ANTICANCER RESEARCH 37 : 489-498 (2017) doi:10.21873/anticanres.11341 Clinical Significance of Methylation and Reduced Expression of the Quaking Gene in Colorectal Cancer NORIKO IWATA 1, TOSHIAKI ISHIKAWA 2, SATOSHI OKAZAKI 1, KAORU MOGUSHI 3, HIRONOBU BABA 1, MEGUMI ISHIGURO 4, HIROTOSHI KOBAYASHI 1, HIROSHI TANAKA 3, TATSUYUKI KAWANO 1, KENICHI SUGIHARA 2 and HIROYUKI UETAKE 2 1Division of Surgical Gastroenterology, 2Division of Surgical Specialties, and 4Department of Translational Oncology, Graduate School of Medical and Dental Sciences, and 3Department of Systems Biology, Graduate School of Biochemical Science, Tokyo Medical and Dental University, Tokyo, Japan Abstract. Background: This study investigated abnormal Colorectal cancer (CRC) is one of the best understood methylation in colorectal cancer (CRC) and the potential neoplasms from a genetic perspective, yet it remains the role of the Quaking RNA-binding protein (QKI) gene in second most common cause of cancer-related death. tumorigenesis. Materials and Methods: Oligonucleotide Although the diagnosis and treatment of CRC has microarray expression profiling was carried out on a panel significantly improved during recent decades, leading to a of primary CRC specimens (n=17) and CRC cell lines (n=5), substantial reduction in cancer-related mortality (1), CRC followed by methylation analysis using methylation-specific continues to be a major public health problem worldwide. polymerase chain reaction. QKI expression levels were It is now well established that widespread epigenetic assessed in 156 primary CRCs by qRT-PCR and changes, including alterations in DNA methylation profiles immunohistochemistry. Results: Low QKI expression was relative to non-neoplastic tissue, are a characteristic of many observed in 47.7% in CRCs. QKI promoter methylation was cancer types (2, 3). Epigenetic alterations, such as aberrant detected in 32.1% of patients with CRC, and in these patients DNA hypermethylation are now recognized as one of the mRNA expression in tumor tissue was significantly down- crucial mechanisms underlying tumor-suppressor gene regulated compared to matched normal tissues (p=0.049). inactivation in cancer (4). In addition to identifying genes There was a significant relationship between low QKI with a potential role in oncogenesis, methylation of specific expression and recurrence after surgery (p=0.004). Low QKI gene promoters is a hallmark of different cancer types and expression was an independent risk factor for recurrence can be used in cancer diagnosis and classification (5). For after surgery in 153 patients with CRC without distant example, in CRC, coordinated methylation of a specific set metastases (p=0.036). Conclusion: Patients with tumors of genes classifies cancers as the CpG island methylator expressing low levels of QKI experienced significantly higher phenotype (CIMP), and this classification is associated with rates of tumor recurrence after curative surgery and worse B-Raf proto-oncogene BRAF mutations (6). prognoses. Methylation of the QKI promoter and Advances in genome-wide methylation analysis concomitant reduced expression of QKI mRNA may be technology have revealed that methylation regulates many important for CRC initiation and progression. Loew QKI genes that control apoptosis, and cell-cycle regulation of expression may be a useful clinical biomarker for predicting other fundamental cellular processes (7, 8). DNA microarray recurrence and prognosis. and high-throughput DNA sequencing are two powerful techniques that enable the large-scale analysis of DNA methylation (9-12). In this study, we searched for clinically significant tumor-suppressor genes in CRC by screening for Correspondence to: Toshiaki Ishikawa, Division of Surgical candidate genes suspected to be silenced by DNA Specialties, Tokyo Medical and Dental University, Graduate School methylation using microarray analysis. We focused our of Medical and Dental Sciences, 1-5-45 Yushima, Bunkyo-ku, study on the Quaking RNA-binding protein gene ( QKI ) Tokyo, 113-8519, Japan. Tel: +81 358035261, e-mail: [email protected] gene, which encodes a RNA-binding protein that was reported to be inactivated in CRC (13), and demonstrated Key Words: Colorectal cancer, Quaking RNA-binding protein gene, the clinical significance of QKI expression in CRC QKI , tumor-suppressor gene, methylation, prognostic factor. specimens. 489 ANTICANCER RESEARCH 37 : 489-498 (2017) Materials and Methods Transcription Polymerase Chain Reaction (qRT-PCR). Total RNA was extracted using an RNeasy mini kit (Qiagen) and cDNA was Clinical sample collection. We collected primary CRC specimens synthesized using the High Capacity cDNA Reverse Transcription kit and matched corresponding normal mucosa from 156 patients who (Applied Biosystems, Foster City, CA, USA) according to the had undergone surgery at Tokyo Medical and Dental University manufacturer’s instructions. cDNA was then amplified by polymerase Hospital (Tokyo, Japan) between 2005 and 2009. All patients chain reaction PCR using a fluorescence-based real-time detection provided written informed consent, and the study was approved by method on an ABI Prism 7300 Real-time PCR System according to the Institutional Review Boards of all institutions involved the manufacturer’s protocol. TaqMan QKI and β- actin ( ACTB ) Gene (approval number: 831). Clinical data were obtained from the Expression Assays-on-demand ( QKI Assay ID Hs00287641_m1, medical records of each patient. The patients were monitored every ACTB Assay ID Hs99999903_ml; Applied Biosystems) were used for 3 months after surgery, and their median follow-up was 60 months. qRT-PCR. QKI mRNA expression levels in each sample were normalized to those of ACTB as an internal standard. Relative Cell culture and demethylation treatment of cell lines. We used five quantification of QKI mRNA expression was calculated by the ΔΔ Ct CRC cell lines (RKO, HCT116, HT29, colo320DM, SW480). These method using SDSv1.2 with RQv1.0 software (Applied Biosystems). were purchased through the American Type Culture Collection Each analysis was performed in triplicate. (Manassas, VA, USA) or Cell Resource Center for Biomedical Methylation analysis. Total genomic DNA was isolated from both Research, Tohoku University (Miyagi, Japan), minimum essential tumor and normal colon epithelium samples using the medium, Dulbecco’s modified Eagle’s medium, or McCoy’s 5A phenol/chloroform method. Bisulfite treatment was performed using medium (Gibco, Carlsbad, CA, USA) containing 10% heat- an EpiTect Plus DNA Bisulfite kit (Qiagen) according to the inactivated fetal bovine serum, 100 units/ml of penicillin, 100 μg/ml manufacturer’s instructions. Bisulfite-modified DNA was then used of streptomycin, 10 mM of HEPES and 1.0 mM of sodium pyruvate as template DNA for PCR amplification with the PCR primers and incubated at 37˚C in 5% CO . Cells were plated in culture 2 corresponding to the region affected by the methylation. Methylated plates on day −2. On day 0, the culture medium was removed and primer sequences were based on previous reports (13). Methylation- new medium containing 0.5 μM of the DNA methylase inhibitor 5- specific PCR (MSP) was performed using an EpiTect MSP kit aza-2-deoxycytidine (5-aza-DC) was added. The cells were treated (Qiagen). PCR conditions were as follows: 95˚C for 5 min, 35 with 0.5 μM of 5-aza-DC for 72 h, according to a previous report cycles of 95˚C for 30 s, 56˚C for 30 s, 72˚C for 30 s, and finally 5 (14). The cells were then rinsed twice with fetal bovine serum-free min at 72˚C. Epitect control DNA (human), methylated/ medium and collected with trypsin-ethylenediaminetetra-acetic acid unmethylated and bisulfite-converted gDNA (Qiagen, Hilden, after 72 h of 5-aza-DC treatment. Germany) was used as a positive control for the methylated/unmethylated determinations. After amplification, PCR Oligonucleotide microarray analysis . RNA from five human CRC cell products were electrophoresed on a 1.5% agarose gels. lines (RKO, HCT116, HT29, colo320DM, SW480) was extracted before and after treatment with 5-aza-DC. Matched samples of primary Immunohistochemistry. IHC studies of QKI expression were cancer and adjacent normal tissues were obtained from 17 patients conducted using FFPE tissue blocks from each of 156 patients. with CRC, and total RNA was extracted from each using a RNeasy Paraffin-embedded tumor tissue sections were deparaffinized in mini-kit (Qiagen, Hilden, Germany). The integrity of the total RNA xylene and rehydrated in graded alcohol. Antigen retrieval was was assessed using an Agilent 2100 BioAnalyzer (Agilent performed by autoclave sterilization in 0.01 M sodium citrate buffer Technologies, Palo Alto, CA, USA). All samples had an RNA integrity (pH 9.0) for 30 min. Endogenous peroxidase activity was quenched number of at least 5.0 prior to gene-expression analysis. with 3% hydrogen peroxide in methanol for 15 min. Sections were Complementary RNA was prepared from 2 μg cell line total RNA, and blocked in goat serum for 30 min at room temperature, and 100 ng tissue sample total RNA by one-cycle and two-cycle target incubated with a 1:100 dilution of rabbit antibody to QKI (IHC- labeling, respectively, using a control reagents kit (Affymetrix, Santa 00574, 9444, AAH19917.1; BETHYL, Montgomery, AL, USA) at Clara, CA, USA). Hybridization to Human Genome (HG) U133 Plus 4˚C overnight, then sections were incubated with Histofine simple 2.0 arrays (Affymetrix) and signal detection were performed according