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Rapalog-Mediated Repression of Tribbles Published OnlineFirst April 3, 2020; DOI: 10.1158/0008-5472.CAN-19-2366 CANCER RESEARCH | MOLECULAR CELL BIOLOGY Rapalog-Mediated Repression of Tribbles Pseudokinase 3 Regulates Pre-mRNA Splicing Bojana Stefanovska1,2,3, Cecile Edith Vicier1,2,3, Thibault Dayris2,4, Vasily Ogryzko5,†, Veronique Scott1,2,3, Ibrahim Bouakka1,2,3, Suzette Delaloge6, Anna Rocca1,2,3, Olivia Le Saux7, Olivier Tredan 7, Thomas Bachelot7, Fabrice Andre1,2,6, and Olivia Fromigue1,2,3 ABSTRACT ◥ Rapalogs have become standard-of-care in patients with induced deregulation of RNA splicing. Conversely, overexpres- metastatic breast, kidney, and neuroendocrine cancers. Never- sion of TRIB3 in a panel of cancer cell lines abolished theless, tumor escape occurs after several months in most the cytotoxic effects of rapalogs. These findings identify TRIB3 patients, highlighting the need to understand mechanisms of as a key component of the spliceosome, whose repression resistance. Using a panel of cancer cell lines, we show that contributes significantly to the mechanism of resistance to rapalogs downregulate the putative protein kinase TRIB3 (trib- rapalog therapy. bles pseudokinase 3). Blood samples of a small cohort of patients with cancer treated with rapalogs confirmed down- Significance: Independent of mTOR signaling, rapalogs induce regulation of TRIB3. Downregulation of TRIB3 was mediated cytoxicity by dysregulating spliceosome function via repression of by LRRFIP1 independently of mTOR and disrupted its inter- TRIB3, the loss of which may, in the long term, contribute to action with the spliceosome, where it participated in rapalog- therapeutic resistance. Introduction development of some derivatives overcame issues related to poor solubility and pharmacokinetics of the original rapamycin molecule. Tumor progression is a complex process resulting from the accu- In that way, temsirolimus and everolimus (also known as rapalogs) mulation of genetic alterations like chromosomal rearrangements, improved patient outcome in a broad range of cancers. As illustration, mutations, silencing of transcription through methylation, or defects the phase III trials BOLERO-2, RECORD-1, and RADIANT-3 dem- in posttranscriptional modifications such as mRNA splicing, mRNA onstrated that everolimus could improve progression-free survival of stabilization, and translational regulation. Thus, tumor cells synthesize patients with metastatic hormone receptor–positive breast cancer, and accumulate proteins altered in their structure or function, mod- metastatic renal cell carcinoma, or advanced pancreatic neuroendo- ifying some signaling pathways. One of the key proteins involved in crine tumors, respectively (6–8). Rapalogs are currently administered tumor progression is mTOR (mechanistic target of rapamycin), to patients presenting hormone receptor–positive breast cancers, because it integrates signals from nutrients, hormones, and growth metastatic kidney cancers, and neuroendocrine tumors (9). While the factors to regulate cell metabolism, growth, proliferation, and surviv- benefit for patients is matter of consensus, resistance to therapy occurs al (1). The mTOR signaling pathway is hyperactivated in up to 70% of usually after several months of treatment. Resistance can often occur human tumors (2). The macrolide rapamycin, originally identified for through the activation of mTOR-regulated feedback loops or by the its antifungal properties, forms a gain-of-function complex with the acquisition of new mutations (10, 11). Nevertheless, these mechanisms immunophilin FKBP12 (FK506-binding protein 1A, 12 kDa) that acts explain only a few percentage of resistance, and there is a need to as an allosteric inhibitor of mTOR (3). Rapamycin exhibits immuno- identify new mechanisms of treatment escape. suppressive properties, but is also used as anticancer therapy (4, 5). The In this study, we aim to identify new mechanisms of action for rapalogs and we have discovered that tribbles pseudokinase 3 (TRIB3) 1Inserm, UMR981, Villejuif, France. 2Gustave Roussy, Villejuif, France. 3Universite encoded by the tribbles pseudokinase-3 gene mediates effects of Paris Sud, Orsay, France. 4Inserm, US23, CNRS, UMS3655, Villejuif, France. rapalogs on splicing, in an mTOR-independent manner. 5CNRS, UMR8126, Villejuif, France. 6Department of Medical Oncology, Gustave Roussy, Villejuif, France. 7Department of Medical Oncology, Centre Leon Berard, Materials and Methods Lyon France. Chemicals and reagents Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Rapamycin was purchased from Sigma-Aldrich. Everolimus [40-O-(2-hydroxy)ethyl-rapamycin] was purchased from Novartis. B. Stefanovska and C.E. Vicier contributed equally to this article as co-first authors. Rabbit anti-TRIB3, anti-GFP, anti-CDC5L, anti-FKBP25, and mouse anti-LRRFIP1 antibodies were purchased from Abcam. F. Andre and O. Fromigue contributed equally to this article as co-last authors. Mouse anti-b-actin antibody was purchased from Sigma-Aldrich. † Deceased. Rabbit anti-phospho-Akt and anti-Akt antibodies were purchased Corresponding Author: Olivia Fromigue, Inserm UMR981, Gustave Roussy, Rue from Cell Signaling Technology. Camille Desmoulins, Villejuif 94805, France. Phone: 331-4211-4211; Fax: 331-4211- 6094; E-mail: [email protected] Plasmids Lentiviral vector for TRIB3 overexpression was generated by Gate- Cancer Res 2020;80:2190–203 way cloning from pDONR223-TRIB3 into pLX303, both purchased doi: 10.1158/0008-5472.CAN-19-2366 from Addgene. Prof. I.C. Eperon (University of Leicester, Leicester, Ó2020 American Association for Cancer Research. United Kingdom), and Dr. Y. Itoh (Nagoya City University, Nagoya, AACRJournals.org | 2190 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst April 3, 2020; DOI: 10.1158/0008-5472.CAN-19-2366 Rapalogs Regulate Pre-mRNA Splicing Japan) kindly provided the pTN23 (12, 13) and pTRB3-Luc1 reporter trypan blue solution. Experiments were conducted with 6 Â 106 viable vector, respectively. The derived plasmid pTRB3-Luc1 D136-145 was cells per condition. generated by the QuikChange II XL Site-Directed Mutagenesis Kit Whole blood samples were derived from the ongoing clinical trial (Stratagene). The mutagenic oligonucleotides were: 50-TGGAGGCT- NCT02536625 (RAPANK; Supplementary Table S1). Blood samples CTGGCGCCCGGGCC-30 (forward) and 50-GGCCCGGGCGCCA- were collected before and one month after everolimus initiation. GAGCCTCCA-30 (reverse). The pEZX-PG02-TRIB3 promoter Written informed consent was collected from all patients, according reported plasmid (HPRM13929) was purchased from Tebu-Bio. The to the principles of the Declaration of Helsinki. pCH110 plasmid was purchased from Addgene. RNA extraction Minigene reporter assay Total RNAs were isolated using TRIzol reagent (Thermo Fisher Cells at 65%–70% confluence were cotransfected with pCH110 and Scientific) for cell lines or PBMC, and using TRIzol-LS reagent pTRB3-Luc1, pEZX-PG02-TRB3 or pTRB3-Luc1 D136-145 using (Thermo Fisher Scientific) for whole blood samples. The quality of Lipofectamine2000 reagent (Thermo Fisher Scientific). Transcription- RNA samples was checked using Bioanalyzer. Mean values of RNA al activity was evaluated using Luciferase Assay Kit (Promega) or integrity numbers were 9.1 Æ 0.3. Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific) and transfection efficiency by b-gal gene Reporter Assay Kit (Roche). Comparative microarray analyses Transcriptional profiles were determined using Agilent SurePrint Cell culture G3 Human GE 8 Â 60K V2 microarrays (AMADID 39494; Agilent All cell lines (except SUM52PE cells, kindly provided by Dr Turner Technologies), and analyzed with Agilent Feature Extraction software from The Institute of Cancer Research, London, UK) were purchased version 10.7.3.1 using the default settings. Raw data files were pro- from the ATCC (LGC Standards) between 2007 and 2011. Each ATCC cessed as follows: control probes were systematically removed and cell line was first amplified to generate a cell master bank. All flagged probes were set to NA. Arrays were normalized by quantile experiments were carried out from this master bank with cells kept normalization. Missing values were inferred using KNN algorithm in continuous culture for less than 6 months. No further authentication (package “impute” from R). was performed. HCC827, SKBR3, MKN45, and SUM52PE cells were cultured in GlutaMAX containing RPMI1640 medium supplemented RNA-seq analysis with 10% heat-inactivated FBS (Thermo Fisher Scientific), 1 mmol/L Libraries were made using the TruSeq Stranded mRNA Library sodium pyruvate, and 1% HEPES. A375 cells were cultured in Glu- Preparation Kit (Illumina). Equimolar pool of libraries were sequenced taMAX containing DMEM supplemented with 10% FBS, 1 mmol/L on a Illumina HiSeq2500 machine using paired-end reads (2 Â 75 bp), sodium pyruvate, and 1% HEPES. MDA-MB-468 cells were cultured and high-output run mode allowing to get 40 millions of raw reads per in GlutaMAX containing DMEM/Nutrient Mix F-12 medium sup- sample. The quality of reads was assessed using FastQC. Quality plemented with 10% FBS, 1 mmol/L sodium pyruvate, and 1% HEPES. reports were gathered with MultiQC. Abundance estimation was MCF7 and T47D cells were cultured in DMEM/F12 medium supple- achieved with Salmon (15) with the arguments keepDuplicates mented with 10% FBS, 1 mmol/L sodium pyruvate, and 10 mg/mL
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