365-384 30/3/2009 09:55 Ì Page 365 MOLECULAR MEDICINE REPORTS 2: 365-384, 2009 365 Genome-wide gene expression profiles of ovarian carcinoma: Identification of molecular targets for the treatment of ovarian carcinoma DRAGOMIRA NIKOLAEVA NIKOLOVA1,4, NIKOLAI DOGANOV2, RUMEN DIMITROV2, KRASIMIR ANGELOV3, SIEW-KEE LOW1, IVANKA DIMOVA4, DRAGA TONCHEVA4, YUSUKE NAKAMURA1 and HITOSHI ZEMBUTSU1 1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; 2University Hospital of Obstetrics and Gynecology ‘Maichin Dom’, Sofia 1431; 3National Hospital of Oncology, Sofia 1233; 4Department of Medical Genetics, Medical University of Sofia, Sofia 1431, Bulgaria Received October 31, 2008; Accepted January 7, 2009 DOI: 10.3892/mmr_00000109 Abstract. This study aimed to clarify the molecular mecha- in women. As there are no specific indicators or symptoms of nisms involved in ovarian carcinogenesis, and to identify ovarian cancer during the early stages of the disease, the candidate molecular targets for its diagnosis and treatment. majority of patients with epithelial ovarian cancer (EOC) are The genome-wide gene expression profiles of 22 epithelial diagnosed at an advanced stage, with involvement of other ovarian carcinomas were analyzed with a microarray represent- sites such as the upper abdomen, pleural space and paraaortic ing 38,500 genes, in combination with laser microbeam lymph nodes. The cancer antigen 125 assay (CA-125) has been microdissection. A total of 273 commonly up-regulated used to screen for ovarian cancer, but is not specific; only 50% transcripts and 387 down-regulated transcripts were identified of patients with early ovarian cancer test positive using this in the ovarian carcinoma samples. Of the 273 up-regulated assay (1). The prognosis for ovarian cancer patients remains transcripts, only 87 (31.9%) were previously reported as up- poor and largely dependant on the stage at diagnosis, with a regulated in microarray studies using bulk cancer tissues and 5-year survival rate of 45% (2). Chemotherapy is the most normal ovarian tissues for analysis. CHMP4C (chromatin- common choice of treatment for patients at advanced stages, modifying protein 4C) was frequently overexpressed in ovarian but often causes severe adverse reactions without affecting carcinoma tissue, but not expressed in the normal human the tumor. Hence, the identification of new markers for early tissues used as a control. Our data should contribute to an diagnostics and molecular targets that can be applied to the improved understanding of tumorigenesis in ovarian cancer, development of new treatment is eagerly anticipated. and aid in the development of diagnostic tumor markers and Genome-wide microarray analysis enabled us to obtain molecular-targeting therapy for patients with the disease. comprehensive gene expression profiles related to the phenotypic and biological information of cancer cells (3,4). Introduction We identified multiple targets that may prove useful in the development of novel anti-cancer drugs and/or diagnostic Ovarian cancer is the second most common gynecologic biomarkers. In ovarian carcinoma, this approach can also be malignancy and the fifth leading cause of cancer-related death applied to identify unknown molecules involved in the car- cinogenic pathway. Through the gene expression profile analysis of 22 _________________________________________ epithelial ovarian cancers coupled with purification of the cancer cell population by laser microbeam microdissection Correspondence to: Dr Yusuke Nakamura, Laboratory of Molecular (LMM) on a microarray consisting of approximately 38,500 Medicine, Human Genome Center, Institute of Medical Science, The transcripts, we identified a number of transcripts that were University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, overexpressed in ovarian cancers. We report important infor- Japan mation regarding the mechanisms of ovarian carcinogenesis, as E-mail: [email protected] well as the discovery of potential targets for the development of diagnostic markers and novel therapeutic strategies for Abbreviations: LMM, laser microbeam microdissection; EOC, ovarian cancer. epithelial ovarian cancer Materials and methods Key words: gene expression profile, microarray, ovarian carcinoma, molecular target gene Tissue samples and microdissection. Twenty-two ovarian cancer tissue samples (median patient age 54 years, range 365-384 30/3/2009 09:55 Ì Page 366 366 NIKOLOVA et al: EXPRESSION PROFILING OF OVARIAN CARCINOMA Table I. Clinicopathological features of ovarian cancer clinical samples used for microarray analysis. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– No. Age Histology Clinical stage ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– BOv.T_3 75 Clear-cell carcinoma III BOv.T_4 61 Serous adenocarcinoma III BOv.T_6 75 Serous adenocarcinoma IV BOv.T_7 27 Mucinous adenocarcinoma III BOv.T_9 57 Serous clear-cell adenocarcinoma III BOv.T_10 62 Poor-to-differentiated carcinoma IV BOv.T_11 57 Cystadenocarcinoma II BOv.T_14 65 Serous adenocarcinoma IV BOv.T_15 37 Papillary serous adenocarcinoma IV BOv.T_16 69 Papillary serous adenocarcinoma IV BOv.T_17 62 Mucinous cystadenocarcinoma I BOv.T_19 49 Moderate adenocarcinoma II BOv.T_21 39 Moderate differentiated adenocarcinoma IV BOv.T_23 53 Moderate adenocarcinoma III BOv.T_27 52 Moderate serous papillary adenocarcinoma III BOv.T_28 46 Moderate serous papillary adenocarcinoma III BOv.T_5 73 Papillary serous adenocarcinoma III BOv.T_6 43 Moderate papillary serous adenocarcinoma I BOv.T_7 30 Well-differentiated papillary serous cystadenocarcinoma III BOv.T_8 52 Poor-to-well differentiated papillary serous cystadenocarcinoma III BOv.T_9 47 Poor-to-well differentiated papillary serous adenocarcinoma III BOv.T_10 61 Moderate serous papillary cystadenocarcinoma I ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 27-75 years; Table I) were obtained with written informed 38,500 genes. For microarray hybridization, we followed the consent. The samples were collected from patients who protocol described in the Affymetrix GeneChip eukaryotic underwent surgical procedures at the University Hospital of two cycle target preparation protocol (Affymetrix). For the Obstetrics and Gynecology (Sofia, Bulgaria) and the General first-round synthesis of double-stranded cDNA, 100 ng of Hospital of Oncology (Sofia, Bulgaria). Cancer tissues were total RNA was reversely transcribed using the Two-Cycle examined by professional pathologists at the hospitals. All 22 cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and T7- samples were determined to be epithelial ovarian carcinomas; oligo-dT primer according to the manufacturer's instructions, one sample was classified as clear cell, two as mucinous followed by IVT amplification with the MEGAscript T7 Kit carcinomas and the remaining 19 cases as serous types. Clinical (Ambion Inc., Austin, TX). After cleanup of the cRNA with a information was obtained from medical records. Clinical GeneChip Sample Cleanup Module IVT column (Affymetrix), stage was judged according to the UICC TNM classification. second-round double-stranded cDNA was amplified using All specimens were embedded in TissueTek OCT medium the IVT Labeling Kit (Affymetrix). A 20 μg aliquot of the (Sakura, Tokyo, Japan) immediately after surgical resection labeled product was fragmented by heat and ion-mediated and stored at -80˚C until use. These frozen tissues were cut into hydrolysis at 94˚C for 35 min in H2O combined with 8 μl of 8-μm sections using a cryostat (Sakura, Tokyo, Japan) and then 5x Fragmentation Buffer (Affymetrix). The fragmented cRNA stained with hematoxylin and eosin (H&E) for histological was hybridized for 16 h at 45˚C in a Hybridization Oven 640 to examination. Ovarian cancer cells and corresponding normal a U133 Plus 2.0 oligonucleotide array (Affymetrix). Washing ovarian epithelial cells were selectively collected using the and staining of the arrays with phycoerythrin-conjugated EZ cut system with a pulsed ultraviolet narrow beam-focus streptavidin (Molecular Probes, Eugene, OR) was completed laser (SL Microtest GmbH, Jena, Germany) according to the in a Fluidics Station 450 (Affymetrix). The arrays were then manufacturer's protocols. The layer of normal epithelial cells scanned using a confocal laser GeneChip Scanner 3000 was collected from eight samples by LMM, and the mixture (Affymetrix). of RNA extracted from them served as a ‘universal control’ for hybridization. Data analysis. Global normalization at a target value of 500 was applied to all 23 arrays (22 cancer arrays and one array Affymetrix GeneChip hybridization. The affymetrix human of the universal control) under consideration using GeneChip genome U133 Plus 2.0 GeneChip array was employed for Operating Software (Affymetrix). Normalized data from text microarray hybridization. This GeneChip comprises more files were imported to a Microsoft Excel spreadsheet. Because than 54,000 probe sets and analyzes the expression level of data derived from low signal intensities are less reliable, we 365-384 30/3/2009 09:55 Ì Page 367 MOLECULAR MEDICINE REPORTS 2: 365-384, 2009 367 Table II. Primer sequences for semi-quantitative RT-PCR experiments. –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––