Musashi1 Impacts Radio-Resistance in Glioblastoma by Controlling DNA-Protein Kinase Catalytic Subunit
2016; Elsevier BV; Volume: 186; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2016.05.020
ISSN1525-2191
AutoresPatricia Rosa De Araujo, Aparna Gorthi, Acarizia E. da Silva, Sonal S. Tonapi, Dat T. Vo, Suzanne Burns, Mei Qiao, Philip J. Uren, Zhi-Min Yuan, Alexander J. R. Bishop, Luiz O. F. Penalva,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA–protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy. The conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA–protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy. Glioblastoma multiforme is the most aggressive form of glioma and is the most common among primary brain tumors. The current standard of care for newly diagnosed glioblastoma is surgical resection, followed by concurrent temozolomide and radiotherapy, then by maintenance chemotherapy with temozolomide. Unfortunately, this route offers a median survival of only approximately 14 months.1Stupp R. Mason W.P. van den Bent M.J. Weller M. Fisher B. Taphoorn M.J. Belanger K. Brandes A.A. Marosi C. Bogdahn U. Curschmann J. Janzer R.C. Ludwin S.K. Gorlia T. Allgeier A. Lacombe D. Cairncross J.G. Eisenhauer E. Mirimanoff R.O. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.N Engl J Med. 2005; 352: 987-996Crossref PubMed Scopus (14644) Google Scholar, 2Van Meir E.G. Hadjipanayis C.G. Norden A.D. Shu H.K. Wen P.Y. Olson J.J. 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Combining molecular targeted agents with radiation therapy for malignant gliomas.Onco Targets Ther. 2013; 6: 1079-1095PubMed Google Scholar In the past decade, much progress has been made in understanding the cellular and molecular heterogeneity in glioblastoma, and its links to clinical aggressiveness, differential response to chemotherapy and radiation treatments, and different patient outcomes. Despite the advances, it is clear that many alterations implicated in glioblastoma initiation and development remain unknown. In particular, the role of aberrant post-transcriptional regulation in gliomagenesis, mediated primarily by RNA-binding proteins, is underexplored and deserving of extra attention. The stem cell–related RNA-binding protein Musashi1 (MSI1) is emerging as an important oncogenic factor in numerous tumor types, including glioblastoma multiforme, in which it is frequently up-regulated.5Glazer R.I. Vo D.T. Penalva L.O. Musashi1: an RBP with versatile functions in normal and cancer stem cells.Front Biosci. 2012; 17: 54-64Crossref PubMed Scopus (48) Google Scholar MSI1 is evolutionarily conserved, displays important functions during nervous system development and embryogenesis, and serves as an important regulatory molecule in neural stem cells by balancing self-renewal and differentiation.6Siddall N.A. McLaughlin E.A. Marriner N.L. Hime G.R. The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity.Proc Natl Acad Sci U S A. 2006; 103: 8402-8407Crossref PubMed Scopus (97) Google Scholar Widespread gene regulatory activities both as a repressor and activator of translation7Imai T. Tokunaga A. Yoshida T. Hashimoto M. Mikoshiba K. Weinmaster G. Nakafuku M. Okano H. The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA.Mol Cell Biol. 2001; 21: 3888-3900Crossref PubMed Scopus (378) Google Scholar, 8de Sousa Abreu R. Sanchez-Diaz P.C. Vogel C. Burns S.C. Ko D. Burton T.L. Vo D.T. Chennasamudaram S. Le S.Y. Shapiro B.A. Penalva L.O. Genomic analyses of musashi1 downstream targets show a strong association with cancer-related processes.J Biol Chem. 2009; 284: 12125-12135Crossref PubMed Scopus (78) Google Scholar, 9Charlesworth A. Wilczynska A. Thampi P. Cox L.L. MacNicol A.M. Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation.EMBO J. 2006; 25: 2792-2801Crossref PubMed Scopus (136) Google Scholar, 10Battelli C. Nikopoulos G.N. Mitchell J.G. Verdi J.M. The RNA-binding protein Musashi-1 regulates neural development through the translational repression of p21WAF-1.Mol Cell Neurosci. 2006; 31: 85-96Crossref PubMed Scopus (147) Google Scholar, 11MacNicol M.C. Cragle C.E. MacNicol A.M. Context-dependent regulation of Musashi-mediated mRNA translation and cell cycle regulation.Cell Cycle. 2011; 10: 39-44Crossref PubMed Scopus (54) Google Scholar, 12Kuwako K. Kakumoto K. Imai T. Igarashi M. Hamakubo T. Sakakibara S. Tessier-Lavigne M. Okano H.J. Okano H. Neural RNA-binding protein Musashi1 controls midline crossing of precerebellar neurons through posttranscriptional regulation of Robo3/Rig-1 expression.Neuron. 2010; 67: 407-421Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar suggest that MSI1 promotes and potentiates tumorigenesis in multiple ways. In glioblastoma multiforme, MSI1 influences numerous cancer-relevant processes. Our functional genomic analysis showed that MSI1 controls hundreds of targets that are preferentially located in pathways such as focal adhesion, adherens junction, Wnt signalling pathway, Janus kinase/signal transducers and activators of transcription, p53, mitogen-activated protein kinase, and ErbB and suggests that MSI1 could be an interesting therapeutic target.13Uren P.J. Vo D.T. de Araujo P.R. Potschke R. Burns S.C. Bahrami-Samani E. Qiao M. de Sousa Abreu R. Nakaya H.I. Correa B.R. Kuhnol C. Ule J. Martindale J.L. Abdelmohsen K. Gorospe M. Smith A.D. Penalva L.O. RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma.Mol Cell Biol. 2015; 35: 2965-2978Crossref PubMed Scopus (41) Google Scholar However, it remains to be determined how high levels of MSI1 impact glioblastoma therapy, as its overexpression correlates with poor prognosis.14Dahlrot R.H. Hansen S. Herrstedt J. Schroder H.D. Hjelmborg J. Kristensen B.W. Prognostic value of Musashi-1 in gliomas.J Neurooncol. 2013; 115: 453-461Crossref PubMed Scopus (40) Google Scholar Here, we elucidate the role that MSI1 has on DNA damage repair and radio-resistance. We demonstrate that MSI1 mediates glioblastoma cell survival after radiation through increased DNA damage repair by end-joining (EJ), presumably via its stabilization of DNA-dependent protein kinase catalytic subunit (PKcs). U251 and U343 glioblastoma cells were obtained from ATCC (Rockville, MD), and U2OS cells with stably integrated EJ5–Green fluorescent protein (GFP)15Gunn A. Bennardo N. Cheng A. Stark J.M. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.J Biol Chem. 2011; 286: 42470-42482Crossref PubMed Scopus (86) Google Scholar were obtained from Dr. Jeremy Stark (City of Hope, Duarte, CA). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% Pen/Strep (Life Technologies, Carlsbad, CA). glioblastoma transcriptome data performed in the study by Uren et al13Uren P.J. Vo D.T. de Araujo P.R. Potschke R. Burns S.C. Bahrami-Samani E. Qiao M. de Sousa Abreu R. Nakaya H.I. Correa B.R. Kuhnol C. Ule J. Martindale J.L. Abdelmohsen K. Gorospe M. Smith A.D. Penalva L.O. RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma.Mol Cell Biol. 2015; 35: 2965-2978Crossref PubMed Scopus (41) Google Scholar were used for authenticating the U251 and U343 cell lines. Cells were transiently transfected with control siRNA or MSI1 siRNA using Lipofectamine RNAiMAX (MSI1HSS106732, MSI1HSS106733, MSI1HSS106734; Life Technologies). MSI1 or DNA-PKcs transgenic expression was achieved after transfecting U251 cells with pcDNA3.1-MSI1 or pCMV-F2_k_DNA-PKcs (obtained from Dr. David Chen, University of Texas Southwestern, Dallas, TX) vectors and GeneJammer transfection reagent (Agilent Technologies, Santa Clara, CA). For sustained suppression of MSI1 expression, short hairpin RNA (shRNAmir) GIPZ lentiviral vector carrying shRNA targeting MSI1 (shMSI1) and a nonspecific shRNA control (shCtrl) (Thermo Fisher Scientific, Rochester, NY) were used for transducing glioblastoma cells, according to the manufacturer's protocol. Two MSI1 target sequences (5′-CACCGTGGGGCGCGTCAGTCTCCAT-3′/5′-CACCGCGAATACTTCGGCCAGTTCG-3′) were cloned into lenti–clustered regularly interspaced short palindromic repeat v2 plasmid (catalog number 52961; Addgene, Cambridge, MA) and used for co-transfecting U251 cells. After selection using puromycin, cells were submitted to cell cloning by serial dilution in 96-well plates. Single colonies were transferred to a 12-well plate and allowed to grow. Three different clones were analyzed in this work. Cells were plated after appropriate dilution and ionizing radiation (IR) treatment was performed on the next day at a dose ranging from 0.15 to 5 Gy. A cabinet X-ray system (CP-160 Cabinet X-Radiator; Faxitron X-Ray Corp., Tucson, AZ) was used for all treatments. After IR, cells were cultured for 10 to 14 days. Then, cells were stained with crystal violet and all colonies of 50 or more cells were manually counted. The survival fraction, expressed as a function of IR, was estimated according to the following formula: Survival fraction = Colonies formed/(Cells seeded × Plating efficiency of the control group/100). Alternatively, crystal violet was dissolved from stained plates, and optic density was measured with a microplate reader at 570 nm. All experiments were performed in triplicate. Cells were plated, subjected to IR, and collected at different time points. The alkaline comet assay was performed according to Singh et al,16Singh N.P. McCoy M.T. Tice R.R. Schneider E.L. A simple technique for quantitation of low levels of DNA damage in individual cells.Exp Cell Res. 1988; 175: 184-191Crossref PubMed Scopus (9016) Google Scholar with some modifications. Briefly, after cell lysis, the slides were washed three times (5 minutes each) with electrophoresis buffer (300 mmol/L NaOH/1 mmol/L EDTA, pH ≥13.0) and placed in horizontal electrophoresis tank filled with electrophoresis buffer to allow for DNA unwinding for 20 minutes. Electrophoresis was performed for 20 minutes at 25 V (300 mA). Subsequently, the slides were neutralized three times (5 minutes each) with 400 mmol/L Tris–HCl (pH 7.5), fixed with 100% ethanol, and dried at room temperature. All steps were conducted under dim light to prevent the occurrence of additional DNA damage. Each slide was stained with 20 μg/mL ethidium bromide and covered with a coverslip before analysis. Images were captured on an Eclipse TE2000-U microscope (Nikon Instruments, Melville, NY) at ×40 magnification. Data are presented as the mean values of tail moment ± SD. Tail moment values of 50 randomly selected cells per slide from duplicate slides were scored using the Scion Image software package version 4.0.3.2 (NIH, Bethesda, MD) and used as the index of DNA damage. Total RNA was extracted using the TRIzol reagent (Life Technologies), following the manufacturer's instructions. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with random priming. After reverse transcription, quantitative PCR was performed using the Gene Expression Assay (Applied Biosystems) in TaqMan Gene Expression Master Mix (Applied Biosystems) for mRNA analysis. Real-time PCRs were performed on a 7500 Real-Time PCR System (Applied Biosystems). Data were acquired using the SDS software package version 2.0.1 (Applied Biosystems), and analyzed using the 2-ΔΔCt method using β-actin as an endogenous control. Cells were lysed in lysis buffer and subjected to Western blot analysis with the following primary antibodies: anti-MSI1 (1:1000; EMD Millipore, Billerica, MA), anti–replication protein A 32 (1:1000; Bethyl Laboratories, Montgomery, TX), anti–phospho-replication protein A 32(S4/S8) (1:1000; Bethyl), anti–CCCTC-binding factor (1:4000; Abcam, Cambridge, UK), anti–DNA-PKcs (1 μg/mL; Abcam), and anti–α-tubulin (1:5000; Sigma-Aldrich St. Louis, MO). Densitometry was performed using ImageJ analysis software version 1.47v (NIH; http://imagej.nih.gov/ij). Samples were normalized to α-tubulin content and results are expressed as the means ± SD values of integrated optical density. U251 cells were grown on fibronectin-coated coverslips. After MSI1 knockdown, cells were treated with 5 Gy IR. Thirty minutes after IR, cells were fixed and permeabilized. Cells were then blocked with 1% bovine serum albumin/4% goat serum followed by an overnight incubation with a 1:3000 dilution of 53BP1 primary antibody (Bethyl). Anti-rabbit IgG, Alexa Fluor 568–conjugated was used as secondary antibody (InvitroGen, Carlsbad, CA). Cells were then stained with DAPI and coverslips were mounted on slides using Vectashield (Vector Laboratories, Burlingame, CA). Images were captured on an Axiovert 200 M microscope (Carl Zeiss, Oberkochen, Germany) at ×40 magnification. A minimum of 100 nuclei were counted for each condition performed in triplicate. Briefly, EJ5-GFP U2OS cells were seeded in a 24-well plate and transfected with relevant transfections (siCtrl, siMSI1). Twenty-four hours later, cells were transfected with either ISceI expression vector alone or in combination with DNA-PKcs. After 72 hours, cells were harvested and GFP-positive cells were evaluated by flow cytometry on a BD flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ). Appropriate controls were used, and all experiments were performed in triplicate. Data were analyzed with the t-test and are presented as means ± SD. Several studies have described elevated MSI1 expression in different cancer/tumor types and subsequent association with a poor prognosis.5Glazer R.I. Vo D.T. Penalva L.O. Musashi1: an RBP with versatile functions in normal and cancer stem cells.Front Biosci. 2012; 17: 54-64Crossref PubMed Scopus (48) Google Scholar However, whether a targeted reduction in MSI1 expression contributes to treatment outcome remains unknown. We looked into the connection between high MSI1 expression and radio-resistance. Initially, to determine the impact of IR on MSI1 expression, U251 glioblastoma cells were treated with a single dose of IR (5 Gy). After treatment, the cells showed an increase in MSI1 expression at both the protein and RNA levels (Figure 1, A–C). To confirm the stress-induced expression of MSI1, cells were also treated with doxorubicin, an intercalating chemotherapeutic agent that blocks topoisomerase II activity, thereby causing double-strand breaks in the DNA. Protein levels of MSI1 were evaluated and, similar to the radiation results, we observed an increase in MSI1 expression caused by doxorubicin in an exposure time–dependent manner (Figure 1D). Thus, our data indicate that MSI1 expression is induced in response to DNA damage. Next, we assessed MSI1 impact on cellular response to radiotherapy. To determine the survival rate after IR, U251 shMSI1 and shCtrl cells were treated with different doses of IR and evaluated in a clonogenic assay. MSI1 knockdown cells (Figure 2A) showed increased sensitivity to IR (Figure 2B). Similar results were obtained with U343 cells (Supplemental Figure S1). We also directly measured the presence of broken DNA using the alkaline comet assay and observed that MSI1 knockdown led to a significant increase in DNA damage levels at early time points after radiation (Figure 2C). Finally, we evaluated IR-induced DNA damage levels in MSI1-silenced and control cells by measuring levels of 53BP1 foci, an established marker of double-strand breaks. Based on spontaneous levels of 53BP1 foci in U251 cells, a frequency of 15 foci, indicating higher levels of DNA damage (Figure 2, D and E). Cross-linking immunoprecipitation analysis of U251 cells, previously performed by our group,13Uren P.J. Vo D.T. de Araujo P.R. Potschke R. Burns S.C. Bahrami-Samani E. Qiao M. de Sousa Abreu R. Nakaya H.I. Correa B.R. Kuhnol C. Ule J. Martindale J.L. Abdelmohsen K. Gorospe M. Smith A.D. Penalva L.O. 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Protein kinase, DNA-activated, catalytic polypeptide encodes DNA-PKcs, which is the key enzyme involved in the classic nonhomologous (NH) EJ pathway of DNA double-strand break repair in mammalian cells. DNA-PKcs promotes NHEJ while suppressing homologous recombination20Allen C. Kurimasa A. Brenneman M.A. Chen D.J. Nickoloff J.A. DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination.Proc Natl Acad Sci U S A. 2002; 99: 3758-3763Crossref PubMed Scopus (153) Google Scholar and facilitates repair of genotoxic and replication stress associated damage via phosphorylation of the single-stranded DNA-binding protein replication protein A 2.21Serrano M.A. Li Z. Dangeti M. Musich P.R. Patrick S. Roginskaya M. Cartwright B. Zou Y. DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.Oncogene. 2013; 32: 2452-2462Crossref PubMed Scopus (72) Google Scholar Similar to MSI1, DNA-PKcs gene expression is increased on radiation (Supplemental Figure S2B). Moreover, we observed a replication protein A 2 hyperphosphorylation pattern after MSI1 overexpression in U251 cells (Figure 3A) concordant with the induction of DNA damage response in a DNA-PK–dependent manner.22Liaw H. Lee D. Myung K. DNA-PK-dependent RPA2 hyperphosphorylation facilitates DNA repair and suppresses sister chromatid exchange.PLoS One. 2011; 6: e21424Crossref PubMed Scopus (51) Google Scholar Most importantly, MSI1 knockdown via siRNA decreased the expression of DNA-PKcs in U251 glioblastoma cells (Figure 3B). Due to the high molecular weight of DNA-PKcs protein (approximately 460 kDa), we used CCCTC-binding factor protein as loading control (83 kDa). To ascertain that DNA-PKcs is a critical MSI1 target in the context of DNA repair, we generated MSI1-knockout cells using the clustered regularly interspaced short palindromic repeat/Cas9 system23Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346 (doi:10.1126/science.1258096): 1258096Crossref PubMed Scopus (3580) Google Scholar and performed rescue experiments. MSI1 knockout was confirmed by Western blot (Figure 3C). DNA-PKcs levels were dramatically reduced in the clones analyzed, confirming that DNA-PKcs expression is indeed controlled by MSI1 (Figure 3C). Then, MSI1-knockout glioblastoma lines were transfected with vectors containing empty control, MSI1, or DNA-PKcs coding sequence, subjected to a single dose of IR (2.5 Gy), and evaluated in clonogenic assays. MSI1 (Figure 3D) and DNA-PKcs (Figure 3E) knockout-rescued cells showed higher radio-resistance compared to cells transfected with an empty vector (Ctrl). MSI1 rescue was more efficient compared to DNA-PKcs, possibly because MSI1 might regulate other genes involved in DNA repair. Finally, the number of endogenous 53BP1 foci was quantified in MSI1-knockout cells after transfection with MSI1 or DNA-PKcs coding sequences (Figure 3F). Endogenous 53BP1 foci levels were higher in MSI1-knockout compared to wild-type cells. Most importantly, the percentage of cells containing multiple 53BP1 foci decreased after overexpression of MSI1 or DNA-PKcs. These results suggest the association of MSI1 expression with lowering levels of DNA damage caused by IR and its role in the double-strand break repair, most likely by regulating DNA-PKcs expression and activity. Given the high levels of persistent DNA damage with MSI1 knockdown and its regulation of DNA-PKcs, we asked whether MSI1 plays a role in the repair of double-strand breaks. DNA-PKcs is a key player in the NHEJ pathway to repair double-strand breaks.24Uematsu N. Weterings E. Yano K. Morotomi-Yano K. Jakob B. Taucher-Scholz G. Mari P.O. van Gent D.C. Chen B.P. Chen D.J. Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.J Cell Biol. 2007; 177: 219-229Crossref PubMed Scopus (310) Google Scholar We therefore asked whether MSI1 modulated EJ repair via its control of DNA-PKcs expression. To evaluate the frequency of EJ, we used the EJ5-GFP reporter assay integrated into U2OS cells.15Gunn A. Bennardo N. Cheng A. Stark J.M. Correct end use during end joining of multiple chromosomal double strand breaks is influenced by repair protein RAD50, DNA-dependent protein kinase DNA-PKcs, and transcription context.J Biol Chem. 2011; 286: 42470-42482Crossref PubMed Scopus (86) Google Scholar, 25Mao Z. Bozzella M. Seluanov A. Gorbunova V. Comparison of nonhomologous end joining and homologous recombination in human cells.DNA Repair (Amst). 2008; 7: 1765-1771Crossref PubMed Scopus (387) Google Scholar This reporter (Figure 4A) consists of a promoter and a GFP-coding cassette that is interrupted by a puromycin (puro) gene. The puro gene is flanked by ISceI homing endonuclease recognition sites in the same orientation. The expression of ISceI allows for site-specific double-strand breaks, resulting in excision of the puro gene and in the presence of functional NHEJ, leading to restoration of wild-type GFP gene. Interestingly, we observed a reduction in NHEJ frequency in MSI1-knockdown cells (Figure 4B), and this phenotype was rescued with the overexpression of DNA-PKcs (Figure 4B). Few recent studies have reported on the biological consequence of MSI1 in cancer. In colon cancer, the silencing of MSI1 induced apoptosis, mitosis, G2/M arrest, and tumor regression.26Sureban S.M. May R. George R.J. Dieckgraefe B.K. McLeod H.L. Ramalingam S. Bishnupuri K.S. Natarajan G. Anant S. Houchen C.W. Knockdown of RNA binding protein musashi-1 leads to tumor regression in vivo.Gastroenterology. 2008; 134: 1448-1458Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar In breast cancer, MSI1 knockdown resulted in decreased tumor mammosphere formation, decreased proliferation, and reduced breast cancer xenograft growth.27Wang X.Y. Penalva L.O. Yuan H. Linnoila R.I. Lu J. Okano H. Glazer R.I. Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival.Mol Cancer. 2010; 9 (doi:10.1186/1476-4598-9-221): 221Crossref PubMed Scopus (114) Google Scholar In lung cancer, MSI1 silencing reduced spheroid colony formation with inhibition of the Wnt and Notch pathways.28Wang X.Y. Yu H. Linnoila R.I. Li L. Li D. Mo B. Okano H. Penalva L.O. Glazer R.I. Musashi1 as a potential therapeutic target and diagnostic marker for lung cancer.Oncotarget. 2013; 4: 739-750Crossref PubMed Scopus (37) Google Scholar Silencing of MSI1 in DAOY medulloblastoma cells decreased proliferation and neurosphere formation, and induced differentiation and apoptosis.29Sanchez-Diaz P.C. Burton T.L. Burns S.C. Hung J.Y. Penalva L.O. Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.BMC Cancer. 2008; 8: 280-292Crossref PubMed Scopus (59) Google Scholar In the particular case of glioblastoma, we observed that a reduction in MSI1 expression increased apoptosis, decreased proliferation, affected cell cycle regulation, and interfered with adhesion-related functions such as invasion and migration.13Uren P.J. Vo D.T. de Araujo P.R. Potschke R. Burns S.C. Bahrami-Samani E. Qiao M. de Sousa Abreu R. Nakaya H.I. Correa B.R. Kuhnol C. Ule J. Martindale J.L. Abdelmohsen K. Gorospe M. Smith A.D. Penalva L.O. RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma.Mol Cell Biol. 2015; 35: 2965-2978Crossref PubMed Scopus (41) Google Scholar However, the impact of MSI1 expression on treatment outcome is poorly understood. Here, we demonstrated the induced expression of MSI1 by radiation in glioblastoma cell lines, which correlates with increased cell survival. Knockout of MSI1 increased radiosensitivity by affecting DNA damage repair through a mechanism involving protein kinase, DNA-activated, catalytic polypeptide expression. Consistent with the role of cancer stem cells in radio-resistance, different studies have reported that radiation was able to induce the expansion of glioma cells that express stem cell markers, such as MSI1.30Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4858) Google Scholar Increased DNA repair capacity of cancer stem cells has been associated with resistance to radiotherapy.31Hussein D. Punjaruk W. Storer L.C. Shaw L. Othman R. Peet A. Miller S. Bandopadhyay G. Heath R. Kumari R. Bowman K.J. Braker P. Rahman R. Jones G.D. Watson S. Lowe J. Kerr I.D. Grundy R.G. Coyle B. Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.Neuro Oncol. 2011; 13: 70-83Crossref PubMed Scopus (53) Google Scholar We suggest in this work that the functional involvement of MSI1 in the efficient repair of double-strand breaks by the NHEJ pathway is a possible mechanism of the observed chemoresistance. NHEJ is the predominant repair pathway in the mammalian system during the G1 and M phases, although it is active throughout the cell cycle. Increased protein expression of DNA-PKcs has been reported in a variety of tumor types, such as nasopharyngeal cancer, colorectal cancer, and non–small cell lung cancer, and overexpression has been correlated with tumor grade and poor survival.32Hsu F.M. Zhang S. Chen B.P. Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment.Transl Cancer Res. 2012; 1: 22-34PubMed Google Scholar In glioma specimens, hyperactivity of this protein was associated with tumor grading33Shao C.J. Fu J. Shi H.L. Mu Y.G. Chen Z.P. Activities of DNA-PK and Ku86, but not Ku70, may predict sensitivity to cisplatin in human gliomas.J Neurooncol. 2008; 89: 27-35Crossref PubMed Scopus (22) Google Scholar and radio-resistance,34Mukherjee B. McEllin B. Camacho C.V. Tomimatsu N. Sirasanagandala S. Nannepaga S. Hatanpaa K.J. Mickey B. Madden C. Maher E. Boothman D.A. Furnari F. Cavenee W.K. Bachoo R.M. Burma S. EGFRvIII and DNA double-strand break repair: a molecular mechanism for radioresistance in glioblastoma.Cancer Res. 2009; 69: 4252-4259Crossref PubMed Scopus (203) Google Scholar and its up-regulation after radiation treatment has been associated with radio-resistance in recurrent oral squamous cell carcinomas.35Shintani S. Mihara M. Li C. Nakahara Y. Hino S. Nakashiro K. Hamakawa H. Up-regulation of DNA-dependent protein kinase correlates with radiation resistance in oral squamous cell carcinoma.Cancer Sci. 2003; 94: 894-900Crossref PubMed Scopus (106) Google Scholar Using a DNA-PK kinase assay and assessing the phosphorylation status of replication protein A 2, a previous study demonstrated IR-induced DNA-PK activity in glioblastoma cell lines.36Shen Y. Wang Y. Sheng K. Fei X. Guo Q. Larner J. Kong X. Qiu Y. Mi J. 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Moreover, MSI1 is specifically expressed in tumor cells and not in adjacent and differentiated cells such as neurons and astrocytes, leaving collateral damage from local inhibition of MSI1 to a minimum. Therapeutic targeting of MSI1 could be accomplished using a myriad of methodologies that could include small-molecule inhibitors (in a similar strategy for drug discovery for DNA-binding proteins),38Berg T. Inhibition of transcription factors with small organic molecules.Curr Opin Chem Biol. 2008; 12: 464-471Crossref PubMed Scopus (104) Google Scholar RNA-based decoys mimicking MSI1 binding motifs,39Keefe A.D. Pai S. Ellington A. Aptamers as therapeutics.Nat Rev Drug Discov. 2010; 9: 537-550Crossref PubMed Scopus (1261) Google Scholar, 40Symensma T.L. Baskerville S. Yan A. Ellington A.D. Polyvalent Rev decoys act as artificial Rev-responsive elements.J Virol. 1999; 73: 4341-4349Crossref PubMed Google Scholar and miRNA mimics.41Bader A.G. Brown D. Winkler M. 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Vo D.T. Penalva L.O. Musashi1: an RBP with versatile functions in normal and cancer stem cells.Front Biosci. 2012; 17: 54-64Crossref PubMed Scopus (48) Google Scholar strategies to modulate MSI1 function or levels of expression could have a large impact on cancer treatment. We thank Dr. Pei Wang for the lenti-CRISPR v2 vector used for generating MSI1 knockout cell lines by CRISPR technology. Download .pdf (145.13 MB) Help with pdf files Supplemental Figure S1Decreased U343 cell survival after knockdown (KD) of Musashi1 (MSI1). A: Immunoblot analysis of MSI1 expression in U343 cells after transfection with sort-hairpin control (shCtrl) or shMSI1. B: Clonogenic assay of U343 MSI1-KD after exposure to increasing doses of X-rays. ∗P < 0.05, ∗∗∗P < 0.001. Download .pdf (183.57 MB) Help with pdf files Supplemental Figure S2A: The University of California at Santa Cruz (UCSC) genome browser (https://genome.ucsc.edu, accessed October 10, 2015) tracks showing individual-nucleotide resolution UV cross-linking immunoprecipitation (iCLIP) read coverage in the 3′ untranslated region (UTR) of protein kinase, DNA-activated, catalytic polypeptide (PRKDC). Vertical red lines indicate occurrences of the trimer UAG, which forms the core of the Musashi1 (MSI1) binding site. The density of iCLIP reads, which mark MSI1 binding activity, correlates with regions showing high occurrence of UAG trimers. Reads were mapped to hg19 using RMAP.17Smith A.D. Chung W.Y. Hodges E. Kendall J. Hannon G. Hicks J. Xuan Z. Zhang M.Q. Updates to the RMAP short-read mapping software.Bioinformatics. 2009; 25: 2841-2842Crossref PubMed Scopus (132) Google Scholar Read-coverage wiggle tracks were produced using a combination of in-house scripts and the UCSC genome browser toolchain. Visualization of read coverage and identification of UAG trimers were performed using the UCSC genome browser. B: Immunoblot analysis showing increased DNA–protein kinase catalytic subunit (PKcs) expression in different times after radiation (5 Gy). CTCF, CCCTC-binding factor.
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