Cells lines

Pancreatic cell lines PANC-1 and MIA PaCa-2, glioblastoma cell lines T98 and U87, colorectal carcinoma cells HCT-116 and DLD-1 and embryonic kidney cell lines HEK293, 293T and RPE (retinal pigment epithelial) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). RWP-1 and CP15 cells were derived from human pancreatic adenocarcinoma biopsies perpetuated as xenograft in nude mice37,38. Non-tumour fibroblasts were kindly provided by Dr Eva Vaquero (Institut d’investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain). RWP-1, CP15, PANC-1, MIA PaCa-2, T98, U87, fibroblasts and RPE cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco-BRL, Carlsbad, CA, USA). DLD-1 and HCT-116 cells were maintained in RPMI-1640 (Gibco-BRL) or McCoy’s 5A (Gibco-BRL) medium, respectively, supplemented with 10% fetal bovine serum. Immortal human pancreatic duct epithelial HPDE cells, kindly provided by Dr F.X. Real (CNIO, Madrid, Spain), were cultured and maintained as reported39. Human hepatocytes were obtained from Biopredic International (St Gregoire, France) and maintained according to the manufacturer’s instructions. RWP-1 shNT, sh2 and sh4 were described previously14. HCT-116 and T98 shNT and sh4 cells were generated by transducing parental cells with shNT- and sh4-expressing lentiviruses14. HPDE-CPEB4-expressing cells were established by transducing parental cells with CPEB4 recombinant lentivirus. Mycoplasma contamination was tested by PCR routinely.

Lentiviral constructs with a modified 3′-UTR

The polyadenylation sequences (4,193–4,199 and 4,423–4,429) from the pLS-CG lentiviral vector (Addgene; no. 12161) were mutagenized with the help of the QuickChange II Site-Directed Mutagenesis Kit (Stratagene, Wilmington, NC, USA) according to the manufacturer’s instructions (see Supplementary Table 1 for primers). Mutagenized sequences were verified by DNA sequencing.

d2EGFP and d2RFP genes were cloned by PCR with specific primers incorporating the AgeI and XhoI restriction sites. The 3′-UTR of tPA was PCR amplified using specific primers with sequences for the XhoI restriction enzyme at both ends. The 3′-UTR of cB1 was designed in the reverse primer of d2EGFP gene with a sequence for the XhoI restriction enzyme. The 3′-UTR with the TNF-α ARE sequences was generated by amplifying the previously generated cB1 3′-UTR with a primer containing the TNF-α main ARE sequence upstream of the CPE sites.

All primer sequences are listed in Supplementary Table 1. The indicated sequences were cloned into the pLS-CG lentiviral vector. The newly generated vectors were sequenced to confirm the DNA sequence.

Lentiviral production

Lentiviral particles were generated by cotransfection of plasmids pCMVAR8.91, pVSV-G and the pLS-CG-derived constructs, or a lenitiviral plasmid expressing CPEB4, in 293T cells by the calcium/phosphate DNA precipitation method (Clontech, Mountain View, CA, USA). Supernatants from 293T-transfected cells were collected at 24 and 48 h, filtered and processed for purification by ultracentrifugation for 2 h at 12 °C at 20,200 r.p.m. After ultracentrifugation, the pellet was resuspended in PBS for 16 h at 4 °C under constant agitation, aliquoted and frozen at –80 °C for later use.

Viral titration was performed by quantitative PCR (qPCR) with specific primers against the PBC-Psi region of the pLS-CG vector (listed in Supplementary Table 1). A standard curve was generated using serial dilutions of pLS-CG-d2EGFP-3′-UTR_tPA construct mixed with genomic DNA. The number of molecules was calculated using the formula: M=(C·6.02 × 1023)/(660·bp), where C is the concentration of the plasmid, bp the number of base pairs and M the number of vector molecules.

Flow cytometry assay

Flow cytometry assays were performed in HPDE, RWP-1, PANC-1 and MIA PaCa-2 cells. Forty thousand cells were plated per well in 12-multiwell plates and, 24 h postseeding, cells were transduced with the indicated lentiviruses at 12 multiplicity of infection per virus. Transduction was facilitated by spinning for 2 h at 12,000 r.p.m. Two days post-transduction, d2EGFP and dRFP levels were analysed by flow cytometry using BD LSR II (Becton Dickinson). Flow cytometry results were analysed using FlowJo 8.7 for Macintosh.

Adenovirus generation

The AdCPE genome was generated by the following steps: first, the E1A polyA sequence of the pEND-K plasmid was mutated from 5′-AATAAA-3′ to 5′-ACTCGA-3′, generating a new XhoI restriction site with the primer 5′-GCTGAATGAGATTGATGTAAGTTTACTCGAGGGTGAGATAATGTTTAACTTGC-3′ using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene, Wilmington, NC, USA). Second, the construct containing E1A and the 3′-UTR of cB1 with the three CPE sequences was generated directly by PCR, by amplifying Adwt E1A with the primers 5′-CCTTGGGTCCGGTTTCTATGCC-3′ and 5′-CGTCTCGAGGCTTTATTAAAACCAGTAAAACATTAAAAACACAATACACTATTTACAGAAGCACATGGTGCAACACTTATGGCCTGGGGCGTTTACAGC-3′. Third, the E1A from the pEND-K with the mutated polyA sequence was replaced by the PCR product, using AgeI and XhoI restriction sites, to generate pEND-K-E1A-cB1. Finally, AdCPE was generated by homologous recombination of pEND-K-E1A-cB1 with the genome of the serotype 5 wild-type adenovirus in E. coli BJ5183 cells as described40.

Adwt was obtained from ATCC. AduPAR was described previously40. AdDUC genome was generated by incorporating the CPE containing 3′-UTR of E1A into the BoxI and AflII restriction sites of the pSH-DM-UPAR-E1A plasmid followed by recombination of the resulting plasmid with the Adwt genome in BJ5183 cells as described previously40.

Adwt, AdCPE, AduPAR and AdDUC were propagated in A549 cells and purified by cesium chloride banding. The concentration of viral particles (vp ml−1) was determined by means of optical density, and infectious particles (pfu ml−1) were determined by hexon immunostaining in HEK293 cells41. Both viruses presented equal vp per pfu ratio.

Western blot analysis

Protein extracts were obtained with lysis buffer (50 mM Tris-HCl at pH 6.8, 2% SDS) containing 1% Complete Mini Protease Inhibitor (Roche Diagnostics, Basel, Switzerland). BCA Protein Assay Kit (Pierce-Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration, and total proteins (35 μg) were resolved by electrophoresis on 7.5% gels and transferred to nitrocellulose membranes by standard methods. Membranes were immunoblotted with rabbit anti-adenovirus-2/5 E1A polyclonal antibody (1:200; clone 13S-5; Santa Cruz Biotechnology, Dallas, TX, USA) or anti-CPEB4 antibody (1:200; Abcam, Cambridge, UK) or anti-CPEB1 (1:200; 13274-1-AP; ProteinTech, IL, USA) 1 h at room temperature. Blots were rinsed with TBS-T and incubated for 45 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (DakoCytomation, Glostrup, Denmark). Antibody labelling was detected by the enhanced chemiluminescent method (Amersham Biosciences, Amersham, UK). Western blot expression data for E1A, CPEB1 and CPEB4 were normalized to glyceraldehyde 3-phosphate dehydrogenase. Uncropped scans of western blots presented in the main figures are provided in Supplementary Fig. 11.

cDNA synthesis and real-time qPCR

RNA was obtained and isolated using RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). A total of 1 μg was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random decamers (Ambion, Carlsbad, CA, USA). One microlitre of the reaction was used as a template for the qPCR amplification reaction (LightCycler 480SYBER Green I Master Mix; Roche Diagnostics) in a thermocycler (ViiA 7 Real-Time PCR System; Applied Biosystems), using the following set of primers: E1A Fw, 5′-CGGCCATTTCTTCGGTAATA-3′ and E1A Rev, 5′-CCTCCGGTGATAATGACAAG-3′; Hexon Fw, 5′- GTCTACTTCGTCTTCGTTGTC-3′ and Hexon Rev, 5′-TGGCTTCCACGTACTTTG-3′; and Fibre Fw, 5′-CTCCAACTGTGCCTTTTC-3′ and Fibre Rv, 5′-GGCTCACAGTGGTTACATT-3′. Quantitative expression data were normalized to Gdx Fw, 5′-GGCAGCTGATCTCCAAAGTCCTGG-3′ and Gdx Rev, 5′-AACGTTCGATGTCATCCAGTGTTA-3′. d2EGFP and dRFP were detected with the primers: d2EGFP Fw, 5′-CAACAGCCACAACGTCTATATCAT-3′ and d2EGFP Rv, 5′-ATGTTGTGGCGGATCTTGAAG-3′; and dRFP Fw, 5′-GCCCTTCGCCTGGGACAT-3′ and dRFP Rv, 5′-GGTGCTTCACGTACACCTTGGA-3′. Quantitative expression data were normalized using the primers ACTB Fw, 5′-CTGGAACGGTGAAGGTGACA-3′ and ACTB Rv, 5′-GGGAGAGGACTGGGCCATT-3′.

RNA-immunoprecipitation and RT–qPCR

RPE cells were transfected at 80% confluence with 25 μg of pCMV-lucRenilla plasmid (cB1-3′-UTR WT or cB1-3′-UTR Mut)27 using Lipofectamine LTX and PLUS Reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h. After transfection, cells were crosslinked with 0.5% formaldehyde in free DMEM for 5 min at room temperature. Then, cells were lysed with RIPA buffer supplemented with EDTA-free protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO, USA) and 200 U ml−1 RiboLock RNAse Inhibitor (Thermo Fisher Scientific). Cell lysates were sonicated for 5 min at low intensity and centrifuged for 30 min at 13,200 r.p.m. and at 4 °C. Eight hundred micrograms of lysates were precleared with 20 ml of Dynabeads protein A (Thermo Fisher Scientific) for 30 min at 4 °C. Finally, they were incubated with 5 mg of anti-CPEB4 antibody (Abcam; ab83009) or anti-HuR antibody (Santa Cruz Biotechnology, sc-5261) or anti-immunoglobulin G antibody (Sigma-Aldrich) coupled to 50 μl of Dynabeads protein A overnight at 4 °C. One-fourth of the immunoprecipitates were eluted with Laemmli sample buffer by heating at 65 °C for 20 min, resolved in SDS–polyacrylamide gel electrophoresis and analysed by western blotting. Three-fourth of remaining immunoprecipitates were digested with proteinase K during 1 h at 65 °C, and the RNA was isolated by phenol extraction. All the RNAs were treated with DNAse (Thermo Fisher Scientific) and retrotranscribed with random hexamers and Oligo d(T)20, using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and subjected to qPCR. The resulting cDNAs were used for gene-specific qPCR, using the following primers: pLucORF Fw, 5′-ACTGGGACGAAGACGAACAC-3′; pLucORF Rv, 5′-GGCGACGTAATCCACGATCT-3′; Renilla S, 5′-GATAACTGGTCCGCAGTGGT-3′; Renilla AS, 5′-ACCAGATTTGCCTGATTTGC-3′. Fold enrichment of luciferase mRNA in the immunoprecipitated fraction was calculated after normalization with the gene expression from the inputs.

Viral genome quantification

Viral DNA was obtained from supernatants, cellular extracts or frozen tissues using the UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. Viral genomes were determined by real-time qPCR using the SYBER Green I Master plus mix (Roche Diagnostics) and the primers Hexon Fw, 5′-GCCGCAGTGGTCTTACATGCACATC-3′ and Hexon Rv, 5′-CAGCACGCCGCGGATGTCAAAG-3′. Adenoviral copy number was relativized to the cellular DNA content using the albumin intron 12 primers of Fw, 5′-CTGTCATCTCTTGTGGGCTGT-3′ and Rv, 5′-GGCTATCCAAACTCATGGGAG-3′.

In vitro cell survival studies

Dose–response curves were constructed for all assessed cells transduced with doses ranging from 0.001 vp per cell to 10,000 vp per cell of Adwt or AdCPE. Cell viability was measured 3 days postinfection by a colorimetric assay following the manufacturer’s instructions (MTT Ultrapure; USB, Cleveland, OH, USA).

Polyadenylation assays

Polyadenylation patterns were evaluated using a modified version of the RNA ligation-coupled RT–PCR presented previously42. One microgram of total RNA was ligated to 0.1 μg of P1 anchor primer (5′-P-GGTCACCTTGATCTCAAGC-NH2-3′) in 10 μl reaction using T4 RNA ligase I (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Half of the reaction product was used in a 50 μl reverse transcription reaction with PrimeScript RT–PCR (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s instructions, using 0.1 μg of P1′ as a reverse primer (5′-GCTTCAGATCAAGGTGACCTTTTT-3′). An aliquot (2.5 μl) of this cDNA preparation was used for a first pre-PCR step with Fw-E1A-polyA primer (5′-GGTGTAAACCTGTGATTGCG-3′). One microlitre of this reaction was later used in each 25 μl PCR with the primers Fw-E1A-polyA and P1′.

Toxicity analysis

PBS or 2 × 1010 vp of Adwt or AdCPE were injected intravenously into the tail vein of 6- to 8-week-old male immunocompetent C57BL/6 mice. Animals were weighed and examined daily for any clinical signs of toxicity. Three days later, mice were killed, organs were isolated and blood samples were collected by intracardiac puncture. Serum aspartate aminotransferase and alanine aminotransferase were determined on an Olympus AU400 Analyser (Olympus, Tokyo, Japan) at the Clinical Biochemistry Service, School of Veterinary Medicine, Autonomous University of Barcelona. All animal procedures met the guidelines of European Community Directive 86/609/EEC and were approved by the ethical committee (CEEA-University of Barcelona) and by the local authorities of the Generalitat de Catalunya.

Mouse xenografts

RWP-1, MIA PaCa-2 and PANC-1 cells (2.5 × 106), embedded in Matrigel 1:1 (BD Biosciences, San Jose, CA, USA), were subcutaneously injected into each flank of male, 7- to 8-week-old, athymic nu/nu mice (Harlan, Sant Feliu de Codines, Spain). Tumours were measured at least three times weekly, and their volumes were calculated using the formula V=larger diameter × (smaller diameter)2 × pi÷6. Mice were randomly assigned to either group for treatment. Virus was administered once tumours achieved a median volume of 100 mm3. The experimenter was blinded until the conclusion of the study.

Statistical analyses

The descriptive statistical analysis was performed on GraphPad Prism v5.0a (GraphPad Software, La Jolla, CA, USA). Statistical differences were evaluated using nonparametric two-tailed Mann–Whitney test. P<0.05 was taken as the level of significance.

Statistical analyses for the d2EGFP/dRFP quantification were performed using R (version 2.10.0) and the ARM package, to perform the mixed model by REML and multcomp package for multiple comparison analysis of means by the Tukey–Kramer test.

Sample size calculation for animal studies took into consideration to have more than five mice in each group.

The in vivo tumour growth statistical analysis was evaluated using R v2.14.1 software (R: a language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria) with a linear mixed-effect model using the lme4 package. We associated a random-effects term with the day of measurement43. Statistical differences were evaluated using a multiple comparison of means by Tukey’s contrasts.

Data availability

All relevant data are available from the authors on reasonable request.