Osteosarcoma mouse model
Four-week-old female Rj:NMRI-nude mice (Janvier, Le Genest Saint Isle, France) were maintained under pathogen-free conditions at the Experimental Therapy Unit (Faculty of Medicine, Nantes, France) in accordance with the institutional guidelines of the French Ethical Committee (CEEA Pays de la Loire n°06 : project authorization 8405). Mice received an intramuscular injection of 1.106 K-HOS parental or mutant cells in close proximity to the tibia. Five days after cell injections, groups of mice received or not the molecules verteporfin (20 mg/kg), CA3 (10 mg/kg) or control vehicle twice a week by intraperitoneal injection. The tumor volume (V) was calculated from the measurement of three perpendicular diameters using a caliper according to the following formula: V = (length x height x depth)/2.
Cell culture and reagents.
K-HOS (CRL-1544), HOS-MNNG (CRL-1543), MG-63 (CRL-1427) and G292 (CRL-1423) osteosarcoma cell lines were purchased from ATCC (LGC Standards, Molsheim, France). HEK293 cells were purchased from Invitrogen (Thermo Fisher, Courtaboeuf, France). Cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (Hyclone Perbio, Bezons, France).
To generate mutant YAP-S94A and YAP-S127A expressing cells, retrovirus infection was performed by transfecting 293 Phoenix retrovirus packaging cells with empty vector, pQCXIH-Myc-YAP-S94A and pQCXIH-Flag-YAP-S127A. pQCXIH-Myc-YAP-S94A and pQCXIH-Flag-YAP-S127A were gifts from Kunliang Guan (respectively Addgene plasmid #33094 and #33092; http://n2t.net/addgene:33094 and 33092; RRID:Addgene-33094 and -33092). pQCXIH CMV/TO DEST (w382-1) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid #17394; http://n2t.net/addgene:17394; RRID:Addgene-17394). 24 hours post-transfection, retroviral supernatant was supplemented with 5 µg/ml polybrene (Santa Cruz Biotechnology, CA, USA), filtered through a 0.45-µm filter and used to infect K-HOS cells. 48 hours after infection, cells were selected with 200 µg/ml hygromycin-B (Invitrogen, Courtaboeuf, France). Verteporfin and CA3 were respectively purchased from Tocris (Bristol, UK), and InvivoChem (Libertyville, IL, US)
Luciferase reporter assay and plasmid constructs
Transient cell transfections were performed with jetPEI™ (Polyplus transfection, Illkirch, France). The pRLTK-Renilla luciferase expression vector was co-transfected in all experiments to monitor transfection efficiencies. Luciferase activity was determined with the Dual-Luciferase reporter assay system (Promega, Charbonnieres, France). The 8xGTIIC-Luc (gift from Stefano Piccolo, Addgene plasmid #34615; http://n2t.net/addgene:34615; RRID:Addgene-34615, (TEAD)8-lux in the text) construct was used as a specific reporter construct specific for TEAD-driven signaling.
Real time proliferation and Annexin V assays
In vitro cell proliferation assays were assessed using xCELLigence Real-Time cell-Analyzer (ACEA Biosciences, San Diego, CA). The experiment was done in triplicate and repeated three times.
Annexin V assay : OS cells were cultured and treated with or without verteporfin or CA3 for 72h. Cells undergoing apoptosis were identified by flow cytometry (Fortessa, BD Biociences, San Jose, CA) using the FITC Annexin V Apoptosis Detection Kit I (BD Biociences).
Cells were seeded onto Ibidi µ-Slide 8 Well overnight and treated with or without verteporfin or CA3 for 48h, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton. Samples were incubated with Anti-YAP antibody (Cell signaling Technology, Leiden, The Netherlands). F-actin and nucleus were stained using respectively Alexa-fluor 488 phalloidin and DAPI. Images were acquired using a confocal microscope (NIKON A1 N-SIM) and processed using ImageJ.
RNA extraction and Real-time polymerase chain reaction
RNA was extracted from cells and tumors using NucleoSpin®RNAplus (Macherey Nagel, Duren, Germany) and reverse transcribed using the Maxima H minus first stand cDNA synthesis kit (Thermo Fisher, Courtaboeuf, France). Real-time monitoring of PCR amplification of complementary DNA was performed using DNA primers (primer sequences are available in Table 1) using QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher) with SYBR® Select Master Mix (Life Technologies, Carlsbad, CA). Target gene expression was normalized to glyceraldehyde 3-phosphatedehydrogenase (GAPDH) and β-actin (ACTB) levels in respective samples as an internal standard.
Western blot analysis
Equal amounts of total protein extracts (lysis buffer: SDS 1%, Tris pH 7.4 10mM, Sodium orthovanadate 1 mM). were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF Transfer membrane (Thermo Scientific, Illkirch, France). Antibodies used for western blotting were YAP1 (Proteintech, Manchester, UK), anti-Flag (Sigma Aldrich), HA-tag (Cell signaling), β-actin (Cell signaling), anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA), anti-rabbit IgG-HRP (Santa Cruz). Antibody binding was visualized with the enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Illkirch, France). For quantification, luminescence was detected with a Charge Couple Device (CCD) camera and analyzed using the GeneTools program (Syngene, Cambridge, United Kingdom).
HEK239FT cells were transfected with different vectors: pPGS-3HA-TEAD1, pCMV-Flag-YAP-S94A and pCMV-Flag-S127A-YAP were gifts from Kunliang Guan (Addgene plasmids #33055, #33102 and #27370; RRID: Addgene-33055, -33102 and -27370).
24 hours after transfection, media were changed with fresh DMEM containing 1% FCS for 24 hours. Transfected cells were then rinsed with ice-cold PBS and lysed in IP-lysis buffer (Invitrogen, Courtaboeuf, France). Equal amounts of proteins were precleared overnight at 4 °C using Protein-A/G-agarose (Santa Cruz Biotechnology, CA). Supernatants were incubated with primary antibody against Flag (Sigma Aldrich) and HA-tag (Cell signaling), for 2 h at 4 °C. 50 µL of Protein-A/G-agarose was then added and incubated overnight at 4 °C. Beads were washed three times with IP-lysis buffer; thereafter, 30 µL of Laemmli buffer was added and boiled for 5 min. After centrifugation, supernatants were harvested and processed for SDS-PAGE and Western blot as described above.
In situ proximity ligation assay (PLA), immunofluorescence and confocal microscopy
Duolink PLA ®: 5x103 OS cells were seeded in Ibidi µ-Slide VI 0.4. 24 later, media was changed to DMEM with 1% FBS. Cells were then fixed with 4% PFA for 15 min at room temperature and incubated overnight at 4 °C with primary antibody against YAP (Cell signaling or Santa Cruz Biotechnology), TEF-1 (Santa Cruz). In situ PLA was performed using DuoLink in Situ Reagents (Sigma-Aldrich) according to the manufacturer’s instructions.
Immunofluorescence assays: cells were seeded onto Ibidi µ-Slide 8 Well overnight, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton. Samples were incubated with Anti-Vinculin−FITC antibody (Sigma-Aldrich). F-actin and nucleus were stained using respectively Alexa-fluor 488 phalloidin and DAPI. Images were acquired using a confocal microscope (NIKON A1 N-SIM) and processed using ImageJ.
RNAseq analysis was performed by Active Motif (Carlsbad, CA). Differential gene expression analysis was performed using DESeq2 package. The p-values obtained were corrected for false positives by using Independent Hypothesis Weighting (package IHW) multiple testing adjustment method. Genes were considered significantly differentially expressed if log2 fold-change was over 1 or less than -1 and FDR was less than 0.05. For the differentially expressed genes, over-representation and gene set enrichment analysis (GSEA) were done using clusterProfiler package, and results were plotted using enrichPlot . GSEA was performed using GSEA software (http://software.broadinstitute.org/gsea/). Gene sets used are described in table 2.
Histogram and data are shown as mean +/- S.D. of a minimum of three independent experiments.
Statistical analyses were performed using GraphPad Prism version 6 for Windows (GraphPad Software, La Jolla, CA), www.graphpad.com. The Wilcoxon matched test was used to compare the expression levels between OS and matched normal tissue. The Mann-Whitney test was used to compare the difference between two groups. A p-value under or equal to 0.05 was considered statistically significant.
RNA sequencing data of OS patient and matched normal tissue were downloaded from the Gene Expression Omnibus database (GSE99671, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99671).
Kaplan Meier analysis of osteosarcoma patient tumor samples was performed using the R2 Genomics Analysis and Visualization Platform. Genome-wide gene expression analyses of high-grade osteosarcoma are from GSE42352 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE42352).