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Targeting the Deregulated Spliceosome Core Machinery in Cancer Cells Triggers Mtor Blockade and Autophagy

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Targeting the Deregulated Spliceosome Core Machinery in Cancer Cells Triggers Mtor Blockade and Autophagy Published OnlineFirst January 28, 2013; DOI: 10.1158/0008-5472.CAN-12-2501 Cancer Therapeutics, Targets, and Chemical Biology Research Targeting the Deregulated Spliceosome Core Machinery in Cancer Cells Triggers mTOR Blockade and Autophagy Virginie Quidville1,2,3, Samar Alsafadi1,2,3, Aïcha Goubar1,2,3,Fred eric Commo1,2,3,Veronique Scott1,2,3, Catherine Pioche-Durieu2,3, Isabelle Girault1,2,3, Sonia Baconnais2,3, Eric Le Cam2,3, Vladimir Lazar2,3, Suzette Delaloge1,2, Mahasti Saghatchian1,2, Patricia Pautier2, Philippe Morice2,3, Philippe Dessen2,3, Stephan Vagner1,2,3, and Fabrice Andre1,2,3 Abstract The spliceosome is a large ribonucleoprotein complex that guides pre-mRNA splicing in eukaryotic cells. Here, we determine whether the spliceosome could constitute an attractive therapeutic target in cancer. Analysis of gene expression arrays from lung, breast, and ovarian cancers datasets revealed that several genes encoding components of the core spliceosome composed of a heteroheptameric Sm complex were overexpressed in malignant disease as compared with benign lesions and could also define a subset of highly aggressive breast cancers. siRNA-mediated depletion of SmE (SNRPE) or SmD1 (SNRPD1) led to a marked reduction of cell viability in breast, lung, and melanoma cancer cell lines, whereas it had little effect on the survival of the nonmalignant MCF-10A breast epithelial cells. SNRPE or SNRPD1 depletion did not lead to apoptotic cell death but autophagy, another form of cell death. Indeed, induction of autophagy was revealed by cytoplasmic accumulation of autophagic vacuoles and by an increase in both LC3 (MAP1LC3A) protein conversion and the amount of acidic autophagic vacuoles. Knockdown of SNRPE dramatically decreased mTOR mRNA and protein levels and was accompanied by a deregulation of the mTOR pathway, which, in part, explains the SNRPE-dependent induction of autophagy. These findings provide a rational to develop new therapeutic agents targeting spliceosome core components in oncology. Cancer Res; 73(7); 2247–58. Ó2013 AACR. Introduction the most enriched pathway in breast cancer samples, when Targeted therapies have been shown to improve outcome in compared with benign lesions (1). breast cancers. Molecular targeted agents usually inhibit key The aim of the present study was to determine whether the oncogenic pathways involved in cancer progression or resis- spliceosome could be a relevant therapeutic target in cancer. tance to conventional treatments. Most of these new molecular The spliceosome is a dynamic macromolecular ribonucleopro- therapies target kinase pathways and are usually highly effec- tein (RNP) complex that catalyses the splicing of nuclear pre- tive in a specific molecular segment. In the recent years, mRNA (precursor mRNA) into mRNAs. Nuclear pre-mRNA accumulative evidence has been reported that beyond kinase splicing is essential to remove internal noncoding regions of and DNA repair defects, many pathways are crucial for cancer pre-mRNA (introns) and to join the remaining segments (exons) progression. For instance, metabolism, immune system, and into mRNA before export to the cytoplasm and translation. The epigenetics have been reported as targetable in oncology. spliceosome does the 2 primary functions of splicing, that is, Looking for novel pathways involved in cancer progression, recognition of the intron/exon boundaries and catalysis of the we previously identified spliceosome assembly components as cut-and-paste reactions that remove introns and join exons. The spliceosomal machinery complex is formed from 5 RNP sub- units, termed uridine-rich (U-rich) small nuclear RNP (snRNP), Authors' Affiliations: 1Institut National de la Sante et de la Recherche transiently associated to a range of protein factors (2, 3). Each Medicale (INSERM) U981, Paris; 2Institut de cancerologie Gustave Roussy, Villejuif; and 3Universite Paris-Sud XI, Kremlin-Bicetre,^ France snRNP (U1, U2, U4/U6, and U5) consists of a uridine-rich small nuclear RNA (snRNA) complexed with a set of 7 proteins known Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). as Sm or SNRP proteins. The Sm proteins (B/B', D1, D2, D3, E, F and G) form a 7-member ring core structure that encompasses V. Quidville and S. Alsafadi contributed equally to this work and are joint first RNA. All Sm proteins share a conserved Sm domain, consisting authors. of 2 sets of conserved sequences (Sm1 and Sm2) that are S. Vagner and F. Andre are co-senior authors. responsible for the assembly of snRNAs in an ordered manner, Corresponding Author: Fabrice Andre, INSERM U981, Institut Gustave to form the Sm core of the spliceosomal snRNPs (4, 5) and are Roussy, 114 rue Edouard Vaillant, Villejuif 94805, France. Phone: 33-1- thereby involved in pre-mRNA processing (6). 42114293; Fax: 33-1-42115217; E-mail: [email protected] The splicing process has a crucial role in the control of the doi: 10.1158/0008-5472.CAN-12-2501 expression of many genes (such as those involved in cell cycle, Ó2013 American Association for Cancer Research. signal transduction, angiogenesis, apoptosis, and invasion; www.aacrjournals.org 2247 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst January 28, 2013; DOI: 10.1158/0008-5472.CAN-12-2501 Quidville et al. refs. 7–15). Mutations of splicing factors or alterations of the of 1 mL. For controls, cells were either exposed to Hiperfect expression of the components of the splicing machinery can reagent alone (mock) or transfected with a nontargeting affect the splicing pattern of genes and contribute to the control siRNA. Transfection medium was replaced by fresh development of tumors (16–19). Deregulated alternative splic- culture medium every 48 hours until the end of experiment. ing can lead to the production of novel mRNA isoforms coding After 96 hours of transfection, cells were harvested, counted for protein variants with key functions in cancer using the trypan blue exclusion method or crystal violet (7, 10, 13, 20, 21). A substantial fraction of alternative splicing staining, and processed for the various biochemical assays. events in humans can also result in mRNA isoforms that harbor siRNA transfection effect and siRNA concentration effect on a premature termination codon (PTC). These transcripts are cell number were estimated by 2-way ANOVA. predicted to be degraded by the nonsense-mediated mRNA For NMD inhibition experiments, cells were transfected with decay (NMD) pathway which constitutes a mechanism of RNA SNRPE siRNA or nontargeting control siRNA. Three days later, surveillance that detects and eliminates aberrantly spliced cells were treated for 8 hours with 100 mg/mL cycloheximide mRNAs harboring a PTC to prevent the synthesis of nonfunc- (Sigma-Aldrich) or with an equivalent amount of dimethyl tional or deleterious truncated proteins (22, 23) and to regulate sulfoxide (DMSO) as a control. RNA was then isolated and the abundance of mRNA transcripts (24). mRNA expression was detected by real-time PCR (RT-PCR). In the present study, we have evaluated whether compo- Quantifications of the RT-PCR signals were calculated from nents of the spliceosome machinery complex were overex- RT-PCR assays conducted on samples from 2 independent pressed in malignant tumors and whether blocking spliceo- transfections. some could lead to antitumor effects. Immunoblot analysis Cells were lysed in radioimmunoprecipitation assay (RIPA) Materials and Methods buffer, and proteins were quantified using a microBCA assay Cell culture and siRNA transfection (Pierce, ThermoScientific). Equal amounts were separated on All cell lines were supplied by American Type Culture SDS-PAGE gels. Proteins were transferred to polyvinylidene Collection (ATCC, LGC Standards) between 2007 and 2011. fluoride (PVDF) membranes followed by immunoblot with Each ATCC cell line was first amplified to generate a cell master antibodies specific for mTOR (Cell Signaling, Ozyme), 4E-BP1 bank. All experiments were carried out from this cell bank. All (Cell Signaling), phospho-4E-BP1 (Ser 65; Cell Signaling), anti- the cell lines used were tested negative for mycoplasma LC3B (Sigma-Aldrich), SNRPD1 (C-15) and SNRPE (L-17; Santa contamination using VenorGeM Advance PCR Kit (Biovalley). Cruz Biotechnology). Immunolabeled proteins were detected Human mammary carcinoma cell lines SKBr-3 (ATCC HTB- by a chemiluminescence detection system (Super Signal West 30), MDA-MB 468 (ATCC HTB-132), HCC1937 (ATCC CRL- Dura Signal, Pierce, ThermoScientific). Membranes were then 2336), lung carcinoma A549 (ATCC CCL-185), and malignant washed in 0.1% TBS-Tween-20 and incubated with rabbit anti- melanoma A375 (ATCC CRL-1619) were cultured as mono- GAPDH or mouse anti-actin (C4; Chemicon, Millipore) to layers at 37 C with 5% CO2 and maintained by regular passage quantify and normalize results. in complete media consisting of RPMI-1640 GlutaMaxI or Dulbeccos' Modified Eagle's Media (DMEM):F12 (Gibco BRL, Cell-cycle analysis Invitrogen Life Technology) supplemented with 10% heat- Cell-cycle distribution was measured in SKBr-3 cells trans- inactivated FBS (PAN Biotech, Dutscher), 1 mmol/L sodium fected with 50 nmol/L SNRPE-specific validated siRNA or pyruvate (Invitrogen), and 10 m mmol/L HEPES (Invitrogen). nontargeting control siRNA. Cells were harvested at 72 hours MCF-7 (ATCC HTB-22) was maintained in completed DMEM/ after transfection, suspended in PBS, and fixed in
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