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Identification of RNA-Binding Protein LARP4B As a Tumor Suppressor in Glioma

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Identification of RNA-Binding Protein LARP4B As a Tumor Suppressor in Glioma Published OnlineFirst March 1, 2016; DOI: 10.1158/0008-5472.CAN-15-2308 Cancer Molecular and Cellular Pathobiology Research Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma Hideto Koso1, Hungtsung Yi1, Paul Sheridan2, Satoru Miyano2, Yasushi Ino3, Tomoki Todo3, and Sumiko Watanabe1 Abstract Transposon-based insertional mutagenesis is a valuable patient-derived glioma stem cell populations. The expression method for conducting unbiased forward genetic screens to levels of CDKN1A and BAX were also upregulated upon identify cancer genes in mice. We used this system to elucidate LARP4B overexpression, and the growth-inhibitory effects were factors involved in the malignant transformation of neural partially dependent on p53 (TP53) activity in cells expressing stem cells into glioma-initiating cells. We identified an RNA- wild-type, but not mutant, p53. We further found that the La binding protein, La-related protein 4b (LARP4B), as a candi- module, which is responsible for the RNA chaperone activity date tumor-suppressor gene in glioma. LARP4B expression was of LARP4B, was important for the growth-suppressive effect consistently decreased in human glioma stem cells and cell and was associated with BAX mRNA. Finally, LARP4B deple- lines compared with normal neural stem cells. Moreover, tion in p53 and Nf1-deficient mouse primary astrocytes pro- heterozygous deletion of LARP4B was detected in nearly moted cell proliferation and led to increased tumor size and 80% of glioblastomas in The Cancer Genome Atlas database. invasiveness in xenograft and orthotopic models. These data LARP4B loss was also associated with low expression and poor provide strong evidence that LARP4B serves as a tumor-sup- patient survival. Overexpression of LARP4B in glioma cell pressor gene in glioma, encouraging further exploration of the lines strongly inhibited proliferation by inducing mitotic RNA targets potentially involved in LARP4B-mediatd growth arrest and apoptosis in four of six lines as well as in two inhibition. Cancer Res; 76(8); 2254–64. Ó2016 AACR. Introduction The La-related proteins (LARP) are characterized by the pres- ence of a La motif (LaM), which is a highly conserved domain Glioblastoma is the most common form of malignant brain similar in structure to the winged-helix motif commonly found in cancer in adults. Patients with glioblastoma invariably have a bad DNA-binding transcriptional factors (6). The La motif is imme- prognosis, with a mean survival of around 1 year (1). Thus, more diately followed by one or several RNA-recognition motifs (RRM), studies are needed to better combat this disease. Genome and this tandem arrangement is known as the La module. The sequencing has revealed a plethora of mutations in glioblastoma functions of the La module have been best studied in the genuine (2); however, the pathologic relevance of these mutations is 0 La proteins (7). The genuine La protein La module binds to 3 unclear. Transposon-based insertional mutagenesis is useful as 0 oligo (uridylic acid) stretches (UUU-3 OH), which are commonly it allows for an unbiased forward genetic screen for cancer genes in found in newly synthesized RNA polymerase III transcripts, such mice (3, 4), thereby providing a functional readout to comple- as tRNA and 5S rRNA precursors. La engagement is thought to ment cancer genome-sequencing studies. We used transposon 0 protect RNA trailers from 3 exonuclease digestion and to assist mutagenesis to better understand the genes and pathways that are their folding via its RNA chaperone activity. able to transform neural stem cells (NSC) into glioma-initiating Seven different LARPs have been identified in humans (LARP1, cells (5), and identified La-related protein 4b (Larp4b)asa 1B, 4, 4B, 6, and 7) based on their homology with genuine La candidate tumor-suppressor gene in glioblastoma. proteins (8). However, the functions and binding modes of the La module in LARPs are not well understood. One of the best- characterized human LARPs is LARP7. By stabilizing 7SK RNA, 1Division of Molecular and Developmental Biology, The Institute of Medical Science,The University of Tokyo,Tokyo, Japan. 2Laboratory LARP7 inhibits the cyclin-dependent kinase (CDK) activity of of DNA Information Analysis, Human Genome Center,The Institute of p-TEFb (9, 10), and functions as a tumor-suppressor gene in Medical Science, The University of Tokyo, Tokyo, Japan. 3Division several types of human cancer (11, 12). LARP4B is a cytosolic of Innovative Cancer Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. protein, which interacts with the cytosolic polyA-binding protein 1 (PABPC1) and the receptor for activated C kinase (RACK1), a Note: Supplementary data for this article are available at Cancer Research component of the 40S ribosomal subunit (13). Schaffler and Online (http://cancerres.aacrjournals.org/). colleagues also showed that overexpression of LARP4B stimulated H. Koso and H. Yi contributed equally to this article. protein synthesis, whereas a knockdown of LARP4B impaired Corresponding Author: Sumiko Watanabe, Institute of Medical Science, translation of a large number of mRNAs, suggesting a stimulatory The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. role for LARP4B in translation (13). In the present study, we þ Phone: 81-3-5449-5664; Fax: 81-3-5449-5474; E-mail: identified LARP4B as a potential tumor-suppressor gene in glio- [email protected] mas. An analysis of cancer database information suggested that doi: 10.1158/0008-5472.CAN-15-2308 LARP4B participates in the pathogenesis of human glioma, and Ó2016 American Association for Cancer Research. cellular analyses revealed its function as a tumor suppressor. 2254 Cancer Res; 76(8) April 15, 2016 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 1, 2016; DOI: 10.1158/0008-5472.CAN-15-2308 LARP4B Acts as a Tumor Suppressor in Glioma Materials and Methods Retroviral infection Retrovirus was generated with Platinum-A packaging cells Retroviral expression vectors (Cell Biolabs, Inc.) and used to infect target cells as described pMXs-IRES-Puro (pMXs-IP; Cell Biolabs, Inc.) and MSCV- previously (5). Infected cells were selected with puromycin LTRmiR30-PIG (LMP; Thermo Scientific) were used for the over- (2 mg/mL) for 2 days and used for proliferation assays. Details expression and knockdown experiments, respectively. Details on on the method used to serially infect target cells with differ- the vector construction strategies are described in Supplementary ent retroviruses are provided in Supplementary Materials and Materials and Methods. Methods. Glioma cell lines Five human glioma cell lines were obtained from the ATCC: Cell proliferation assay LARP4B Â 4 U-87MG (ATCC HTB-14), U-118MG (ATCC HTB-15), U- -transduced or control cells (5 10 ) were plated in 6- 138MG (ATCC HTB-16), T98G (ATCC CRL-1690), and A172 well plates in triplicate. The total number of cells was counted on (ATCC CRL-1620). U-251MG was obtained from the JCRB Cell day 5, and the fold change was calculated. Viable cell counting was Bank (IFO50288). All cell lines tested negative for mycoplasma performed using the Trypan Blue exclusion method. contamination. The lines were authenticated by standard mor- phologic examination using microscopy. The glioma cell lines Western blot analysis were cultured in DMEM containing 10% FBS and penicillin– HEK293T cells (ATCC) were transfected using GeneJuice trans- streptomycin. fection reagent (Millipore). After 48 hours, the transfected cells were collected and used for Western blotting. Primary antibodies Glioma stem cells against HA (Covance), actin (Sigma), Larp4b (Abcam), and Information regarding the patient-derived glioma stem cells horseradish peroxidase–linked secondary antibodies (GE Health- (TGS-01, TGS-02, and TGS-04; ref.14) is provided in Supple- care) were used. Band intensities were quantified using ImageJ mentary Table S1. Glioma stem cells were cultured in DMEM/ software. F12 supplemented with B27 (Invitrogen), 20 ng/mL EGF, and 20 ng/mL bFGF (both from PeproTech). Real-time PCR analysis To analyze the expression levels of the candidate genes Preparation of primary astrocytes in Table 1, total RNA was extracted from NSCs, glioma stem cells, Primary astrocytes were harvested from the brains of neo- and cell lines using Sepazol (Nacalai Tesque, Inc.). cDNA was natal C57BL6/J mice as described previously (15). After 1 to 2 synthesized with ReverTra Ace (Toyobo), and qPCR was per- weeks, growing cells were retrovirally transduced with domi- formed using TaqMan assays (Invitrogen; Supplementary Table nant-negative p53 (p53DN; ref.15) and used as immortalized S2). All other qPCR experiments were performed with the primers astrocytes. shown in Supplementary Table S3 using the LightCycler 96 Table 1. Genomic alterations in candidate tumor-suppressor genes in human glioblastoma Gene %Homo_Dela %Hetero_Delb %Diploidc %Low_Ampd %High_Ampe %Mutf Expression P val (adjusted)g Cox P val (adjusted)h PTEN 9.7 79.4 10.6 0.2 0.2 34.3 N/A 4.5E–03 VTI1A 0.2 88.4 10.9 0.3 0.2 0.0 4.0E–05 1.1E–02 DDX50 0.3 86.8 12.5 0.3 0.0 0.0 2.6E–04 1.5E–03 MLLT10 0.3 82.1 14.7 2.1 0.7 0.0 8.3E–04 4.0E–04 LARP4B 0.7 80.9 15.6 2.1 0.7 0.4 <1.0E–05 1.1E–03 IFT74 6.6 48.4 39.7 5.2 0.2 0.4 <1.0E–05 0.99 BNC2 1.6 48.9 42.6 5.9 1.0 1.6 5.8E–02 0.95 MPDZ 1.7 45.6 45.2 6.9 0.5 0.4 <1.0E–05 0.55 UHRF2 1.6 40.7 49.9 7.5 0.3 0.4 <1.0E–05 0.97 ATXN10 0.5 35.9 57.9 5.5 0.2 0.0 <1.0E–05 0.99 QKI 2.6 29.8 63.3 4.3 0.0 2.0 <1.0E–05 0.38 MNAT1 0.5 29.1 66.2 4.2 0.0 0.0 <1.0E–05 0.99 ARID1B 0.7 28.1 67.1 4.0 0.2 0.4 <1.0E–05 0.40 MAPK1 0.2 30.3 62.7 6.4 0.3 0.8 N/A 0.90 RCOR1 0.3 27.4 67.1 5.0 0.2 0.4 <1.0E–05 0.99 TRAF3 0.3 27.2 67.2 5.0 0.2 0.0 N/A 0.99 CYFIP1 0.0 21.1 74.7 4.2 0.0 0.0 1.8E–02 0.72 Abbreviation: N/A, data not available in the TCGA dataset.
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