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Significance of Serine Threonine Tyrosine Kinase 1 As a Drug Resistance Factor and Therapeutic Predictor in Acute Leukemia

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Significance of Serine Threonine Tyrosine Kinase 1 As a Drug Resistance Factor and Therapeutic Predictor in Acute Leukemia INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1867-1874, 2014 Significance of serine threonine tyrosine kinase 1 as a drug resistance factor and therapeutic predictor in acute leukemia Shinya Nirasawa, DAISUKE Kobayashi, TAKASHI KONDOH, KAGEAKI Kuribayashi, MAKI TANAKA, NOZOMI YANAGIHARA and NAOKI Watanabe Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan Received June 13, 2014; Accepted July 30, 2014 DOI: 10.3892/ijo.2014.2633 Abstract. Alterations in the mRNA expression or the tutively in patients before therapy and that promote natural mutation of previously reported tyrosine kinases have been resistance. detected only in a limited number of patients with acute The overexpression and mutation of various tyrosine leukemia. In this study, we examined whether the widely kinases contributes to the development of acute leukemia. For expressed serine threonine tyrosine kinase 1 (STYK1)/novel example, the overexpression or mutation of tyrosine kinases, oncogene with kinase domain (NOK) acts as a drug resis- including Flt3, c-kit, platelet-derived growth factor receptor tance factor in acute leukemia. The transfection of leukemic and Bcr-Abl has been reported (1-3). Once these tyrosine HL-60 cells with an STYK1 expression vector resulted in kinases are activated, they transmit major molecules such as the resistance to doxorubicin and etoposide and decreased phosphatidylinositol 3-kinase (PI3K) and mitogen-activated drug-induced caspase-3/7 activity and sub-G1 popula- protein kinase (MAPK) (1-3). This not only induces prolif- tion. To investigate the mechanism of STYK1-induced eration of leukemia cells, but also renders them resistant to drug resistance, microarray analysis was performed using various anticancer drugs. Therefore, several kinase inhibi- HL-60 cells transfected with control or STYK1 expression tors are used clinically and some were reported to serve as vectors. Three tyrosine kinases (EphA4, FLT4 and STK31), predictive factors for the patient response to treatment or prog- two NF-κB inducers (MAPK4 and TNF-RSF11A), and two nosis (4-6). However, these abnormalities are detected only in a genes essential for stem cell replication (SALL4 and NOV) limited number of patients with acute leukemia. Furthermore, were identified as novel STYK1-induced genes. In addition energy-dependent, rapid drug efflux and multidrug-resistance to the data using cell line, a comparison of the leukemic molecules are the major factors for the resistance of several patients who did and did not respond to therapy revealed that leukemic cells; however, drug retention does not always STYK1 expression before therapy was significantly higher correlate with cytotoxicity and these molecules are not always in the non-responder group compared with the group that expressed before therapy. responded completely. These results suggest that STYK1 is We recently reported that serine threonine tyro- a novel drug resistance factor and could be a predictor of the sine kinase 1 (STYK1)/novel oncogene with kinase therapeutic response in acute leukemia. domain (NOK), a receptor protein tyrosine kinase (RPTK)- like protein, is widely expressed in patients with leukemia (7). Introduction We also demonstrated that STYK1 expression was decreased after chemotherapy in all patients, and was important in Anticancer drug resistance is evoked by various mechanisms, the proliferation of leukemia cells. STYK1 was reported to but there is no universal marker that plays a role in drug be a tumorigenesis-inducing factor by Liu et al (8). STYK1 resistance and that is useful to predict the therapeutic effect overexpression results in the growth factor-independent in different disease types. To characterize such markers, it proliferation of murine bone-marrow-derived lymphoid BaF3 is important to identify candidates that are expressed consti- cells and surface adhesion-independent growth and colony formation in NIH3T3 and BaF3 cells (8). STYK1 shares 20-30% homology with members of the fibroblast growth factor receptor and platelet-derived growth factor receptor families (8). RPTKs usually bind to specific ligands to stimu- Correspondence to: Dr Naoki Watanabe, Department of Clinical late tyrosine phosphorylation and growth signal transduction. Laboratory Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-Ku, Sapporo 060-8543, Japan Interestingly, STYK1 has a single putative transmembrane E-mail: [email protected] domain and an intracellular tyrosine kinase domain, but it lacks an extracellular domain. Therefore, STYK1 expression Key words: serine threonine tyrosine kinase 1, mRNA expression, may itself trigger self-phosphorylation and transmit growth chemo-resistance, prediction, acute leukemia signals without ligand binding. PI3K and MAPK are reported as activated growth signals by STYK1, suggesting that these signals might induce the expression of various molecules 1868 Nirasawa et al: Role of STYK1 in leukemia such as inhibitor of apoptosis proteins (IAPs) and NF-κB Microarrays. Total RNA was prepared from transfected cells, signals to stimulate resistance against anticancer drugs. and then subjected to industrial analysis. Quality control Based on these observations, we hypothesized that the widely check and global gene expression profiling was performed expressed STYK1 may play key roles in the drug resistance by TakaraBio. Inc. (Otsu, Japan) using SurePrint G3 Human of hematopoietic malignancies. However, STYK1 expres- GE 8x60 K Microarrays (both from Agilent Technologies, sion has not yet been reported to act as a resistance factor in Santa Clara, CA, USA) following the Agilent one-color leukemic cells; therefore, whether its expression is related to microarray-based gene expression analysis protocol. The therapeutic outcome in patients with acute leukemia remains slides were scanned using an Agilent Technologies Microarray unclear. Scanner, and the images were processed using Agilent Feature In this study, to determine whether STYK1 functions as Extraction software, version 10.7.3.1. a resistance factor against anticancer drugs, we transfected cells with either a control or STYK1 expression vector and RNA extraction and quantification of STYK1 mRNA expres- compared drug sensitivity and the activation of signaling sion. The expression of STYK1 mRNA was determined by pathways. We next examined STYK1 mRNA expression levels performing qRT-PCR on an ABI PRISM 7700 sequence in patients with acute leukemia and compared its expression detection system (Applied Biosystems, Foster City, CA, in two groups with completely different therapeutic outcomes. USA). Total RNA was isolated, and the concentration was To ensure the applicability of the mRNA expression measure- determined using the GeneQuant DNA/RNA Calculator ments to clinical practice, we used peripheral blood and bone (Amersham Pharmacia Biotech, Uppsala, Sweden). The marrow blood samples without purifying blasts. gene-specific primers and fluorescent hybridization probes used for quantitative PCR were as follows: STYK1 forward Materials and methods primer, 5'-CAT CTT TCG AGC CAA TAT GAA CAC-3'; reverse primer, 5'-TGG AAT TGG ATT CGC CCT AA-3'; Cell culture and anticancer drugs. The human myelogenous and probe, 5'-(FAM) CCA GCT GGG CTC CAT GAG leukemia cell lines HL-60 and K562 were obtained from GTA CAA GAT (TAMRA)-3'. Quantitative RT-PCR was the American Type Culture Collection (Manassas, VA, performed using the TaqMan One-Step RT-PCR Master Mix USA). The cells were cultured in RPMI-1640 (BioWhittaker, Reagents kit (Applied Biosystems). To compare the STYK1 Walkersville, MD, USA) supplemented with 10% heat-inacti- mRNA expression between different samples, the specific vated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) mRNA was normalized to that of 18S ribosomal RNA at 37˚C in a humidified atmosphere of 5% CO2. Doxorubicin (rRNA) to obtain a ratio. The 18S rRNA expression was and etoposide were purchased from Sigma-Aldrich (St. Louis, determined using TaqMan Ribosomal RNA control reagents MO, USA). (Applied Biosystems), according to the protocol provided by the manufacturer. For each experiment, a calibration curve Patients and samples. A total of 34 peripheral or bone was prepared using control RNA from K562 cells. Briefly, marrow blood samples were examined. Fresh peripheral a computer algorithm was used to analyze the emission blood samples from 22 patients who had been diagnosed with of reporter dye and quenching dye emission during PCR acute leukemia were obtained at Sapporo Medical University amplification, and the intensity of the fluorescence signals Hospital. Fresh bone marrow blood samples were also from each PCR cycle was detected. The amplification curves obtained from 12 patients with acute leukemia. Ten patients obtained from serial dilutions of control RNA were prepared, with acute lymphoblastic leukemia, 20 with acute myeloid and the optimal signal intensity (threshold) was selected leukemia and 4 with acute mixed-lineage leukemia were manually in the exponential phase of the curves. The PCR assessed. A total of 25 patients were newly diagnosed and 9 cycle number (threshold cycle, CT) at each concentration of were relapsed cases. After informed consent was obtained, starting RNA was determined to draw the calibration curve, blood samples were freshly prepared immediately before which was constructed as a xy plot [with the log of the input therapy, hemolyzed using a lysis reagent (Ortho Diagnostic amount
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