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Clinical Significance of FANCD2 Gene Expression and Its Association

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Clinical Significance of FANCD2 Gene Expression and Its Association ANTICANCER RESEARCH 37 : 1083-1090 (2017) doi:10.21873/anticanres.11420 Clinical Significance of FANCD2 Gene Expression and its Association with Tumor Progression in Hepatocellular Carcinoma HISATERU KOMATSU 1,2 , TAKAAKI MASUDA 1, TOMOHIRO IGUCHI 1, SHO NAMBARA 1, KUNIAKI SATO 1, QUINGJANG HU 1, HIDENARI HIRATA 1, SHUHEI ITO 1, HIDETOSHI EGUCHI 1, KEISHI SUGIMACHI 1, HIDETOSHI EGUCHI 2, YUICHIRO DOKI 2, MASAKI MORI 2 and KOSHI MIMORI 1 1Department of Surgery, Kyushu University Beppu Hospital, Beppu, Japan; 2Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan Abstract. Background/Aim: Fanconi anemia complementation identification of novel biomarkers that can predict clinical group D2 (FANCD2) gene is vitally involved in DNA damage outcomes in HCC and investigation of molecules which are responses. We investigated the clinical significance of FANCD2 involved in the tumor progression are both very important for expression in hepatocellular carcinoma (HCC). Patients and patient treatment. Methods: FANCD2 mRNA expression of resected HCC tissues Fanconi anemia (FA) complementation group D2 ( FANCD2 ) was assessed in two HCC cohorts; Our cases (n=111), and The gene encodes the FANCD2 protein, which localizes to DNA Cancer Genome Atlas (TCGA; n=371). Gene set enrichment repair foci and plays crucial roles as a component in the FA analysis (GSEA) was conducted using the TCGA dataset. pathway (4). The FANC genes are critically involved in the FA Proliferation and invasion assays were performed using pathway and regulate DNA damage responses and maintain siRNAs, and the effect of inhibition of the mechanistic target of genomic integrity (5, 6). It is well established that dysfunction rapamycin (mTOR) pathway was evaluated. Results: FANCD2 of the FANC genes confers a high risk of occurrence of certain expression was up-regulated in tumor tissues. Cases with high types of hematological and solid tumor, including liver cancer, FANCD2 expression had poorer prognoses in both cohorts, and due to increased genomic instability (7, 8). Therefore, in were associated with larger tumor size and invasive phenotypes. general, the biological roles of FANC genes, including FANCD2 knockdown attenuated proliferation and invasion of FANCD2 , have mainly been recognized as being tumor- HCC cells. FANCD2 expression was suppressed by mTOR suppressive (9). However, there is increasing evidence that inhibition. GSEA supported these findings. Conclusion: suggests that aberrant FANCD2 expression is associated with Elevated FANCD2 expression in HCC could be a novel tumor aggressiveness in several types of cancers (10-13). biomarker for poor prognosis with potential therapeutic Interestingly, there are seemingly paradoxical findings on relevance. FANCD2 expression even in the same cancer types. In breast cancer, for instance, accelerated epithelial tumor formation was Hepatocellular carcinoma (HCC) is the major histological observed in a FANCD2-deficient mouse model (14), although subtype of primary liver cancer, accounting for approximately there was a strong association between FANCD2 expression 70-90% of all cases (1). HCC has been recognized as one of and the proliferative state of cancer cells (15). the most threatening malignancies because of the limited Taken together, the clinical significance and involvement availability of radical therapeutic options (2, 3). Thus, in cancer progression of FANCD2 have not been fully elucidated in solid types of cancer, including HCC. For this reason, we aimed to determine the significance of FANCD2 expression in HCC. Correspondence to: Koshi Mimori, MD, Ph.D., Department of Surgery, Kyushu University Beppu Hospital, 4546 Tsurumihara, Materials and Methods Beppu, Oita 874-0838, Japan. Tel: +81 977271650, Fax: +81 977271651, e-mail: [email protected] Patients and sample collection. Between 2000 and 2004, 111 patients with HCC who underwent liver resection at the Kyushu Key Words: FANCD2 , hepatocellular carcinoma, prognostic University Beppu Hospital and affiliated hospitals were enrolled in biomarker, mTOR inhibitor. this study. Resected HCC tissues were immediately frozen in liquid 1083 ANTICANCER RESEARCH 37 : 1083-1090 (2017) nitrogen and kept at –80˚C until RNA extraction. Corresponding Research, Institute of Development, Aging, and Cancer, Tohoku noncancerous liver tissues were also stored (available in 65 out of University (Sendai, Japan), respectively. Cells were maintained in 111 cases). Registration of clinicopathological characteristics and a Dulbecco’s modified Eagle’s medium (Gibco, CA, USA) prognostic follow-up were conducted after surgery. Written supplemented with 10% fetal bovine serum and (FBS) 1% informed consent was obtained from each patient. All protocols in streptomycin sulfate. All cells were cultured at 37˚C in a humidified this study met the guidelines of relevant governmental agencies and atmosphere containing 5% CO 2. were approved by the Ethics Review Board of Kyushu University after informed consent was obtained from patients. Transfection with small interfering RNA (siRNA) . A FANCD2 - specific siRNA, a specific siRNA against mechanistic target of RNA preparation, reverse transcription (RT), and quantitative rapamycin ( mTOR ), and a negative control siRNA were obtained polymerase chain reaction (qPCR). Total RNA from frozen tissue from Thermo Fisher Scientific (Waltham, MA, USA). siRNA specimens and HCC cell lines was extracted using ISOGEN (Nippon oligonucleotides were transfected into the cells using Lipofectamine Gene, Tokyo, Japan). The quality assessment of extracted RNA was RNAiMAX (Thermo Fisher Scientific) following the manufacturer’s performed by measuring absorbance, and we confirmed that all instructions. samples were of satisfactory quality. cDNA was synthesized by RT from 8 μg total RNA with M-MLV reverse transcriptase (Invitrogen, Western blot analysis. Total cellular protein (35 μg) was extracted CA, USA). qPCR was performed using a LightCycler 480 Probe from cultured cells with RIPA lysis buffer and electrophoresed on Master kit (Roche Applied Science, Penzberg, Germany). mRNA 10% Tris-Glycine gels (Thermo Fisher Scientific) and then expression of each gene was quantified using the following specific electroblotted onto Immobilon-P Transfer Membranes (Merck oligonucleotide primers: FANCD2 : 5’-AACTTGGAGGAGATTGA Millipore, Billerica, MA, USA) at 70 V for 4 h at 4˚C. Proteins TGGTC -3’ (sense) and 5’-CGCTCTTTAGCAGACATGGA-3’ were detected using primary antibodies as follows. Rabbit (antisense); glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ): 5’- polyclonal antibodies against FANCD2 (Abcam, Cambridge, UK) AGCCACATCGCTCAGACAC-3’ (sense) and 5’-GCCCAATACGA and mTOR (Cell Signaling Technology, Danvers, MA, USA), and CCAAATCC-3’ (antisense). mRNA amplification conditions consisted mouse monoclonal antibodies against β- Actin (Santa Cruz of initial denaturation at 95˚C for 10 min, followed by 40 cycles of Biotechnology, Dallas, TX, USA) were used at a dilution of denaturation at 95˚C for 10 s, annealing at 62˚C for 10 s, and 1:1000. We used horseradish peroxidase-linked anti-rabbit or anti- elongation at 67˚C for 10 s. mouse immunoglobulin (GE Healthcare Japan, Tokyo, Japan) diluted 1:5000 as secondary antibodies. Immobilon Western Acquisition of profiles of gene expression, copy number, and clinical Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, information from The Cancer Genome Atlas (TCGA) dataset. We USA) was used for emission and a FUSION Solo 7S SYSTEM obtained data on RNA sequencing, single nucleotide polymorphism (Vilber Lourmat, Marne-la-Vallée, France) was used for detection (SNP) arrays, and corresponding clinical information on HCC cases of chemiluminescence. from The Cancer Genome Atlas (TCGA), via the Broad Institute’s Firehose (http://gdac.broadinstitute.org/runs/ stddata__2015_11_01/ 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide data/LIHC/20151101/). Of the 371 cases with mRNA expression (MTT) proliferation assays. For cell proliferation assays, cells were 4 profiles, clinical information was available in 370 cases and seeded at 1.0×10 cells/well with 500 μl culture medium per well information on SNPs was available in 364 cases. Expression profiles in 24-well plates 24 h before analysis. On the next day (day 1), cell of 50 paired noncancerous liver samples were also acquired. Gene lines were transfected with each siRNA. MTT assays were copy number alteration profiles calculated from the results of SNP conducted on the days 1, 3, 5, and 7 using a Cell Proliferation Kit I arrays were analyzed to assess the relationships between the copy (Roche Applied Science) following the manufacturer’s protocols. number alteration and expression of FANCD2 . Copy number gain The contents of each well was moved to 96-well plates and their was defined as log-ratios of 0.10 or more. absorbance was measured using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength Gene set enrichment analysis (GSEA). The correlations between of 570 nm corrected to 655 nm. FANCD2 mRNA expression and predefined gene signatures in public datasets listed above by GSEA (16) were investigated using the TCGA In vitro invasion assays. In vitro Invasion assays were conducted dataset. Gene sets extracted from the Broad Institute database were as using BD Biocoat Matrigel Invasion Chambers (pore size: 8 mm, follows: LEE_LIVER_CANCER_SURVIVAL_DN (http://www. 24-well; BD
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