Endocrine-Related Cancer (2004) 11 843–854

Comprehensive expression profiling of anaplastic thyroid cancers with cDNA microarray of 25 344

M Onda, M Emi, A Yoshida 1, S Miyamoto, J Akaishi, S Asaka, K Mizutani, K Shimizu 2, M Nagahama 3, K Ito 3, T Tanaka 4,5 and T Tsunoda 4

Department of Molecular Biology, Institute of Gerontology, Nippon Medical School, 1-396, Kosugi-cho, Nakahara-ku, Kawasaki 211-8533, Japan 1Kanagawa Prefectural Cancer Center, 1-1-2, Nakao, Asahi-ku, Yokohama 241-0815, Japan 2Department of Surgery, Nippon Medical School and 3Ito Hospital, 4-3-6, Jinguumae, Shibuya-ku, Tokyo 150-8308, Japan 4Laboratory for Medical Informatics, RIKEN, 22-7-1, Suehiro-cho, Tsurumi-ku, Kanagawa 230-0045, Japan 5Laboratory of Molecular Medicine, Center, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan (Requests for offprints should be addressed to M Emi; Email: [email protected])

Abstract Little is known about the genetic mechanisms of anaplastic thyroid cancer (ATC). This is the most virulent of all human malignancies, and it is believed to result from transformation of differentiated thyroid cancers. To identify a set of genes involved in the development of ATC, we investigated expression profiles of 11 cell lines derived from ATC using a cDNA microarray representing 25 344 genes. Semi-quantitative RT-PCR experiments carried out for some genes that had shown altered expression on the microarray verified frequent over-expression of destrin, HSPA8, stathmin, LDH-A, ATP5A1, PSMB6, B23, HDP-1 and LDH-B, and frequent under-expression of thyroglobulin, PBP and c-FES/FPS genes among the cell lines and also among ten primary ATCs. In addition to mRNA expression studies, up-regulation of GDI2, destrin and stathmin were confirmed with immunohistochemical analysis. The extensive list of genes identified provides valuable information towards understanding the development of ATC, and provides a source of possible biomarkers for diagnosis and/or molecular targets for the development of novel drugs to treat ATC. Endocrine-Related Cancer (2004) 11 843–854

Introduction 2003), with a mean survival time among patients of less than 1 year after diagnosis, regardless of treatment Thyroid cancers are classified as medullary, papillary, (Passler et al. 1999, Voutilainen et al. 1999). Differences follicular or anaplastic. The medullary type derives from in biological characteristics among thyroid tumors might parafollicular C cells; papillary, follicular and anaplastic be explained by variations in the pattern of sequential cancer originate from follicular cells of the thyroid gland. somatic mutations among genes that participate in the Anaplastic thyroid cancer (ATC) is thought to arise mainly from a background of differentiated (papillary or mechanisms of growth and differentiation. Although follicular) cancer, on the basis of clinicopathological mutation of TP53 (Kitamura et al. 2000) and b-catenin observations that ATCs are often accompanied by such (Garcia-Rostan et al. 1999) are observed in some ATCs, cells, and because anaplastic tumors tend to arise in the former probably inactivating a tumor suppressor and patients who had previously been treated for differen- the latter activating an oncogenic function, the underlying tiated cancer of the thyroid (Nakamura et al. 1992, Ozaki molecular mechanism involved in this type of thyroid et al. 1999, Kitamura et al. 2000). cancer is poorly understood. The clinical behavior of ATC is markedly distinct Using 11 anaplastic cancer cell lines (ACLs) and ten from other types of thyroid cancer. It is one of the most primary ATCs, we investigated gene-expression profiles virulent cancers of all human malignancies (Sherman on a cDNA microarray consisting of 25 344 genes. The

Endocrine-Related Cancer (2004) 11 843–854 DOI:10.1677/erc.1.00818 1351-0088/04/011–843 # 2004 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 10/02/2021 03:38:52AM via free access Onda et al.: cDNA microarray of anaplastic thyroid cancer tumors displayed remarkably characteristic profiles that photometer, and its quality was checked by formalde- should be useful for molecular diagnosis, for prediction of hyde-agarose gel electrophoresis. prognosis and for identifying potential target molecules for novel drugs to treat this type of cancer. Preparation of cDNA microarray

Materials and methods The microarray contains 27 648 genes and expression sequence tags (ESTs) were generated with Spot Ready ATC cell lines DNA for microarray (Amersham Biosciences Corp., Piscataway, NJ, USA). This cDNA panel was spotted Eleven cell lines that were derived from ATCs, 8305c, using microarray spotter generation III (Amersham 8505c, ARO, FRO, TTA1, TTA2, TTA3, KTA1, KTA2, Biosciences Corp.) on microarray slide type 7 (Amersham KTA3 and KTA4, were used for this study. These cell Biosciences Corp.) and it was then cross-linked by u.v. lines were maintained with Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) for 8305c and 8505c, minimum essential medium for ARO and FRO Labeling of aRNA and competitive and RPMI 1640 for the other seven lines. All media hybridization contained 10% fetal bovine serum but no antibiotics. The cells were cultured in a 378C incubator under 5% CO2 To generate hybridization probes, a 3 mg aliquot of each atmosphere. second-round aRNA was rendered fluorescent with the amino allyl cDNA labeling kit (Ambion), following the manufacturer’s protocol. Probes derived from cell lines Patients and specimens and from the normal thyroid gland pool were labeled Primary ATC and non-cancerous thyroid tissues were respectively with Cy5 or Cy3 mono-reactive dye (Amer- excised from ten patients who underwent surgery at the sham International plc, Amersham, Bucks, UK). To Ito Hospital, Tokyo, Japan; the samples were frozen eliminate incorporated dye, the labeled probes were immediately and stored at 808C. All patients had given cleaned up with QIAquick PCR purification kits (Qiagen). informed consent according to guidelines approved by the Fluorescent labeled probe (15 pmol) from each cell Institutional Research Board. All tumor specimens that line was mixed with the Cy3-labeled normal control in we analyzed contained more than 70% tumor cells. To 4microarray hybridization buffer (Amersham Bio- equalize the tumor-associated deviation in gene expres- sciences Corp.) and de-ionized formamide. The probe sion from normal thyroid, an equal amount of a mixture mixtures were hybridized for 12 h at 408C, then washed of RNAs from the ten non-cancerous tissues was used as a once with 0.1SSC, 0.2% SDS for 5 min and twice for normal control for competitive hybridizations on the 10 min in the same washing solution. All procedures were microarray. performed with an automated slide processor system (Amersham Biosciences Corp.) After hybridization, fluor- escent signals were scanned with GenePix 4000 (Amer- RNA extraction and RNA amplification sham International plc and data were collected by Each tissue was homogenized with TRIZOL reagent GenePix Pro 3.0 software (Amersham International plc). (Invitrogen) according to the manufacturer’s instructions Scanned signals were normalized by a global method for RNA extraction. One microgram of extracted RNA (Manos & Jones 2001, Yang et al. 2002). was electrophoresed on a 3.0% formaldehyde denaturing gel to eliminate degenerated RNA. Samples with 28S/18S Analysis of data ratios greater than 1.5 were selected for subsequent purification using RNeasy kits (QIAGEN, Valencia, We performed random permutation tests to distinguish CA, USA) to eliminate contamination with DNA. Total genes that were expressed differently between ACL and RNA was prepared for microarray analysis using T7 normal thyroid gland (Kitahara et al. 2002). The criteria RNA polymerase-based amplification with MessageAmp for selection of discriminating genes were (1) signal/noise aRNA kits (Ambion, Austin, TX, USA). In the first ratio of the gene greater than 3.0 in at least ten cell lines, round, 5 mg aliquots of total RNA were used as templates (2) P value in a random permutation test lower than for amplification, then 2mg aliquots of first-amplified 0.0001 and (3) expression in cancer at least twofold RNA (aRNA) became the templates for second-round stronger than normal (over-expressed) or half that of amplification. After the second-round amplification, normal thyroid tissue (under-expressed). Genes that fitted aRNAs were purified with RNeasy purification kits, the these criteria were considered significant for discrimina- amount of each aRNA was measured by a spectro- tion.

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Semi-quantitative RT-PCR for ACL and ATC were significantly over-expressed with microarray analysis and SQ-PCR in both ACL and ATC cases. All samples To confirm the microarray results we performed semi- were collected at the Ito Hospital, Tokyo, Japan. quantitative RT-PCR (SQ-PCR) analysis of genes selected Antibodies for this assay were available for GDI2 (1:200 according to the following criteria: (1) the P value in dilution; ProteinTech Group, Inc., Chicago, IL, USA), permutation tests after microarray analysis was below Op-18 (stathmin) (1:100 dilution; Santa Cruz Biotechnol- 0.000001 and (2) expression in ACL was either threefold ogy, Inc., Santa Cruz, CA, USA) and destrin/ADF (1:100 stronger or one-third weaker than in the normal thyroid dilution; Sigma, St Louis, MO, USA). Antigens were gland. In addition to these selected genes, expression of microwaved prior to immunostaining with VECTA- p53 gene was evaluated in ATC samples because p53 is STAIN Elite ABC kits (Vector Laboratories Inc., considered to be one of the key genes in ATC Burlingame, CA, USA) and Dako ENVISION kits carcinogenesis. Five normal thyroid samples served as a (Dako Corporation, Carpinteria, CA, USA) following control. cDNA was reverse transcribed from 10 mg of each the manufacturers’ instructions. The sections were coun- total RNA in the usual manner. To adjust the amount of terstained with hematoxylin, and then scanned at low transcribed cDNA, glyceraldehyde-3-phosphate dehydro- power to identify areas that were evenly stained. genase (GAPDH) was selected as an internal control and Estimates of the numbers of positive cells were scored as SQ-PCR experiments were done as previously described, follows: negative, 0%; 1, 1–10%; 2, 11–25%; 3, 26–50%; after adjustment of concentrations (Ono et al. 2000). The 4, >50% positive (Saiz et al. 2002). Two independent primer sequences for GAPDH were 50-GGAAGGT investigators performed the estimation. GAAGGTCGGAGT-30 (forward) and 50-TGGGTGG AATCATATTGGAA-30 (reverse). Sequence information was collected from the NCBI Results GenBank (http://www.ncbi.nlm.nih.gov/), and all primers were designed with primer 3 software (http://www- Over-expressed genes in ACL genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Permutation tests selected 31 genes and ESTs were up- Information about the PCR primers is available upon regulated in ACL compared with normal thyroid gland. request to the corresponding author. SQ-PCR experi- Those genes, along with their accession numbers, specu- ments were performed with 1 ml cDNA for the template, lated function and chromosomal position are shown in 5U Takara EX Taq (Takara, Otsu, Japan), 1PCR buffer Table 1. Twenty-four of these genes have known or (10 mM Tris–HCl, 50 mM KCl and 1.5 mM MgCl2) and suggested functions. Among the over-expressed group reverse primers in 30 ml of total reaction mixture. PCR were genes encoding small nuclear ribonucleoprotein, conditions for each gene were optimized in their respective stathmin (Op-18) and DNA topoisomerase III, all linear phases of amplification. apparently related to mechanisms of cell growth, were For evaluation of differences in gene expression up-regulated in ACL. On the other hand, metabolism- between ACL/ATC and normal thyroid gland, 10 mlof related genes such as ATP5A1, ATP synthase and ODC1 each SQ-PCR product was electrophoresed on a 2.0% were also over-expressed in ACL; however, these result agarose gel and stained with ethidium bromide. After might simply reflect the activated cell dynamics in the staining, the density of each sample spot was measured by immortalized cells. In other categories, several genes AlphaImager 3300 (AlphaIonotech, San Leandro, CA, encoding ribosomal were expressed dominantly USA) with background revision. A 16 bit imaging score in ACL, although a literature search revealed no implied was acquired from each sample. All SQ-PCR experiments connection between expression levels of those genes and were duplicated. We applied Student’s t-tests to the results thyroid cancer, especially in ATC, the anaplastic form in of the SQ-PCR assay; P values smaller than 0.05 were particular. considered statistically significant. All statistical proce- dures were archived by Statview version 5.0 software (SAS Institute Inc., Cary, NC, USA). Under-expressed genes in ACL The 56 genes that were under-expressed in ACL in comparison with the normal thyroid gland are listed in Immunohistochemical analysis Table 2. Fifty-one of these were known genes, with a wide To determine a correlation between RNA expression and distribution of speculated functions. For example, ubi- protein expression, we performed immunohistochemical quitin-activating enzyme E1 is thought to be a tumor- analyses using ten paraffin-embedded samples of primary suppressor gene, while the proto-oncogenes encoding c- ATC in three selected genes. GDI2 was significantly over- fes/fps and human receptor protein tyrosine phosphatase expressed with microarray analysis, stathmin and destrin hPTP-J precursor were unexpectedly under-expressed in

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Table 1 List of over-expressed genes in ACL

Gene name Accession number Function Position

FLJ39361 fis, clone PEBLM 2004733 AK096680 Unknown 5p14 GD12 GDP dissociation inhibitor 2 NM_001494 Miscellaneous 10p15 Ribosomal protein L9 U09953 Ribosomal protein 4p13 DSTN-destrin (actin depolymerizing factor) NM_006870 Cell structure 20p11.23 Small nuclear ribonucleoprotein poylpeptide A (SNPRA) NM_004596.1 Cell proliferation 19q13.1 IMAGE:1141693 50 similar to gb:X 16869 elongation factor 1-a 1 (eef1a1) AA706503 Unknown 9q34 Tyrosine 3-mono-oxygenase/tryptophan 5-mono- oxygenase activation protein, poly peptide NM_003406 Signal transduction 8q23.1 Scaffold attachment factor A (SAF-A) AF068846 Transcription 1q44 IMAGE:47833 30 similar to gb:K00558 tubulin a-1 chain H11622 Unknown 12q13 Hums3 mRNA for 40S ribosomal protein s3 X55715 Ribosomal protein 11q13.3-q13.5 HSP A8 heat shock 70 kDa protein 8 BC016660 Miscellaneous 12q23 Stathmin X53305 Cell proliferation 1p36.1-p35 ELAM-1 ligand fucosyltransferase (ELFT) M58596 Enzyme 11q21 NPD017 AF271783 Unknown Xq22.1 Lactate dehydrogenase-A (LDH-A, EC 1.1.1.27) NM_005566 Metabolism 11p15 MRL3 mRNA for ribosomal protein L3 homolog P09001 Miscellaneous 3q21-q23 Heterogeneous nuclear ribonucleoprotein complex K S74678 Transcription 9q21.32-q21.33 Vascular proton-ATPase subunit M9.2 Y15286 Miscellaneous 5q35.2 NEDD5: neural precursor cell expressed, developmentally down regulated 5 NM_004404 Miscellaneous 2q37 HL23 ribosomal protein homolog X55954 Ribosomal protein 17q NAD+-specific isocitrate dehydrogenase b subunit precursor (IDH3B) U49283 Miscellaneous 20p13 Hums3 mRNA for 40S ribosomal protein s3 X55715 Ribosomal protein 11q13.3-q13.5 ATP5A1 BT007209 Metabolism 18q12-q21 PSMB6, proteasome subunit Y D29012 Prosome 17p13 Annexin A2 D00017 Miscellaneous 15q21-q22 hB23 gene for B23 nucleophosmin M26697 Miscellaneous 5q35 KIAA0683 protein NM_016111 Unknown 16p13.3 Similar to helix-destabilizing protein (HDP-1) XM_210678 Unknown 6p22.1 DNA topoisomerase III U43431 Replication 17p12-p11.2 Lactate dehydrogenase B (LDH-B) Y00711 Metabolism 12p12.2-p12.1 Ornithine decarboxylase (ODC1) M16650 Metabolism 2p25

ACL. These results suggested either that the proposed chosen according to the criteria of (1) a P value below functions of the listed gene are variable, or the genes 0.000001 in the permutation test of microarray results and themselves were altered in ATC cell lines. Moreover, (2) expression levels in tumor at least threefold stronger or genes encoding laminin B2 chain and PLOD3, said to one-third weaker than that of normal thyroid gland tissue, relate to cell structure, were down-regulated in ACL. A as shown in Materials and methods. Figures 2 and 3 show gene expression portrait of all 87 genes with altered SQ-PCR results for 12 of the over-expressed genes (Fig. expression in the cell lines is shown in Fig. 1. 2A for ACL, Fig. 2B for ATC) and 15 under-expressed genes (Fig. 3A for ACL, Fig. 3B for ATC). All of the results corroborated our microarray data, with statistical Validation of microarray data and comparison significance evaluated by Student’s t-test. of ACLs with ATCs Of the 12 genes that were significantly over-expressed To validate the microarray results, we performed SQ-PCR in ACL, destrin, HSPA8, stathmin, LDH-A, ATP5A1, experiments using samples from 11 cancer cell lines and PSMB6, B23, HDP-1 and LDH-B were commonly over- ten primary ATC tumors, as well as tissues from five expressed both in primary ATC tumors and ACL. normal thyroid glands, and evaluated gene expression NPD017, NAD+ specific isocitrate dehydrogenase-b after normalization of signals according to the expression subunit precursor (IDH3B) and ANXA2 were over- of GAPDH. The genes to be tested by SQ-PCR were expressed only in cell lines. Of the 15 significantly

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Downloaded from Bioscientifica.com at 10/02/2021 03:38:52AM via free access www.endocrinology-journals.org Table 2 List of under-expressed genes in ACL

Gene name Accession number Function Position

Carcinoembryonic antigen (CGM2) L31792 Cell adhesion 19q13.2 Cell cycle-regulated factor p78 AF068007 Cell cycle 12q13.12 Proliferating-cell nucleolar protein P120 M32110 Cell proliferation 12p13 Splicing factor, arginine/serine-rich 7 (SFRS7) NM_006276 Cell proliferation 2p22 M-phase phosphoprotein, mpp5 X98261 Cell proliferation 10q11 KH type splicing regulatory protein (KSRP) U94832 Cell proliferation 19p13.3 Aquaporin D63412 Cell structure 18q11.2-q12.1 Laminin B2 chain J03202 Cell structure 1q31 lysylhydoxylase isoform 3 (PLOD3) AF046889 Cell structure 7q22 CD34 S53911 Cell surface antigen 1q32 Histone deacetylase 3 (HDAC3) U66914 structure 5q31 Estrogen sulfotransferase (SULTIA3) L25275 Enzyme 16p11.2 Methylmalonate semialdehyde dehydrogenase (M M SDH) M93405 Enzyme 14q24.3 NAD (H)-specific isocitrate dehydrogenase g subunit (IDH3G) U69268 Enzyme Xq28 Heat shock protein (hsp 70) gene NP_005336 Heat shock protein 6p21.3 Thyroglobulin (TG) X05615 Hormone 8q24.2 IgG Fc receptor hFcRn (FCGRT) U12255 Immune response 19q13.3 Arg/Abl-interacting protein ArgBP2a (ArgBP2a) AF049884 Kinase 4q35.1 Protein tyrosine kinase related mRNA sequence L05148 Kinase 2q12 Phosphatidylethanolamine-binding protein (PBP) X75252 Kinase inhibitor 12q24.23 Glucose-6-phosphate dehydrogenase M24470 Metabolism Xq28 Multiple exostosis-like protein (EXTL) U67191 Metabolism 1p36.1 Cancer Endocrine-Related Carbonic anhydrase precursor (CA 12) AF037335 Metabolism 15q22 Acyl-CoA thioester hydrolase U91316 Metabolism 1p36.31-p36.11 Clones 23667 and 23775 zinc finger protein U90919 Miscellaneous 14q24.3 WD repeat protein HAN11 (AN11) U94747 Miscellaneous 17q24.1 TTF-I interacting peptide 20 AF000560 Miscellaneous 19q13.4 ADP-ribosylation factor NM_001661 Miscellaneous 17q12 nkat9 L76672 Miscellaneous 19q13.4 ATP-driven ion pump (ATP1AL1) U02076 Miscellaneous 13q12.1-q12.3 Secretory protein (P1.B) L15203 Miscellaneous 21q22.3 Downloaded fromBioscientifica.com at10/02/202103:38:52AM Neuronal nitric oxide synthase (NOS1) U17327 Miscellaneous 12q24.2-q24.31 Sex hormone-binding globulin (SHBG) X05403 Miscellaneous 17p13-p12 Metaxin 1 (MTX1) NM_002455 Miscellaneous 1q21 (2004) Butyrophilin (BTF2) U90550 Miscellaneous 6p22.1 Parathymosin M24398 Miscellaneous 17q12-q22

HBB hemoglobin, b AF117710 Miscellaneous 11p15 11 Receptor protein tyrosine phosphatase hPTP-J precursor U73727 Oncogene 1p35.3-p35.1 843–854 c-fes/fps proto-oncogene (FES) X06292 Oncogene 15q26.1 Serine-threonine phosphatase (PP5) U25174 Phosphatase 19q13.3 847 PSMC1 proteasome 26S subunit, ATPase, 1 NM_002802 Prosome 14q32.11 26S proteasome subunit p97 D78151 Proteasome Chr.3 via freeaccess nae l:cN iraryo npatctyodcancer thyroid anaplastic of microarray cDNA al.: et Onda 848 Table 2 continued

Gene name Accession number Function Position

BBC1 X64707 Ribosomal protein 16q24.3 Bone morphogenetic protein receptor type I (ALK-6) U89326 Signal transduction 4q23-q24 Autoantigen calreticulin (CALR) M84739 Signal transduction 19p13.2 Guanine nucleotide exchange factor p115-RhoGEF (ARHGEF1) U64105 Signal transduction 19q13.13 TNF receptor associated factor 6 (TRAF6) U78798 Signal transduction 11p12 TRAMP protein X63679 Signal transduction Chr.8 CD1a antigen (CD1a gene) X04450 Tumor antigen 1q21-q23 UCHL3 ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiol esterase) M30496 Ubiquitination 13q21.33 Ubiquitin-activating enzyme E1-related protein L13852 Ubiquitination, tumor suppressor 3p21 IMAGE:343903 W69766 Unknown 16q22 TEX27 testis expressed sequence 27 AL137302 Unknown 6pter-p22.3 C21orf97 chromosome 21 open reading frame 97 NM_021941 Unknown 21q22 HS747E2A hypothetical protein HS747E2A AL035364 Unknown 22q12.1 IMAGE:1715637 30 similar to gb:J00117 choriogonadotropin b chain precursor AI160936 Unknown 19q13.3

Gene in bold locates on chromosomal region where loss of heterozygosity was previously reported in ATC. Downloaded fromBioscientifica.com at10/02/202103:38:52AM www.endocrinology-journals.org via freeaccess Endocrine-Related Cancer (2004) 11 843–854

Genes AF117710 M24398 U90550 AI160936 NM_002455 AL035364 X05403 U94832 L15203 NM_021941 U17327 U02076 U91316 X63679 AL137302 AF046889 L76672 H28951 J03202 AF037335 AF068007 U67191 U69268 S53911 U66914 AF000560 X06292 U78798 D78151 H06378 U25174 M93405 M24470 L31792 L05148 X64707 W69766 X98261 U73727 U64105 M30496 NM_002802 AF049884 X75252 L13852 U94747 L25275 D63412 X04450 U12255 M84739 R39171 M32110 U90919 U89326 X05615 M16650 Y00711 U43431 XM_210678 NM_016111 M26697 BT007432 D29012 BT007209 X55715 U49283 X55954 NM_004404 Y15286 S74678 H05820 NM_005566 AF271783 M58596 X53305 BC016660 X55715 H11622 AF068846 M86400 AA706503 R25535 U09953 NM_006870 NM_001494 AK096680 Cell Line 8305c 8505c ARO FRO TTA1 TTA2 TTA3 KTA1 KTA2 KTA3 KTA4

Under-expressed genes Over-expressed genes

LOW HIGH

Expression level

Figure 1 Expression portrait of 87 genes with significantly altered expression in ACL, according to microarray analysis; 31 of them were up-regulated and 56 were down-regulated in the cell lines. The signal indicator reflects the expression intensity of each gene in each ACL sample. The darkest red at the right-hand end of the bar indicates expression in ACL that is three times as strong as that in normal thyroid; the darkest green at the far left of the bar indicates a level of expression a third of normal thyroid. under-expressed genes in ACL, thyroglobulin, PBP and c- Immunohistochemical analysis for GDI2, fes/fps proto-oncogene, were also under-expressed in destrin and stathmin primary tumor. Down-regulation of calreticulin To evaluate the correlation between microarray profiling (CALR), hFcRn (FCGRT), estrogen sulfotransferase and protein expression, we performed immunohistochem- (SULT1A), HAN11, p115-RhoGEF (ARHGEF1), ical analysis of three antigenic for which anti- methylmalonate semialdehyde dehydrogenase bodies were commercially available (GDI2, stathmin and (MMSDH), TRAF6, TTF-1, CD34, NAD (H)-specific destrin). GDI2 was highly expressed (scored over 3) in isocitrate dehydrogenase-g subunit (IDH3G), C21orf97 and sex hormone-binding globulin (SHBG), were limited seven of the ten primary ATC tumors. Stathmin (Op18) to cancer cell lines. was also highly expressed in primary ATCs, with six With regard to the p53 gene, the expression was tumors scoring over 3. Destrin was stained in five cases, slightly decreased (0.92-fold) in ACLs but it did not reach mainly in cytoplasm. The representative results of these statistical significance with microarray analysis. We also experiments are shown in Fig. 5. On the other hand, all tested p53 gene expression in ten ATC samples with SQ- genes showed lower expression (staining) in part of the PCR. The decreased expression was seen in ATC normal thyroid tissue. Table 3 summarizes the results of compared with normal thyroid tissue ðP ¼ 0:03Þ (Fig. 4). immunostaining analysis of GDI2, stathmin and destrin in

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Normal ACL Normal ACL

M N1 N2 N3 N4 N5 1 2 3 4 5 6 7 8 9 10 11 M N1 N2 N3 N4 N5 1 2 3 4 5 6 7 8 9 10 11 DSTN p=1.97E-9 TG p=2.7E-16 HSPA8 p=6.67E-8 CALR p=1.62E-6 Stathmin p=2.5E-6 FCGRT p=1.06E-6 NPD017 p=5.47E-5 SULT1A3 p=8.76E-5 LDH-A p=2.34E-8 HAN11 p=0.0043 IDH3B p=0.0014 PBP p=9.08E-14 ATP5A1 p=9.81E-10 ARHGEF1 p=0.05 PSMB6 p=3.6E-6 MMSDH p=5.14E-8 ANXA2 p=1.87E-11 TRAF6 p=0.0032 B23 p=8.11E-11 FES p=2.23E-8 HDP-1 p=1.85E-9 TTF-1 p=2.3E-6 LDH-B p=5.7E-6 CD34 p=9.73E-10 IDH3G p=2.35E-5 GAPDH C21orf97 p=0.00014 SHBG p=1.05E-5 Normal ATC

M N1 N2 N3 N4 N5 1 2 3 4 5 6 7 8 9 10 GAPDH DSTN p=0.04 Normal ATC HSPA8 p=0.0004 M N1 N2 N3 N4 N5 1 2 3 4 5 6 7 8 9 10

Stathmin p=0.02 TG p=0.0004

LDH-A p=0.001 PBP p=0.0004 ATP5A1 p=0.006

PSM B6 p=0.0003 FES p=0.008 B23 p=0.001

HDP-1 p=0.001 GAPDH LDH-B p=0.005

GAPDH Figure 3 (A) SQ-PCR assay of 15 genes that were significantly under-expressed in ACL, using GAPDH as an internal control. The intensity of each sample was measured and evaluated with Figure 2 (A) Confirmation by SQ-PCR of up-regulation of 12 a 16 bit image and adjustment of background; P values shown genes in ACL. Expression of GAPDH constituted an internal are the result of t-tests. M, size marker; N1-N5, normal thyroid; control. The intensity of each sample was measured and lane 1, cell line 8305c; lane 2, 8505c; lane 3, ARO; lane 4, FRO; evaluated with a 16 bit image and adjustment of background. lane 5, TTA1; lane 6, TTA2; lane 7, TTA3; lane 8, KTA1; lane 9, P values are the results of t-tests. M, size marker; N1-N5, KTA2; lane 10, KTA3; lane 11, KTA4. (B) SQ-PCR images normal thyroid; lane 1, cell line 8305c; lane 2, 8505c; lane 3, showing down-regulation of three genes in primary ATCs. ARO; lane 4, FRO; lane 5, TTA1; lane 6, TTA2; lane 7, TTA3; Ex, 10x. lane 8, KTA1; lane 9, KTA2; lane 10, KTA3; lane 11, KTA4. (B) SQ-PCR results in primary ATCs. Nine of the 12 genes over- study. In addition, this is the first report of over-expression expressed in ACL were also significantly over-expressed in all of in RNA and protein levels of these three genes in human the primary tumors; P values are the results of t-tests. Ex, 10x. thyroid malignancies as far as we can find in the literature. all ten primary ATC cases. Consistent correlation between Discussion cDNA microarray data, SQ-PCR data and immunohis- Among human cancers, ATC is one of the most aggressive tochemistry provides solid verification for the present and has the highest potential for malignancy; at present,

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Normal ATC of the immortalized cells. Among the 31 genes in this list, M N1 N2 N3 N4 N5 1 2 3 4 5 6 7 8 9 10 GDI2 (located on 10p15) binds and solubilizes several membrane-associated Rab proteins in a GDP/GTP- p53 P=0.03 dependent manner (Chinni et al. 1998). Amplification of GAPDH chromosome 10p has been observed in a small number of head and neck cancers (Speicher et al. 1995). Our study showed up-regulation and over-expression of GDI2 in Figure 4 Results of SQ-PCR experiment for p53 gene both ACL and ATC, suggesting that GDI2 contributes to expression analysis with ATC samples. Expression of p53 in ATC was weakly decreased compared with normal thyroid carcinogenesis of ATC. tissue; P ¼ 0:03 (Student’s t-test). The gene encoding destrin behaved similarly in our experiments. Destrin is important for actin remodeling, endocytosis, polarized cell growth and cellular activation the 1-year survival rate among patients with this disease is (Moriyama et al. 1990, Yahara et al. 1996). Chromo- only a few percent (Passler et al. 1999, Voutilainen et al. some 20p, where the destrin gene locates, is often 1999). Although several clinical approaches have been amplified in ovarian cancer cell lines (Watanabe et al. tried, no effective therapeutic strategy has yet been 2001). Our results suggest that destrin might be a established for ATC. Few studies on the molecular aspect previously unsuspected participant in carcinogenesis. of this disease have been done because ideal biological For its part, stathmin (Op18) is a member of a novel material is hard to obtain, since most ATC tumors are not class of microtubule-destabilizing proteins that regulate subject to surgical intervention. the dynamics of microtubule polymerization and depo- For this study we did have access to 11 established lymerization (Mistry & Atweh 2002). Stathmin protein ACLs for analysis on a cDNA microarray, which allowed appears to have oncogenic potential, because it is widely us to construct a molecular portrait of ATC-derived cells. expressed in various kinds of human cancers, including This is the first report to document a comprehensive gene leukemia (Ghosh et al. 1993), prostate cancer (Friedrich expression profile of ATC. At present, p53 is the most et al. 1995) and breast cancer (Curmi et al. 2000), and well-known gene that might be responsible for ATC because inhibition of stathmin can decrease the rate of carcinogenesis (Kitamura et al. 2000); however, the proliferation of K562 erythroleukemic cells (Luo et al. expression level of p53 did not alter significantly between 1994). In the clinical setting, some anti-cancer drug ACL and normal thyroid tissue with microarray analysis. In ATC samples, expression of p53 was weakly decreased. regimens are designed to inhibit microtubule assembly In addition, point mutations of p53 were found in five and arrest cells in mitosis, or to promote assembly of ACLs (TTA1: codon 72, CGC (wild type)-CTC (mutant); microtubules and stabilize tubulin polymers by prevent- KTA2: codon 158, AAC-GAC; 8305c: codon 248, CGG- ing their depolymerization (Mistry & Atweh 2002). GGG; ARO: codon 273, CGT-CAT; KTA3: codon 276, Over-expression of annexin II (15q21-22) reflects poor GCC-CCC) All mutations were missense mutations but prognosis of colorectal and gastric cancers (Emoto et al. there was no specific mutation spectrum in ACLs. Hence 2001a,b). Our results appear to corroborate an onco- it is difficult to explain the carcinogenic mechanism of genic role of annexin II in ATC. only p53 single gene alterations. It is therefore necessary On the other hand, 56 genes, including hypothetical to examine gene expression alterations widely through the protein and ESTs, were significantly down-regulated in human genome to discover the novel genes responsible for ACLs on our microarray. Although one of them, ACL/ATC carcinogenesis. thyroglobulin, serves as a tumor marker in differentiated Although some discrepancies are likely to exist thyroid cancers (Hoang-Vu et al. 1992), its expression was between cell lines and primary ATCs, in general cell significantly decreased in ACL. Expression of TTF-1 lines are thought to reflect, to a large degree, the (interacting peptide 20) also decreased in this study; the characteristics of their tumors of origin; for example, a TTF-1 gene encodes a transcription factor that contri- relationship between gene expression profiles of cell lines butes to expression of thyroid-specific proteins like and primary tumor has been confirmed in bladder cancer thyroglobulin (Fabbro et al. 1994). These results implied (Sanchez-Carbayo et al. 2002). Our microarray analysis that a drastic abolition of normal thyroidal function had identified the up-regulation of 31 genes in the panel of 11 occurred in the tissue — giving rise to the parent ATCs, ACLs. Some of the over-expressed genes encoded and suggested that thyroglobulin cannot be used as a ribosomal proteins, and several others encoded metabo- marker for anaplastic thyroid tumors. PBP was also lism-related proteins such as lactate dehydrogenase A, B under-expressed. PBP, alternatively known as Raf- or ATP5A1 (ATP synthase); however, these may have mediated activation inhibitor protein (RKIP), is expressed been over-expressed simply as a result of active dynamism in prostate cancers but not in metastatic foci derived from

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GDI2 Stathmin

X 200 X 200

Destrin

X 200

Figure 5 Representative images from immunohistochemical analyses of GDI2, stathmin and destrin, confirming over-expression of these proteins in primary ATC materials. GDI2 protein was diffuse in cytoplasm; stathmin was expressed in cytoplasm and the periphery of nuclei; destrin was expressed only in cytoplasm. those tumors. In other reports, over-expression of RKIP (17p13-p12) and autoantigen CALR (19p13.2) locate on in C4-2B cells decreased cell invasion in vitro and inhibited the chromosomal position which showed frequent LOH in lung metastasis (Fu et al. 2003). Thus PBP has shown a ATC (40%, 44% and 36% respectively) (Kitamura et al. tumor-suppressive function in prostate cancer; our data 2000). These genes should be investigated as to potential suggest that PBP might be a tumor suppressor for human function of tumor suppression in thyroid tissue. ATC as well. Of the 56 under-expressed genes listed in Cell lines generally reflect the character of their tumors Table 2, 22 genes are located in chromosomal regions of origin but it is always possible for the nature of a cell line where we previously detected LOH in > 20% of to change during immortalization. It is therefore necessary informative ATCs. In particular, CD34 (1q32), SHBG to evaluate expression profiles of primary tumors as well,

Table 3 Summary of immunohistochemical analysis. Numbers show the staining score

Case GD12 Stathmin Destrin

ATH-1 2 3 4 ATH-2 4 3 4 ATH-3 Negative 1 2 ATH-4 4 4 3 ATH-5 1 1 1 ATH-6 3 1 1 ATH-7 4 4 1 ATH-10 4 4 4 ATH-11 3 2 3 ATH-13 2 4 2

Percentage of positive staining cells; 0%, negative; 1–10%, 1; 11–25%, 2; 26–50%, 3; > 50% positive, 4.

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Downloaded from Bioscientifica.com at 10/02/2021 03:38:52AM via free access Endocrine-Related Cancer (2004) 11 843–854 but because ATC is so aggressive that there is little chance Foltz for critical reading of this manuscript. Cell lines for surgical intervention which would provide fresh tissue 8305c and 8505c were obtained from the Japanese for analysis. For the study reported here we did have access Collection of Research Bioresources Cell Bank. ARO to ten precious ATC samples, and we were able to perform and FRO, established by Dr G J F Juillard (University of SQ-PCR with selected genes. Of 12 genes that had been California–Los Angeles, Los Angeles, CA, USA), were significantly over-expressed in ACL, nine (destrin, HSPA8, kindly given by Dr H Nanba (Nagasaki University, stathmin, LDH-A, ATP5A1, PSMB6, B23, HDP-1 and Nagasaki, Japan). The others cell lines were established by LDH-B) were also over-expressed in primary ATCs. Of A Y. This work was supported by special grants for those, LDH-A and -B are isoenzymes, and LDH-A is Strategic Advanced Research on Cancer from the reported to increase in tumors of all origins (Liu et al. Ministry of Education, Science, Sports and Culture of 2003). Generally speaking, up-regulation of metabolic Japan and by a Research for the Future Program Grant enzymes such as LDH-A or -B and ATP5A1 is likely to be of the Japan Society for the Promotion of Science. the result of high metabolic activity of ACL, not a cause of carcinogenesis. B23 (alternatively NPM1) is a nucleolar phosphoprotein that is more abundant in tumors than in References normal cells. Functionally, it relates to chromosomal Chinni SR, Brenz M & Shisheva A 1998 Modulation of GDP- translocation, and its over-expression has been confirmed dissociation inhibitor protein membrane retention by the in acute myeloid leukemia by microarray analysis (Jhanwar cellular redox state in adipocytes. Experimental Cell Research et al. 1984). HSPA8, PSMB6 and HDP-1, located at 12q23, 242 373–380. 17p13 and 6p22.1 respectively, do not have any known Curmi PA, Nogues C, Lachkar S, Carelle N, Gonthier MP, Sobel functions, especially in cancer. On the other hand, up- A, Lidereau R & Bieche I 2000 Overexpression of stathmin in regulation of NPD017, IDH3B and ANXA2 was found in breast carcinomas points out highly proliferative tumours. ACLs only, suggesting that the activating changes might British Journal of Cancer 82 142–150. have been acquired in the process of immortalization. Emoto K, Sawada H, Yamada Y, Fujimoto H, Takahama Y, Of the 15 genes that were selected from the list of Ueno M, Takayama T, Uchida H, Kamada K, Naito A et al. under-expressed elements in ACL only three (thyroglo- 2001a Annexin II overexpression is correlated with poor bulin, PBP and FES) were under-expressed in primary prognosis in human gastric carcinoma. Anticancer Research 21 1339–1345. tumors as well. 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