1542 ONCOLOGY REPORTS 30: 1542-1548, 2013 STK10 missense mutations associated with anti-apoptotic function KAZUTAKA FUKUMURA1,2, YOSHIHIRO YAMASHITA4, MASAHITO KAWAZU1, EIRIN SAI1, SHIN-ICHIRO FUJIWARA5, NAOYA NAKAMURA6, KENGO TAKEUCHI7, MIZUO ANDO1, KOHEI MIYAZONO2, TOSHIHIDE UENO4, KEIYA OZAWA5 and HIROYUKI MANO1,3,4,8 Departments of 1Medical Genomics, 2Molecular Pathology and 3Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033; 4Division of Functional Genomics and 5Department of Hematology, Jichi Medical University, Tochigi 329-0498; 6Department of Pathology, Tokai University School of Medicine, Kanagawa 259-1193; 7Pathology Project for Molecular Targets, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo 135-8550; 8CREST, Japan Science and Technology Agency, Saitama 332-0012, Japan Received May 8, 2013; Accepted June 12, 2013 DOI: 10.3892/or.2013.2605 Abstract. Peripheral T-cell lymphoma (PTCL) is an aggres- ALK (2) are, for instance, identified in subsets of non-small sive lymphoma with a 5-year overall survival rate of <30%. To cell lung cancer (NSCLC), and reagents targeting EGFR or identify carcinogenesis-related genes in PTCL, we conducted ALK are proved clinically effective against NSCLC positive high-throughput resequencing of target-captured cDNA in for the corresponding genetic lesions (3,4). a PTCL specimen, revealing a total of 19 missense muta- Peripheral T-cell lymphoma (PTCL) is a subdivision of tions among 18 independent genes. One of such substitutions, non-Hodgkin's malignant lymphoma (5). In contrast to the c.2201G>A in STK10 cDNA, replaces an arginine residue to improved prognosis for individuals with B-cell lymphoma with a histidine (R634H) in the encoded protein. Of note, while recent treatment modalities, most subtypes of PTCL still have wild-type STK10 suppresses NF-κB activity and potentiates a 5-year survival rate of only <30% (6). Molecular mechanism dexamethasone-induced apoptosis, the R634H change signifi- for PTCL carcinogenesis is mainly enigmatic, except for the cantly decreases such pro-apoptotic activity. This c.2201G>A facts that i) a part of PTCL is associated with infection with change of STK10 was also identified in another PTCL specimen, Epstein-Barr virus (EBV) or human T-lymphotropic virus but now registered as a single nucleotide polymorphism in the type I (HTLV-I), and ii) activating ALK fusions are found in latest dbSNP database. Furthermore, other somatic mutations of the anaplastic large cell lymphoma (ALCL) subtype. Given STK10 have been reported, and we now reveal that some of them the fact that an ALK inhibitor is effective in the treatment of (L85P and K277E) have more profound anti-apoptotic effects ALCL (7), identification of essential growth drivers in other compared to R634H. These results suggest that STK10 functions subsets of PTCL is urgently needed. as a tumor suppressor gene, and that dysfunction of STK10 While exome sequencing of cancer specimens is used for activity either through polymorphism or somatic mutations may the detection of somatic mutations in the cancer genome, such confer anti-apoptotic effects contributing to carcinogenesis. approach fails to detect gene fusions, because gene fusions usually take place at intronic regions. To simultaneously detect Introduction point mutations/indels/gene fusions in a single experiment, we previously reported the ‘cDNA capture system’ that conducts Various genetic alterations such as point mutations, insertions/ massive resequencing on purified cDNAs for cancer-related deletions (indels) and gene fusions directly participate in genes (8). Herein we applied such technology to the cDNAs human carcinogenesis. Some of such changes confer growth isolated from a PTCL specimen, and discovered a STK10 advantage to cancer cells, and targeting their encoded proteins amino acid substitution that turned out to exert anti-apoptotic is one of the most effective means to treat cancer. Activating effects. While this amino acid change was recently deposited mutations in EGFR (1) or the gene fusion between EML4 and as a single nucleotide polymorphism (SNP) in the 1000 genome database (http://www.1000genomes.org), we also confirmed that other nonsynonymous, somatic mutations within STK10 confer marked anti-apoptotic activity. These results suggest Correspondence to: Professor Hiroyuki Mano, Department of that STK10 may contribute to carcinogenesis, either through Cellular Signaling, Graduate School of Medicine, University of polymorphism or somatic mutations, by suppressing apoptotic Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan signaling in cancer. E-mail: [email protected] Materials and methods Key words: tumor suppressor gene, apoptosis, NF-κB Cell lines and specimens. PTCL specimens were collected in Fukushima Medical University and The Cancer Institute. FUKUMURA et al: STK10 IS A NOVEL TUMOR-SUPPRESSOR 1543 A human embryonic kidney 293T (HEK293) cell line was by 60 cycles of 94˚C for 15 sec and 60˚C for 30 sec and 72˚C obtained from American Type Culture Collection (ATCC: for 1 min. Relative expression levels of target mRNAs to Manassas, VA, USA), and maintained in Dulbecco's modi- GAPDH were normalized to those in the mock-transfected fied Eagle's medium-F12 medium (Invitrogen, Carlsbad, CA, cells. The primer sequences used for the PCR reactions were: USA) supplemented with 10% fetal bovine serum (FBS) and 5'-TTCATTCCTGGCAAGTGGATCATT-3' and 5'-ATGG 2 mM L-glutamine (both from Invitrogen). An IL-2-dependent CAGCATCATTGTTCTCATCA-3' for TLR2, 5'-TGACAAC mouse cytotoxic T-cell line, CTLL-2, and a human T-cell line, CTTCTGGTTGGTAGGGA-3' and 5'-CCAAGGTCATGGT Jurkat, were both obtained from ATCC, and maintained in TGTCCAAAGAC-3' for BCL2, 5'-AAGAATCACCAGCA RPMI1640 medium (Invitrogen) supplemented with 10% FBS. GCAAGTGTCC-3' and 5'-TTGGGTTGTGGAGTGAGTG Mouse recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA) TTCAA-3' for CCL2, and 5'-CCAGGTGGTCTCCTCTGAC was added to the culture medium of CTLL-2 at the concentra- TTCAA-3' and 5'-CACCCTGTTGCTGTAGCCAAATTC-3' tion of 2 ng/ml. Total RNA was extracted from cell lines and for GAPDH. a PTCL specimen with an RNeasy mini kit (Qiagen, Valencia, CA, USA), and was subjected to reverse transcriptase (RT) Antibodies, immunoprecipitation and immunoblotting. with an oligo-dT primer. Written informed consent was Antibodies used in this study were: anti-FLAG M2 (Sigma- obtained from the subjects who provided cancer specimens, Aldrich, St. Louis, MO, USA), anti-PLK1 (Santa Cruz and the study was approved by the human ethics committee of Biotechnology, Santa Cruz, CA, USA), anti-phospho-PLK1 the University of Tokyo, Jichi Medical University, Fukushima (BD Pharmingen, San Jose, CA, USA), anti-ACTB (Cell Medical University, and The Cancer Institute of the Japanese Signaling Technology, Danvers, MA, USA), anti-mouse IgG Foundation for Cancer Research. and anti-rabbit IgG (both from GE Healthcare, Piscataway, NJ, USA). HEK293 cells were transfected with the appropriate Resequencing of target-captured cDNA in a PTCL specimen. expression vectors, and then lysed in lysis buffer [1% NP-40, Resequencing with a custom cDNA-capture system was 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM NaF, 1 mM performed as previously described (8). In brief, RNA probes Na3VO4, 1 mM phenylmethylsulfonyl fluoride and aprotinin]. of 120 bases were designed to interrogate cDNAs of 906 Target proteins were purified by incubation of the cell lysates human protein-coding genes, and were synthesized by with appropriate antibodies and Protein G Sepharose Fast Agilent Technologies (Santa Clara, CA, USA). Hybridization Flow (Sigma-Aldrich) for 3 h at 4˚C. Immunoblot analyses of cDNA fragments isolated from a PTCL specimen to the were visualized with SuperSignal West Femto Maximum RNA probes was performed according to the protocols recom- Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA). mended for the SureSelect Target Enrichment system (Agilent Technologies). Purified cDNA fragments were then subjected In vitro kinase assay. Immunoprecipitates were washed to deep sequencing with a Genome Analyzer IIx (GAIIx; with kinase buffer [50 mM NaCl, 50 mM Tris-HCl (pH 7.4), Illumina, San Diego, CA, USA) for 76 bases from both ends 10 mM MgCl2, 10 mM MnCl2 and 0.1 mM Na3VO4], and then by the paired-end sequencing system. incubated in kinase buffer containing [γ-32P]ATP (Perkin- Elmer, Boston, MA, USA) and histone H2A protein (New Plasmid construction. The full-length cDNA for the wild-type England BioLabs, Ipswich, MA, USA) for 30 min at room or the R634H mutant of STK10 was amplified by PCR from temperature. cDNA of a PTCL specimen. The cDNAs for other mutant forms of STK10 were further generated by a polymerase chain Dexamethasone-induced apoptosis assay. CTLL-2 cells were reaction (PCR)-based mutagenesis approach. The cDNA for infected with retrovirus expressing STK10 and the blasticidin- PLK1, IKK-α, IKK-β or IKK-γ was amplified by PCR from resistant gene, and cultured under the presence of 10 µg/ml our cancer cell lines. blasticidin (InvivoGen, San Diego, CA, USA). Blasticidin- resistant cells were then treated with 1 µM dexamethasone Luciferase-based reporter assay. HEK293 cells were trans- (Sigma-Aldrich), and cell number was determined every fected with the expression plasmids, a promoter-less Renilla 24 h with CellTiter-Glo Luminescent Cell Viability Assay luciferase plasmid pGL-4.70 (Promega, Madison, WI, USA), (Promega). At 72 h after treatment with dexamethasone, and firefly luciferase-based reporter plasmid for Fos (pFL700) CTLL-2 cells were collected and subjected to apoptosis (9), Myc (pHXL) (10), NF-κB (Stratagene, La Jolla, CA, USA), assay with flow cytometry (FACSCanto II; BD Biosciences, Bcl-xL (11), Notch (pGa981-6) (12), Wnt (TOP-flash, Upstate San Jose, CA, USA) using Annexin V/propidium iodide (PI) Biotechnology, Lake Placid, NY, USA) or Rho (pSRE.L) (13) staining kit (eBioscience, San Diego, CA, USA). signaling pathway. Luciferase activities were determined with the Dual-Luciferase Reporter Assay System (Promega), and Results the firefly luciferase activities were normalized to the Renilla luciferase activities.