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Genome-Wide Gene Expression Profiles of Thyroid Carcinoma: Identification of Molecular Targets for Treatment of Thyroid Carcinoma

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Genome-Wide Gene Expression Profiles of Thyroid Carcinoma: Identification of Molecular Targets for Treatment of Thyroid Carcinoma 105-121 10/6/08 12:41 Page 105 ONCOLOGY REPORTS 20: 105-121, 2008 105 Genome-wide gene expression profiles of thyroid carcinoma: Identification of molecular targets for treatment of thyroid carcinoma DRAGOMIRA NIKOLAEVA NIKOLOVA1,3, HITOSHI ZEMBUTSU1, TANIO SECHANOV2, KALIN VIDINOV2, LOW SIEW KEE1, RADINA IVANOVA2, ELITZA BECHEVA3, MIRIANA KOCOVA4, DRAGA TONCHEVA3 and YUSUKE NAKAMURA1 1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; 2Clinic of Endocrine Surgery, Medical University of Sofia, Sofia 1303; 3Department of Medical Genetics, Medical University of Sofia, Sofia 1431, Bulgaria; 4Department of Endocrinology and Genetics, Medical Faculty, Skopje 1000, The Former Yugoslav Republic of Macedonia Received January 21, 2008; Accepted March 12, 2008 Abstract. In order to clarify the molecular mechanism better understanding of the tumorigenesis of thyroid cancer involved in thyroid carcinogenesis and to identify candidate and could contribute to the development of diagnostic tumor molecular targets for diagnosis and treatment, we analyzed markers and molecular-targeting therapy for patients with genome-wide gene expression profiles of 18 papillary thyroid thyroid cancer. carcinomas with a microarray representing 38,500 genes in combination with laser microbeam microdissection. We Introduction identified 243 transcripts that were commonly up-regulated and 138 transcripts that were down-regulated in thyroid Thyroid carcinoma represents 1% of all malignant diseases carcinoma. Among these 243 transcripts identified, only 71 and accounts for nearly 90% of neuroendocrine malignancies. transcripts were reported as up-regulated genes in previous Papillary thyroid carcinoma (PTC) is the most common microarray studies, in which bulk cancer tissues and normal malignant thyroid tumor, representing 80-90% of thyroid thyroid tissues were used for the analysis. We further selected malignancies. Papillary, follicular and anaplastic thyroid genes that were overexpressed very commonly in thyroid carcinomas are known to arise from follicular epithelial carcinoma, though were not expressed in the normal human cells while medullary thyroid carcinomas arise from the tissues examined. Among them, we focused on the regulator parafollicular C cells of the thyroid. PTC is usually well- of G-protein signaling 4 (RGS4) and knocked-down its differentiated; however, the clinical characteristics of PTC expression in thyroid cancer cells by small-interfering RNA. vary widely. For example, incidental microcarcinomas are The effective down-regulation of its expression levels in often noninvasive or minimally invasive and grow very thyroid cancer cells significantly attenuated viability of slowly. However, invasive PTC often causes metastasis and thyroid cancer cells, indicating the significant role of RGS4 can be lethal. PTCs often recur many years after their surgical in thyroid carcinogenesis. Our data should be helpful for a operation. The prognosis for PTC is frequently favorable, though ~20% of patients with PTC experience recurrence and some of them die of the disease (1,2). A postoperative _________________________________________ follow-up for monitoring recurrence is critically important for a favorable outcome. Although PTC can be diagnosed by a Correspondence to: Dr Yusuke Nakamura, Laboratory of cytological examination of fine-needle aspiration materials, Molecular Medicine, Human Genome Center, Institute of Medical this method is not applicable for the prediction of a post- Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, operative clinical outcome. Serum thyroglobulin (TG) Tokyo 108-8639, Japan immunoassay has been used for monitoring the recurrence of E-mail: [email protected] differentiated thyroid carcinoma, though measurement of serum TG is sometimes hindered by the presence of Abbreviations: PTC, papillary thyroid cancer; LMM, laser circulating factors, particularly anti-TG antibodies and a microbeam microdissection; TG, thyroglobulin; ATC, anaplastic residual normal thyroid gland tissue that produces TG (3). thyroid carcinoma The genome-wide microarray analysis enabled us to obtain Key words: gene expression profile, microarray, thyroid carcinoma, comprehensive gene expression profiles related to phenotypic molecular target gene and biological information in cancer cells (4-10). We have identified multiple applicable targets for the development of novel anti-cancer drugs and/or diagnostic markers. For thyroid 105-121 10/6/08 12:41 Page 106 106 NIKOLOVA et al: EXPRESSION PROFILING OF THYROID CARCINOMA carcinoma, this approach is useful for identifying unknown Table I. Clinical features of PTC clinical samples used for molecules involved in the carcinogenic pathway (5,11-14). microarray analysis. Through gene expression profile analysis of 18 papillary ––––––––––––––––––––––––––––––––––––––––––––––––– thyroid cancers coupled with the purification of the cancer No Age Gender Clinical Stage cell population by laser microbeam microdissection (LMM) ––––––––––––––––––––––––––––––––––––––––––––––––– on a microarray consisting of ~38,500 transcripts, we TH_T_1 37 Female III identified a number of transcripts that were overexpressed in TH_T_3 69 Female III thyroid cancers. We report important information regarding TH_T_4 54 Female II the mechanisms of thyroid carcinogenesis as well as the TH_T_6 22 Female III discovery of potential targets for the development of diagnostic markers and signal-suppressing therapeutic TH_T_7 60 Female II strategies for thyroid cancer treatment. TH_T_8 58 Female II TH_T_9 34 Female III Materials and methods TH_T_10 71 Male III TH_T_11 64 Female II Tissue samples and microdissection. Eighteen thyroid cancer TH_T_13 49 Female I tissue samples (17 female and 1 male patients; median age, TH_T_14 28 Female III 46 years; range, 20-71 years; Table I) were obtained with TH_T_15 47 Female I written informed consent along with adjacent normal thyroid tissue samples from patients undergoing surgery at the Clinic TH_T_16 26 Female III of Endocrine Surgery (Sofia, Bulgaria). These cancer tissues TH_T_20 44 Female II were histopathologically diagnosed as papillary thyroid TH_T_21 36 Female II carcinoma. Clinical information was obtained from medical TH_T_26 45 Female II records. Clinical stage was judged according to the UICC TH_T_28 20 Female II TNM classification. Each speciman was embedded in TH_T_31 59 Female II TissueTek OCT medium (Sakura, Tokyo, Japan) immediately ––––––––––––––––––––––––––––––––––––––––––––––––– after surgical resection and stored at -80˚C until use. These frozen tissues were cut into 8-μm sections using a cryostat (Sakura) and then stained with hematoxylin and eosin IVT labeling kit (Affymetrix). A 20 μg aliquot of the labeled (H&E) for histological examination. Thyroid cancer cells product was fragmented by heat and ion-mediated hydrolysis and corresponding normal thyroid epithelial cells were at 94˚C for 35 min in H2O and 8 μl of 5x fragmentation buffer selectively collected using the EZ cut system with a pulsed (Affymetrix). The fragmented cRNA was hybridized for 16 h ultraviolet narrow beam-focus laser (SL Microtest GmbH, at 45˚C in a hybridization oven 640 to a U133 Plus 2.0 Jena, Germany) according to the manufacturer's protocols. oligonucleotide array (Affymetrix). The washing and staining RNA isolated from the microdissected normal thyroid of the arrays with phycoerythrin-conjugated streptavidin follicular epithelial cells of 13 individuals was mixed and (Molecular Probes, Eugene, OR) was completed in a used as a ‘universal control’ for each of the 18 cancer samples. fluidics station 450 (Affymetrix). The arrays were then scanned using a confocal laser GeneChip scanner 3000 Cell lines. The human thyroid carcinoma cell line, FTC-133, (Affymetrix). was used in this study. FTC-133 was grown in a monolayer in appropriate media supplemented with 10% fetal calf serum Data analysis. Global normalization at a target value of 500 (FCS) and 1% antibiotic/antimycotic solution (Sigma- was applied to each of the 19 arrays (18 cancer arrays and one Aldrich). FTC-133 was maintained at 37˚C in an atmosphere array of the universal control) under consideration using of humidified air with 5% CO2. GeneChip operating software (Affymetrix). Normalized data from text files were imported to a Microsoft Excel spread Affymetrix GeneChip hybridization. The Affymetrix human sheet. Since data derived from low signal intensities are less genome U133 Plus 2.0 GeneChip arrays were used for reliable, we excluded transcripts with low intensities from microarray hybridizations. This GeneChip comprises of further analysis when the signal intensities of both normal >54,000 probe sets and analyzes the expression level of and cancer cells were lower than that of the cut-off. For the >47,000 transcripts. For microarray hybridization, we other genes, we calculated the signal intensities of cancer/ followed the protocol described in the Affymetrix GeneChip normal ratio using the raw data of each sample. We selected eukaryotic two cycles target preparation protocol (Affymetrix). up-regulated or down-regulated genes based on their signal For the first-round synthesis of double-stranded cDNA, 100 ng intensities of cancer/normal ratios (r): up-regulated (r>5.0) and of total RNA were reverse transcribed using the two-cycle down-regulated (r<0.2). cDNA synthesis kit (Affymetrix, Santa Clara, CA) and T7- oligo-dT primer according to the
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