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Identification of Molecular Targets in Head and Neck Squamous Cell Carcinomas Based on Genome-Wide Gene Expression Profiling

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Identification of Molecular Targets in Head and Neck Squamous Cell Carcinomas Based on Genome-Wide Gene Expression Profiling 1489-1497 7/11/07 18:41 Page 1489 ONCOLOGY REPORTS 18: 1489-1497, 2007 Identification of molecular targets in head and neck squamous cell carcinomas based on genome-wide gene expression profiling SATOYA SHIMIZU1,2, NAOHIKO SEKI2, TAKASHI SUGIMOTO2, SHIGETOSHI HORIGUCHI1, HIDEKI TANZAWA3, TOYOYUKI HANAZAWA1 and YOSHITAKA OKAMOTO1 Departments of 1Otorhinolaryngology, 2Functional Genomics and 3Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Received May 21, 2007; Accepted June 28, 2007 Abstract. DNA amplifications activate oncogenes and are patients and metastases develop in 15-25% of patients (1). hallmarks of nearly all advanced cancers including head and Many factors, such as TNM stage, pathological grade and neck squamous cell carcinoma (HNSCC). Some oncogenes tumor site, influence the prognosis of HNSCC but are not show both DNA copy number gain and mRNA overexpression. sufficient to predict outcome. In addition, treatment often Chromosomal comparative genomic hybridization and oligo- results in impairment of functions such as speech and nucleotide microarrays were used to examine 8 HNSCC cell swallowing, cosmetic disfiguration and mental pain. These lines and a plot of gene expression levels relative to their inflictions significantly erode quality of life. To overcome this position on the chromosome was produced. Three highly situation, there is a need to find novel biomarkers that classify up-regulated genes, NT5C3, ANLN and INHBA, were patients into prognostic groups, to aid identification of high- identified on chromosome 7p14. These genes were subjected risk patients who may benefit from different treatments. to quantitative real-time RT-PCR on cDNA and genomic Comparative genomic hybridization (CGH) has facilitated DNA derived from 8 HNSCC cell lines. ANLN and INHBA chromosomal characterization of solid tumors as it can showed a strong positive correlation between mRNA provide detailed information on gains and loss of tumor DNA expression and genomic DNA levels and a similar relationship throughout the entire genome. CGH has been widely used to was shown for the known oncogene, EGFR, at 7p11.2. In analyze many types of tumors, including HNSCC (2,3). The clinical samples, ANLN and INHBA showed a significantly pattern of aberration, which comprises the numbers and types higher expression in tumors than in normal tissues. Patients of aberrations and the regions that are recurrently altered, is with high expression levels of INHBA had a shorter disease- characteristic for each tumor type. Most findings based on free survival rate. Therefore, INHBA may be a promising CGH, however, have shown only slight DNA copy number prognostic marker of HNSCC. aberrations because of the large chromosomal areas detected. Also, identification of tumor-related genes is difficult because Introduction an extremely large number of genes are within these regions. In this study, gene expression analyses using DNA microarray Carcinomas of the head and neck represent the sixth most was performed and combined with the results of CGH. Gene common cancer worldwide and at least 90% of them are expression profiling has recently been used to promote rational squamous cell carcinomas. Despite considerable advances in approaches to therapy as well as to improve diagnosis and surgery, radiotherapy and chemotherapy, the 5-year survival prognosis in many tumor types (4-6), including HNSCC (1). rate for HNSCC patients has improved only marginally over Cancers, including HNSCC, are characterized by a complex the past decade. Local tumor recurrence, second primary pattern of cytogenetic and molecular genetic changes. Several tumors and distant metastasis after conventional therapy chromosomal regions are recurrently amplified or deleted in appear to be major contributing factors for restricted survival these tumors, but little is known about specific underlying of HNSCC patients. Local tumor recurrence affects ~60% of genes that could be important mediators in tumor formation or progression. The relationship between change in DNA content _________________________________________ and gene expression is unknown. Therefore, identification of affected genes in these loci, elucidation of their functions and Correspondence to: Dr Naohiko Seki, Department of Functional association of these genes with cancer progression, are Genomics, Graduate School of Medicine, Chiba University, 1-8-1 required to fully understand HNSCC tumorigenesis and Inohana, Chuo-ku, Chiba 260-8670, Japan progression. E-mail: [email protected] Oncogenes can be activated by mutation, structural rearrangement or amplification and tumor suppressor genes Key words: head and neck squamous cell carcinoma, comparative may be inactivated in some tumors by methylation and in genomic hybridization, microarray, 7p14, ANLN, inhibin ß A, others by mutation or physical deletion. DNA amplification activin A is an important and common mechanism for oncogene overexpression in many cancers. Some oncogenes have been 1489-1497 7/11/07 18:41 Page 1490 1490 SHIMIZU et al: IDENTIFICATION OF MOLECULAR TARGETS IN HEAD AND NECK SQUAMOUS CELL CARCINOMAS described as being both amplified and overexpressed, for Lumonics scanArray 4000 (Perkin Elmer, Boston, MA). Data example, EGFR (7p11.2) and CCND1 (11q13) (7). Therefore, were analyzed by DNASISarray software (Hitachi Software to identify prognostic markers of HNSCC, chromosomal CGH Engineering Co. Ltd.), which converted the signal intensity and oligonucleotide microarray analysis was performed on 8 of each spot into text format. Log2-ratios of median local HNSCC cell lines. Up-regulated genes at chromosomal gain background subtracted intensity levels were analyzed. Each regions detected by CGH were identified. Quantitative real- microarray data was normalized by Lowess normalization. time RT-PCR on cDNA and genomic DNA from 8 HNSCC cell lines was performed for 3 selected genes and expression Quantitative real-time RT-PCR on cDNA. For cDNA synthesis, was further analyzed in clinical samples. the QuantiTect® reverse transcription kit (Qiagen) was used. Reaction mixtures contained 1 μl of a 1/50 dilution of cDNA, Materials and methods 0.5 μM of each primer, 8 μl H2O and 10 μl of 2X QuantiTect SYBR Green PCR Master mix (Qiagen). Primers were Cell lines and clinical samples. Eight HNSCC cell lines were designed to amplify products of 100-200 bp within target and used. Six cell lines were derived from oral squamous cell control sequences. Primers used for real-time PCR on cDNA carcinomas (OSCC; Ca9-22, H1, HO1N1, HSC2, HSC3 and were: NT5C3: 5'-aagaatggcagatggagtgg-3' (sense), 5'-acag Sa3) and 2 were from pharyngeal squamous cell carcinomas ttcaattgcacccaca-3' (antisense); ANLN: 5'-caatcttgctgcaa (PSCC; Detroit562 and FaDu). Six OSCC cell lines were ctatttgct-3' (sense), 5'-tgcttaacactgctgctattga-3' (antisense); maintained in DMEM/F12 and 2 PSCC cell lines in RPMI- INHBA: 5'-ggagggcagaaatgaatgaa-3' (sense), 5'-ccttggaa 1640. All cell lines were supplemented with 10% fetal bovine atctcgaagtgc-3' (antisense); EGFR: 5'-aaggaaatcctcgatgaag serum and were cultured under 5% CO2 at 37˚C. Eighteen cct-3' (sense), 5'-tgtctttgtgttcccggacata-3' (antisense); ACTB: nonmalignant specimens and 22 HNSCC samples were used 5'-ggcatgggtcagaaggatt-3' (sense), 5'-aggtgtggtgccagattttc-3' for quantitative real-time RT-PCR experiments in order to (antisense). Reactions were performed under the following validate candidate genes. HNSCC samples (total 49 samples) conditions: 95˚C for 15 min and 40 cycles of 94˚C, 15 sec; were obtained from surgical specimens collected at Chiba 55˚C, 30 sec; 72˚C, 34 sec. PCR products were assayed using University Hospital (Chiba, Japan). the 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). Experiments were performed in duplicate and Genomic DNA extraction and chromosomal CGH. Genomic gene expression levels were normalized to ACTB expression DNA was extracted using the blood and cell culture DNA kit levels. (Qiagen, Valencia, CA) according to the manufacturer's procedures. CGH was performed as described previously (8). Quantitative real-time RT-PCR on genomic DNA. Primers DNA from each cell line was directly labeled with Spectrum were quoted from the NCBI database. Primers used for Green-dUTP (Vysis Inc., Downers Grove, IL) by nick trans- real-time PCR on genomic DNA were: NT5C3: 5'-ggaga lation using a commercial kit (Vysis). Normal, sex-matched atctgaagctctcctg-3' (sense), 5'-tttccccacaaacttcatcc-3' reference DNA was labeled with SpectrumRed-dUTP (Vysis). (antisense); ANLN: 5'-gtgtatttaaaagttgagaccctgc-3' (sense), Labeled cell line and reference DNAs, together with human 5'-aacagtgtctagaaacccattttg-3' (antisense); INHBA: 5'-tgtc Cot-1 DNA, were denatured and hybridized to metaphase cagagaagacagtggc-3' (sense), 5'-ctgtttcatctgtttcatcagg-3' spreads that were prepared using a standard protocol. The (antisense); EGFR: 5'-aagcaattccagggtgagg-3' (sense), 5'-ctc slides were washed and counterstained with DAPI. Threshold agacaaacggtcagaagc-3' (antisense); SUFU (10q24): 5'-gctgct values for detection of genomic imbalances were determined tcgatggaaac-3' (sense), 5'-aacctgtgtcggctgc-3' (antisense). as 0.7 for loss and 1.3 for gains. Reactions were performed with the same conditions as above. Human female genomic DNA (Novagen, Madison, WI) was Oligonucleotide microarray experiment. Total RNA was used as a normal control. Experiments were performed in isolated using TRIzol reagent
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