437727 Pp0.03 Alpha-actininb (ACTN2) 33677590 13787254 P ¼ 0.03

The expression levels of three nuclear genes (NOS3, alpha-actinin and alpha-catenin) and one reference gene (b-actin) were measured on a LightCycler apparatus in five oncocytic follicular carcinomas and seven PTCs, different from those explored on the microarrays for 10/12tumors. The amount of cDNA from the different genes was normalized by quantifying b-actin as described elsewhere (Savagner et al., 2001b). The primers used for quantification were as follows: NOS3S, GAGCAGCTGCTGAGTCAGGC; NOS3R, CACTGTGATGGCCGAGCGAA; actinin S, GATCTGGACCATCATCCTTC; actinin R, GACGTACGTCATGATGGCTC; catenin S, CTGCTATGTTCCCTGAGACA; catenin R, CATGGTACGTACAATAGCAG. The reactions were performed following the manufacturer’s recommendations (Roche, Mannheim, Germany). For each run, a standard curve was generated using four 10-fold serial dilutions of an external standard. The different standards were generated by PCR of nuclear cDNA produced from a histologically normal thyroid, using the primers previously described and following the manufacturer’s recommendations (Eurogentec, Seraing, Belgium). The PCR product was phenol–chloroform purified from low melting point agarose and cloned using the Topo TA Cloning kit (Invitrogen, Life Technologies, Groningen, The Netherlands). The recombinant was quantified by spectrophotometry. All samples were tested twice. A melting curve was systematically analysed to check for the specificity of the PCR products. aMean7s.d. bCopy number/b-actin copy number  10À4

of gene and protein data (Huang et al., 2001; Maeta Most of the genes overexpressed in oncocytic folli- et al., 2001). The RT–PCR and immunochemistry study cular carcinomas are involved in metabolic functions concerns a complementary set of seven samples for (83.33%). In addition, the proportion of genes involved PTCs and three samples for oncocytic follicular in response to stress was greater in these tumors carcinomas. The gene expression fold-change deter- (21.05%) than in papillary carcinomas (7.84%). The mined by quantitative RT–PCR analysis correlated well analysis of the 24 genes overexpressed in oncocytic with the fold-change observed by microarray analysis. carcinomas suggested that the rate of oxidative meta- There were no major discrepancies as alpha-actinin was bolism in these tumors was higher than in PTCs. The significantly overexpressed in oncocytic carcinomas (2.4- GLUL gene involved in glutamine synthesis and several fold) and alpha-catenin was significantly overexpressed genes coding for respiratory chain subunits were over- in papillary carcinomas (1.9-fold) (Table 3). At the expressed in oncocytic carcinomas compared to PTCs. protein expression level, the most important difference Overexpression of the different respiratory chain com- concerns the expression of alpha-catenin. In oncocytic plexes has been previously described for both types of carcinomas, the protein expression was less intense than carcinoma (Haugen et al., 2003; Baris et al., 2004). We in PTCs, either situated in the cytoplasm or at the apical showed that complexes I and II were not differentially membrane, in contrast with a stronger expression in expressed in the two types of carcinoma. However, in PTCs at the cellular border (Figure 2). Concerning spite of the abundance of mitochondria in the ordinary alpha-actinin, a homogeneous cytoplasmic staining was PTCs studied, the overexpression of complexes III and found in oncocytic follicular carcinomas. In contrast, IV clearly distinguishes oncocytic carcinomas from the expression in PTCs was more variable from one case PTCs. to another. Alpha-actinin was either situated in the We postulate that oncocytic follicular carcinomas cytoplasm or condensed on the inside of the cellular tend to modulate ROS production by several mechan- membrane (Figure 2). Then, the imbalance in actinin : - isms such as an increase in the electron flow rate through catenin ratio may either reflect or induce a disturbed the overexpression of complexes III and IV, as well as cytoskeleton in PTCs. the induction of glutathione, NO synthase and UCP2.

Oncogene Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4160

Figure 2 Immunoperoxidase staining of oncocytic follicular carcinomas and ordinary papillary carcinomas sections. A total of 26 formalin-fixed, paraffin-embedded thyroid tissue samples (12tumors: five oncocytic follicular carinomas, seven papillary carcinomas, 14 normal) were used for tissue microarray construction as described (Kononen et al, 1998). The oncocytic and the papillary carcinomas were arrayed separately. For each case, the array was composed of a double triplet (the tumor, the normal counterpart). Standard indirect immunoperoxidase procedures were used for immunohistochemistry. The primary antibodies were anti-nitric oxide synthase NOS3 (dilution 1 : 500, Calbiochem, San Diego, CA, USA), anti-alpha-actinin (dilution 1 : 400, Sigma, Saint-Louis, USA), anti-alpha-catenin (dilution 1 : 50, Calbiochem, San Diego, CA, USA). Diaminobenzidine was used as the chromogen and hematoxylin as the nuclear counterstain. Replacing the primary antibody by buffer performed negative controls. The analysis takes into account the cellular localization of the signal in addition to the intensity and the percentage of positive cells scored as 0, no staining; 1, less than 25%; 2, between 25 and 75%; and 3, equal or more than 75%. Magnification (a–f): Â 400. (a) NOS3 in oncocytic carcinomas, homogeneous staining of nearly all the tumor cells. Score 2, four samples and score 3, one sample. (b) NOS3 in papillary carcinomas, dense brown immunostaining at the apical pole of some cells. Score 2, four samples and score 3, three samples. (c) Alpha-actinin in oncocytic carcinomas, the staining occupied the whole cytoplasm in a homogeneous manner. Some brown granules are visible at the apical border. Score 2, three samples and score 3, two samples. (d) Alpha-actinin in papillary carcinomas, the staining is irregularly distributed all over the cytoplasm and concentrated at the inner part of the cellular membrane. Score 2, five samples and score 3, two samples. (e) Alpha-catenin in oncocytic carcinomas, a thin brown membrane staining. Score 1, four samples and score 2, one sample. (f) Alpha-catenin in papillary carcinomas, the cellular membrane staining is sharply defined. Score 2, two samples and score 3, five samples

The upregulation of complex IV may be associated with to UCP2overexpression (Savagner et al., 2001a, 2003). its specific function of regulating the production of free Increased expression of NRF-1 is confirmed in all the radicals. It has been shown that the potential-dependent cases of oncocytic carcinomas in the present study. slip in cytochrome c oxidase prevents the increase of However, further studies on the effect of the phosphor- ROS at the cellular level (Papa et al., 1997). UCP2has ylation level of the NRF-1 protein and of the interaction been reported to limit the production of ROS (Arseni- of NRF-1 with NRF-2on the expression of these jevic et al., 2000). The expression of UCP2, as well as subunits would be necessary to elucidate the specific that of the subunits of complexes III and IV, is overexpression of the complexes III and IV of the dependent on PRC, which is related to the PGC-1 respiratory chain. family. We have shown that the overexpression of PRC Recently, the induction of mitochondrial biogenesis in oncocytic tumors is related to the increased mito- has been associated with NOS3 overexpression through chondrial biogenesis induced by NRF-1 and TFAM and the induction of PGC-1 expression (Nisoli et al., 2003).

Oncogene Gene profiling of mitochondrial-rich thyroid tumors O Baris et al 4161 NO synthesis may mediate mitochondrial and protein turnover or a strong involvement of this protein oncocytic tumor proliferation. As NO is able to inhibit in the mitochondrial biogenesis of oncocytic follicular the activity of complex IV by competing with O2 carcinomas. (Cleeter et al., 1994), increasing the electron flow In conclusion, oxidative metabolism and efficient through complexes III and IV may favor the binding ROS detoxification seem to characterize oncocytic of O2, and thus the oxidative metabolism. In our follicular carcinomas compared to PTCs. In spite of study, we measured the level of NOS3 expression the mitochondrial richness of both types of carcinoma, since this gene was suspected of being involved in the mechanisms of tumor genesis are dissociated and oncocytic development in comparison with normal independent of the mitochondrial metabolism for PTCs. surrounding tissue (Baris et al., 2004). We would like The overexpression of the respiratory chain complexes to specify its role in the two types of mitochondrial-rich III and IV seems to be specific to the development of carcinomas. We found that NOS3 expression was oncocytic follicular carcinomas. increased in oncocytic compared to papillary carcino- mas (Table 3). At the protein level, NOS3 immunostain- ing was highly variable: in oncocytic follicular Acknowledgements We thank Christophe Savagner for the statistical analysis, and carcinomas, the staining was cytoplasmic, and in PTCs, Kanaya Malkani for the critical reading of this paper. This it was either homogeneously distributed in the cyto- work was supported by grants from the French Ministry of plasm or as large focal cytoplasmic dots (Figure 2). The Research, the Institut National de la Sante´ et de la Recherche difference between the protein expression and distribu- Me´ dicale (INSERM) and the Academic Hospital of Angers tion and the transcript results may reflect a difference in and the University of Angers (PHRC 01-10).

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