Role of the splicing factor SRSF4 in cisplatin-induced modifications of pre-mRNA splicing and apoptosis
- Maude Gabriel1Email author,
- Yves Delforge1,
- Adeline Deward2,
- Yvette Habraken2,
- Benoit Hennuy3,
- Jacques Piette2,
- Roscoe Klinck4,
- Benoit Chabot4,
- Alain Colige†1 and
- Charles Lambert†1
© Gabriel et al.; licensee BioMed Central. 2015
Received: 17 November 2014
Accepted: 25 March 2015
Published: 7 April 2015
Modification of splicing by chemotherapeutic drugs has usually been evaluated on a limited number of pre-mRNAs selected for their recognized or potential importance in cell proliferation or apoptosis. However, the pathways linking splicing alterations to the efficiency of cancer therapy remain unclear.
Next-generation sequencing was used to analyse the transcriptome of breast carcinoma cells treated by cisplatin. Pharmacological inhibitors, RNA interference, cells deficient in specific signalling pathways, RT-PCR and FACS analysis were used to investigate how the anti-cancer drug cisplatin affected alternative splicing and the cell death pathway.
We identified 717 splicing events affected by cisplatin, including 245 events involving cassette exons. Gene ontology analysis indicates that cell cycle, mRNA processing and pre-mRNA splicing were the main pathways affected. Importantly, the cisplatin–induced splicing alterations required class I PI3Ks P110β but not components such as ATM, ATR and p53 that are involved in the DNA damage response. The siRNA-mediated depletion of the splicing regulator SRSF4, but not SRSF6, expression abrogated many of the splicing alterations as well as cell death induced by cisplatin.
Many of the splicing alterations induced by cisplatin are caused by SRSF4 and they contribute to apoptosis in a process requires class I PI3K.
KeywordsCancer therapy Alternative splicing PI3K Apoptosis Drug efficiency Cisplatin SRSF4
Chemotherapy with platinum-based compounds is used extensively for the treatment of a wide range of solid tumours, including breast cancers resistant to first line therapy, ovarian, non-small cell lung, testis, endometrial, head and neck and colorectal cancers. Cisplatin (cis-diamine platinum (II) dichloride), the founding member of this class of agents, covalently binds to DNA and induces the formation of bulky DNA adducts consisting of intra-strand cross-links preferentially formed between adjacent guanine residues and, to a lower extent, inter-strand DNA lesions [1,2]. Cell toxicity is linked to these adducts that interfere with DNA replication and transcription. Intra-strand cross-links are mainly processed by removal of platinum adducts via the nucleotide excision repair, and inter-strand cross-links are removed via nucleotide excision repair, translesion polymerase and homologous recombination. Cisplatin activates various signalling pathways that include the DNA damage response (DDR) and the PI3K-Akt pathways . The DDR pathway detects and corrects DNA defects. However, when alterations are too numerous or too severe, cells are committed to death and eliminated. The DDR machinery relies on the activity of three enzymes that belong to the phosphatidyl inositol-3 kinases (PI3K) family: DNA-protein kinase (DNA-PK), Ataxia and Telangectasia Mutated (ATM) and Ataxia Telangiectasia and Rad 3-Related (ATR) . These kinases trigger specific and overlapping cascades of signalling events that result in cell cycle arrest, DNA repair or cell death .
Alternative splicing (AS) occurs in more than 90% of multi-exons primary transcripts [4,5]. Proteins produced through AS can have markedly different and sometimes opposite functions, as exemplified by a number of factors involved in apoptosis or cell survival . In other instances, AS controls the level of proteins by producing transcripts carrying premature termination codons that are degraded by non-sense mediated RNA decay (NMD) . Splicing decisions result from an interplay between highly degenerated cis-acting sequences and a large number of trans-acting factors that include the arginine- and serine-rich proteins (SR-proteins) and the heterogenous nuclear ribonucleoproteins (hnRNPs) families . The participation of these factors in splicing control is often regulated by post-translational modifications such as phosphorylation and acetylation which affect their localisation and their interaction with other proteins .
Aberrant AS occurs in cancer and a growing number of studies have reported a functional link between splicing anomalies and the evolution of the disease [9-12]. Several groups, including ours, have shown that chemotherapeutic drugs can affect the AS of a large number of transcripts [13-16]. However, the impact of these changes on the cancer cell is still poorly understood. Here, we analyse the transcriptome of cisplatin-treated cancer cells, and use AS changes to identify pathways that link cisplatin with the cellular response.
Cell culture, authentication, reagent and survival assay
MCF7, MDA-MB-231, HT1080, BT549, RD, HDF1 and HDF2, MG-63, MSU and AT5BIVA (deficient in ATM, Coriell Cell Repository, Camden, NJ, USA) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Verviers, Belgium) supplemented with non-essential amino-acids (NEAA) (1%), penicillin and streptomycine (1%), gentamycin (0.1%), fungizone (0.1%) and 10% FCS (Lonza). Ishikawa cells (human endometrial adenocarcinoma cell line) were cultured in RPMI 1640-glutamax (Lonza) supplemented with NEAA (1%), sodium pyruvate (1%), penicillin and streptomycine (1%), fungizone (0.1%) and 10% FCS, GM09607 cells (deficient in ATM, Coriell Cell Repository) in EMEM (Lonza) supplemented with 10% FCS and 1% NEAA, and MO59J cells (glioblastoma cell line, deficient in the catalytic subunit of DNA-PK) in DMEM/F12 supplemented as DMEM.
The study conforms to the principles outlined in the Declaration of Helsinki and was approved by the ethic committee of Liège University Hospital (B707201110973).
MCF7 and Ishikawa cells were authenticated by DSMZ (Braunschweig, Germany). Although no authentication of the other cell lines was made, the deficiency in ATM of GM09607 and AT5BIVA was ascertained by western blotting, and that of p53 in MG-63 was confirmed by RT-PCR.
Cisplatin (cis-diamine platinum (II) dichloride), wortmannin, caffein, and triciribine were from Sigma-Aldrich (St-Louis, MO, USA), oxaliplatin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), ATM kinase inhibitor from Calbiochem EMD biosciences (La Jolla, CA, USA), NU7026 from Merck Millipore (Darmstadt, Germany), TGX221, IC87114 and MK2206 from Selleckchem (Munich, Germany) and PX866 from LC Laboratories (Woburn, MA, USA).
Cell survival and apoptosis/necrosis were measured, respectively, by trypan blue exclusion in blind tests and by FACS analysis as described in .
RNA isolation, RT-PCR and RT-qPCR
RNAs were purified from cultured cells using the High Pure RNA isolation kit (Roche, Mannheim, Germany) and quantified by spectrometry. Gene expression was measured by RT-qPCR. Details according to the Minimum Information for Quantitative RT-PCR Experiment (MIQE) guidelines , are given in Additional files 1 and 2. For analysis of exon inclusion/exclusion, primers were chosen on exons surrounding the sequences potentially alternatively spliced. Primers, protocols and amplification products sizes are detailed in Additional file 2. Splice variants were discriminated by electrophoresis as described .
RNA sequencing analysis
RNA libraries and sequencing were performed on total RNA samples at the GIGA Genomics facility, University of Liège, Belgium. The quality of RNA was checked with BioAnalyser 2100 (Agilent technologies, CA, USA) that indicated a RQI score >8. The libraries were prepared with Truseq® mRNA Sample Prep kit (Illumina, CA, USA) from 1 microgram of total RNA following manufacturer’s instructions. mRNAs were isolated by poly-A selection and fragmented (8 minutes at 94°C). Fragmented mRNAs (around 170 nucleotide-long in average) were used for reverse-transcription in the presence of Superscript II (Invitrogen, Oregon, USA) and random primers. After second strand synthesis, end-repair, A-tailing and purification, the double strand cDNA fragments were ligated to Truseq® adapters containing the index sequences. Fifteen cycles of PCR in the presence of dedicated PCR primers and PCR master mix were applied to generate the final libraries. Libraries were sequenced in pair-end sequencing runs on the Illumina GAIIx in multiplexed 2 × 76 base protocols. The raw data was generated through CASAVA 1.6 suite (Illumina, CA, USA). TopHat (http://ccb.jhu.edu/software/tophat/index.shtml) software was used to align RNA-Seq reads to the reference genome (hg19, UCSA) and discover transcript splice sites. Cufflinks (http://cole-trapnelllab.github.io/cufflinks/) used the resulting alignment files to quantify the gene expression levels, identify up- and down-regulated transcripts and find the alternative splice junctions.
SpliceSeq(1.2) (http://bioinformatics.mdanderson.org/main/SpliceSeq:Overview) was used for a focused AS analysis. Using alignment database and Bowtie, SpliceSeq aligns reads from RNA-Seq data to a reference collection of splice variants [19,20].
Lists of genes modulated in term of expression and splicing were imported in the ToppGene Suite for analysis .
Antibodies and Western blotting
Antibodies directed against Akt, phospho-Akt (ser473) and β-actin were purchased from Cell Signalling (Beverly, MA, USA). Cells were lysed in Laemmli buffer containing 50 mM DTT. Lysates were briefly sonicated, incubated at 65°C for 15 min and analyzed by SDS-PAGE. Proteins were electroblotted and detected as described in . Probing of β-actin was performed as a control of protein loading.
SMARTpool siGENOME (Dharmacon by Thermo Fisher Scientific, Lafayette, CO, USA), consisting of four siRNA duplexes, were used to target SRSF4 and SRSF6 mRNA. siRNA targeting ATR were from Ambion (Life technologies). The 5′-UUGCAUACAGGACUCGUUATT-3′ and 5′-UAACGAGUCCUGUAUGCAATT-3′ oligoribonucleotides were used as control siRNA (siSCR) that does not target any known human transcript . Cells were transfected by siRNAs as previously described .
The means and standard deviation were calculated from three or four independent experiments. The significance of differences was determined using t-test or ratio paired t-test of Student.
Cisplatin alters alternative splicing
Poly A+ RNA from MCF7 cells untreated or treated with 50 μM cisplatin for 24 hours was isolated and prepared for next-generation sequencing analysis. No significant cell death over untreated samples was noted in these conditions (as measured by trypan blue exclusion). The average number of reads approached or exceeded 20 millions in both samples. Alignment of transcripts to the genome indicated that 16733 and 16969 genes were expressed in the control and cisplatin-treated samples, respectively. Sequencing data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2663. The global gene expression in the two conditions was highly correlated, with a Pearson correlation coefficient = 0.835 (p = 0.000000).
Effect of cisplatin on gene expression
Top twenty up- and down-regulated genes by cisplatin in MCF7 cells
serpin peptidase inhibitor, Clade B (Ovalbumin), member 5
POU class 3 homeobox 1
NK1 homeobox 2
lysosomal-associated membrane protein 3
activating transcription factor 3
growth arrest and DNA-damage-inducible, alpha
heparin-binding EGF-like growth factor
hairy and enhancer of split 2 (Drosophila)
nerve growth factor receptor
snail family zinc finger 1
G protein-coupled receptor 3
solute carrier family 52, riboflavin transporter, member 1
platelet-activating factor receptor
proline dehydrogenase (oxidase) 1
chromosome 13 open reading frame, human
phorbol-12-myristate-13-acetate-induced protein 1
huntingtin-associated protein 1
TNF receptor superfamily member 6
guanylate cyclase activator 1B (retina)
leukemia inhibitory factor
roundabout, axon guidance receptor, homolog 1
neuronal growth regulator 1
eyes absent homolog 4
Ca++ − dependent secretion activator 2
solute carrier organic anion transporter family, member 3A1
sterile alpha motif domain containing 12
nuclear factor I/A
membrane associated guanylate kinase, WW and PDZ domain containing 1
heparan sulfate 6-O-sulfotransferase 3
phospholipase C, eta 1
protein phosphatase 1, regulatory subunit 9A
potassium inwardly-rectifying channel, subfamily J, member 8
myeloid/lymphoid or mixed-lineage leukemia translocated to, 3
plexin domain containing 2
v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian)
latent transforming growth factor beta binding protein 1
T-cell lymphoma invasion and metastasis 1
Significantly enriched biological processes affected by cisplatin
Modulated genes in the treated cells
Total genes in the pathway
Enzyme linked receptor protein signaling pathway
Regulation of cell proliferation
Regulation of cell cycle
Negative regulation of cell cycle
Cell cycle process
mRNA metabolic process
RNA splicing, via transesterification reactions
Nuclear mRNA splicing, via spliceosome
RNA splicing, via transesterification reactions with bulged adenosine as nucleophile
Genes regulated by cisplatin and matching annotations for transcription factors E2F
Cisplatin regulates the expression of tumor suppressors and oncogenes
Effect of cisplatin on post-transcriptional events
Potential modifications of splicing by cisplatin were investigated from the RNA-seq data. The SpliceSeq software identified 717 AS events occurring in 619 primary transcripts (Additional file 4). Only 5 genes (UGDH, SLC38A1, RETSAT, PDE8A, NASP) (0.44%) were affected simultaneously at transcriptional and post-transcriptional levels. Changes in splicing were grouped based on the type of events being affected: 79 changes involved cassette exon inclusion events, 166 were cassette exon exclusion events (of which 49% were not annotated as alternative exons in NCBI), 243 changes affected alternative 5′ or 3′ splice site selection events, 144 involved alternative promoters, 83 indicated alternative terminations and 2 were splicing changes attributed to mutually exclusive exon. Significantly affected biological processes identified by ToppFun Suite software on affected genes were “RNA splicing and processing” and “cell cycle” (Table 2).
PI3K pathway, but not DNA damage response and p53, is involved in the alteration of splicing by cisplatin
Cisplatin-induced alteration of splicing involves SRSF4
Cisplatin-induced cell death involves SRSF4
The development of chemotherapeutic agents has enabled tremendous progress in cancer therapy. However, the success of these treatments is offset by the development of drug resistance and by toxic side-effects on healthy cells and tissues. The development of this resistance is encouraged by several processes, including decreased access and increased efflux of the drug from the tumor, altered expression of oncogenes, reduced apoptosis and increased DNA repair . In order to evaluate the role of AS in the efficiency of cisplatin, we performed a transcriptome analysis of breast cancer cell line because platinum-based chemotherapy is used as second and third-line of treatment against resistant metastatic breast cancer [28,29]. Moreover, MCF7 cells are well-characterized notably in terms of their response to chemotherapeutic drugs. Our results indicate that cisplatin affects the expression level (absolute fold change >2) of more than 500 genes and provokes changes in at least 700 splicing events, thereby extending previous observations that chemotherapeutic agents affect AS [6,13,16]. This splicing reprogramming also occurs in other transformed cell lines including the breast cancer cell lines MDA-MB-231 and BT549, the endometrial adenocarcinoma cell line Ishikawa and in primary fibroblasts.
Many of the genes whose expression is altered by cisplatin have functions in cell cycle. Cisplatin-induced changes also affect the expression of tumor suppressor genes, oncogenes and genes involved in determining cell fate (Table 4). Strikingly, the list lacks genes encoding splicing factors, suggesting that the impact on splicing control principally stems from post-transcriptional and/or post-translational events affecting their expression, localization and activity. In contrast, cisplatin affected the AS of many splicing factors. Accordingly, our gene ontology analysis suggests that splicing function may be one of the pathways most affected by cisplatin.
We observed that other chemotherapeutic drugs, namely camptothecin and doxorubicin, induce the same changes in AS as those elicited by cisplatin (unpublished work). As these drugs all induce DNA damage, it is tempting to speculate that activation of the DDR pathway may be involved in promoting these splicing alterations. In contrast to this prediction, the genetic depletion and/or the specific inhibition of p53, ATM, ATR and DNA-PK failed to suppress AS re-programming upon cisplatin treatment. These data contrast with those of Shkreta et al.  who observed that the shift in Bcl-x splicing induced by oxaliplatin or cisplatin in HEK-293 cells was abrogated by inhibiting ATM, ATR or p53. However, no significant change in Bcl-x splicing by cisplatin was recorded here by deep sequencing or RT-PCR in MCF7 cells (not illustrated), consistent with the very small shift previously observed in MCF7 cells . These discrepancies may be related to the different cell lines used, which may display different thresholds to elicit the DNA damage response.
Previous reports indicate that the PI3K/Akt axis can affect the AS of many primary transcripts at least in part by activation of SRPK and the phosphorylation of SR proteins [31-33]. We investigated the role of this pathway in the AS changes induced by cisplatin by using a panel of inhibitors. Our results indicate that cisplatin alters AS in a process that requires the PI3K subunit p110β. The link between p110β and the splicing events altered by cisplatin remains unclear but is independent of Akt. An intriguing possibility is that cisplatin affects the nuclear activity of p110β, which in turn may directly affect the activity of splicing factors. A role for p110β is not totally unexpected since there is mounting evidence indicating that nuclear lipids can regulate nuclear functions including splicing [34,35]. While phosphoinositides associate with nuclear membranes, they also co-localize in nuclear speckles  and interact with various proteins or ribonucleoprotein complexes including the spliceosome components U2 snRNP, U4/U6 snRNP and SF3A1.
We observed that knocking down SRSF4, but not SRSF6, abrogated the cisplatin-induced changes in splicing. CLIP analysis followed by high-throughput sequencing identified GA rich pentamers with G/AAAA/GA sequence as a consensus motif for the binding of SRSF4 to RNA . Moreover, SRSF4 preferentially binds to exons, with a peak of binding ~50 nucleotides upstream of the 5′ splice site. Sequences matching with these sequences are observed in the exons that were skipped in response to cisplatin. However, that they represent binding sites for SRSF4 remains to be tested.
Although SRSF4 may also have an indirect function, for example by regulating the splicing of other splicing factors, we believe that this scenario is unlikely to explain the rapid changes in the steady state levels of splice variants imposed by cisplatin. Nevertheless, portions of the RS-rich regions of SRSF3 and SRSF7 are truncated due to exon skipping (SRSF7) or alternative termination (SRSF3) in response to cisplatin treatment, thereby possibly affecting the phosphorylation of these proteins and their association with other splicing partners.
A link between altered splicing and the efficacy of cancer treatment is suggested by several findings. In lymphocytes of patients with chronic lymphocytic leukemia, mutations in the gene encoding the splicing factor SF3B1 are more frequent after treatment, suggesting a chemotherapy-driven clonal selection for cells being affected in splicing [38,39]. The efficacy of chemotherapetic agents may act at least in part through reprogrammation of AS Consistent with this view, treatments of human 293 cell line with a panel of chemotherapeutic agents induced splicing shifts that encouraged the production of pro-apoptotic variants of Bcl-x, caspase-9 and survivin . Moreover, altering the ratio of splice variants of caspase-9 reduced the resistance of non-small lung cancer cells to various chemotherapeutical agents . On the contrary, splicing switches toward anti-apoptotic versions, as in the conversion from FAS to sFAS, have also been observed ( and personal observation). As high sFAS levels correlate with poor survival in patients with T-cell leukemia and gynecological malignancies [41,42], sFAS may contribute to the acquisition of drug resistance and a chemotherapy designed to revert splicing to FAS may increase treatment efficiency .
GO terms related to apoptosis were not highlighted by hierarchization analysis of the transcripts alternatively spliced upon cisplatin. We compared a list of transcripts related to apoptosis (GSEA [44,45]) with the list of transcripts with splicing affected by cisplatin treatment. Twenty-six actors involved in the regulation of apoptosis were common to both lists, as for example BAX, caspase-6, caspase-8 (pro-apoptotic) and MADD, API5 (anti-apoptotic). These examples illustrate that cisplatin-induced alterations of splicing may have both anti- and pro-apoptotic effects, and the net effect cannot be estimated on a theoretical basis.
Here, we observed that knocking down SRSF4 reduced the impact of cisplatin on cell death, suggesting an overall therapeutic benefit associated with the expression of SRSF4. Thus, while the pharmacological alterations of splicing induced by chemotherapic agents may fuel therapeutic efficiency, preventing these alterations by inhibiting SRSF4-regulated splicing may help cells to resist the cisplatin treatment. This situation is likely to be more complex given the large number of splicing regulators, their combinatorial mode of regulation and the diversity of their targets. A growing list of pharmacological agents that can modulate splicing is now emerging, with some demonstrating anti-tumor activity [46-48]. Pladienolide, spliceostatin and herboxidiene modulate the function of the spliceosome by binding to the SF3B core component protein [49,50]. A link between splicing alterations and inhibition of cancer cell proliferation was established , supporting the concept of using splicing to improve anti-cancer therapy. Another example is provided by the anti-hypertensive agent amiloride that also affects the level and/or the phosphorylation of splicing factors, alters the splicing of cancer genes in various tumor cell lines and sensitizes chronic myelogenous leukemia cells to imatinib . Similarly, dietary agents possessing anticancer activities as curcumin, resveratrol and epigallocatechin-gallate, have been shown to affect splicing, at least in part through modulation of splicing factors levels [52-55].
We showed that the reprogramming of splicing induced by cisplatin makes a large contribution to its anti-cancer property, and that its action requires class I PI3K p110β and the splicing factor SRSF4. In this context, our data have two major implications. They suggest that pharmacologically modulating AS can potentially affect the success of chemotherapy. Moreover, they raise the interesting possibility that molecules or conditions (as drugs used for non-tumoral diseases, food components and redox status) that modify AS may influence the response to anti-cancer treatments.
We thank Dr. Michael C. Ryan (In Silico Solutions, Fairfax, VA) for the help with the SpliceSeq® software. The assistance of Raafat Stefan (Cell Imaging and Flow cytometry platform, GIGA, University of Liège, Belgium) with FACS analysis is acknowledged. We thank Pr. Betty Nusgens for her careful review of the manuscript. B.C. is the Canada Research Chair in Functional Genomics. This work was supported by a grant from the National Fund for Scientific Research, Belgium (F.N.R.S-Télévie, # 7.4634.10), from Belspo, from Research Concerted Action (# ARC 10/15-02) and the Fonds Léon Frédericq of the University of Liège, Belgium, and from the Canadian Institutes of Health Research (MOP136948 to B.C.).
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