srsf1 regulates the alternative splicing of caspase 9 via a novel intronic splicing ... · cell...

Cell Cycle, Cell Death, and Senescence

SRSF1 Regulates the Alternative Splicing of Caspase 9 Via ANovel Intronic Splicing Enhancer Affecting the ChemotherapeuticSensitivity of Non–Small Cell Lung Cancer Cells

Jacqueline C. Shultz1, Rachel W. Goehe1, Charuta S. Murudkar1, Dayanjan S. Wijesinghe1,Eric K. Mayton1, Autumn Massiello1, Amy J. Hawkins2, Prabhat Mukerjee1, Ryan L. Pinkerman5,Margaret A. Park1, and Charles E. Chalfant1,3,4

AbstractIncreasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology

with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlyingmolecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence ofcaspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNAsplicing. This analysis revealed 13 possible RNA cis-elements for interaction with SRSF1 with mutagenesis of theseRNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of theinclusion of the 4 exon cassette. To further determine the biological importance of this mechanism, we employedRNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted inan increase in the IC50 of non–small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. Incontrast, downregulation of caspase 9b induced a decrease in the IC50 of these chemotherapeutic drugs. Finally,these studies showed that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combinationtherapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer thatregulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we showed that the alternativesplicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.Mol Cancer Res;9(7); 889–900. �2011 AACR.

Introduction

Historically, the regulation of the alternative splicing ofcaspase 9 by signaling pathways began with the lipidsecond messenger, ceramide. This lipid second messengeris an important regulator of various stress responses andgrowth mechanisms including apoptosis, and the forma-tion of ceramide from the hydrolysis of sphingomyelin or

from de novo pathways has been observed in response tonumerous apoptotic and stress agonists including che-motherapeutic agents (1–7). On mechanism by whichceramide imparts these biological actions is via a family ofceramide-regulated enzymes designated, ceramide-acti-vated protein phosphatases, which include the serine/threonine-specific protein phosphatases PP1 and PP2A(8–11). Specific substrates of PP1 are the SR proteins (12),a family of arginine/serine-rich domain containing pro-teins that are major players in both constitutive andalternative pre-mRNA processing. Endogenous ceramidehas been found to modulate the phosphorylation status ofSR proteins in a PP1-dependent manner leading to depho-sphorylation of the RS-domain (13). In regard to caspase 9RNA splicing, a pathway linking the generation of de novoceramide and the activation of PP1 to the regulation of theexclusion or inclusion of an exon 3,4,5,6 cassette ofcaspase 9 pre-mRNA was identified by our laboratory(14). Specifically, ceramide treatment resulted in anincrease in the pro-apoptotic caspase 9a MRNA mRNAand protein levels with concomitant decrease in caspase 9b(cassette exclusion) mRNA and protein levels in A549cells (14). This effect required the generation of endo-genous ceramide through the de novo pathway, and

Authors' Affiliations: Departments of 1Biochemistry and 2Human Genet-ics, 3The Massey Cancer Center, Virginia Commonwealth University;4Research and Development, Hunter Holmes McGuire Veterans Admin-istration Medical Center, Richmond, Virginia; and 5Department of Bio-chemistry and Molecular Biology, Medical University of South Carolina,Charleston, South Carolina

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

J.C. Shultz and R.W. Goehe authors contributed equally to the work.

Corresponding Author:Charles E. Chalfant, Department of Biochemistry,Room 2-016, Sanger Hall, Virginia Commonwealth University—Schoolof Medicine, 1101 East Marshall Street, P.O. Box 980614, Richmond,VA 23298-0614. Phone: (804) 828-9526; Fax: (804) 828-1473; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-11-0061

�2011 American Association for Cancer Research.

MolecularCancer

Research

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Published OnlineFirst May 26, 2011; DOI: 10.1158/1541-7786.MCR-11-0061

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more importantly, inhibitors of protein phosphatase-1abolished the ability of ceramide to affect the alternativesplicing of caspase 9 (14). Thus, both the phospho-state ofSR proteins and the alternative splicing of caspase 9 areregulated by the generation of de novo ceramide andsubsequent PP1 activation (14).The involvement of PP1 and endogenous ceramide in the

dephosphorylation of SR proteins and the effects on caspase9 alternative splicing suggested that at least 1 SR proteinisoform regulated the alternative splicing mechanism ofcaspase 9. Indeed, in further mechanistic studies, ourlaboratory identified 1 SR protein, SRSF1 (also known asSRp30a), as a critical splicing factor in the alternativesplicing of caspase 9 pre-mRNA in A549 lung adenocarci-noma cells (15). Furthermore, we showed that SRSF1 is arequired RNA trans-acting factor for ceramide to affect thealternative splicing of caspase 9 pre-mRNA, thereby linkingde novo ceramide generation, SRSF1, and the regulation ofcaspase 9 expression. More recently, our laboratory showedthat the phospho-status of SRSF1 on serine199, 210, 227, and234 mediated the suppression of the exon 3,4,5,6 cassette,suggesting that ceramide activation of PP1 leads to depho-sphorylation of these residues.The linking of ceramide signaling to caspase 9 RNA

splicing has significant relevance to cancer therapeutics asstudies have shown that the ectopic expression of caspase9b confers the opposite effect on apoptosis as full-lengthcaspase 9 (caspase 9a); by inducing resistance to manyapoptotic stimuli (e.g., FAS ligand and UV radiation; refs.16–18). Caspase 9b elicits this biological outcome at leastin part by competing with the full length caspase 9 forbinding to the apoptosome (e.g., APAF-1; refs. 17, 18).Therefore, regulation of the inclusion of this 4 exoncassette is a critical determinant to decide whether a cellis susceptible or resistant to apoptosis (17, 18). Recently,our laboratory expanded upon both the mechanisticregulation of caspase 9 RNA splicing and the sensitivityof non–small cell lung cancer (NSCLC) cells to thechemotherapeutic agent, erlotinib. Indeed, our laboratoryfound that the erlotinib induced an increase in the caspase9a/9b ratio in NSCLC cells, which was important for thesensitivity of these cells to this chemotherapeutic agent(19). Therefore, the alternative splicing of caspase 9 has arole in the mechanism by which a clinically relevanttherapy affects NSCLC cells.In the presented study, we delve further into both the

underlying molecular mechanisms that regulate the alter-native splicing of caspase 9 as well as the biological relevanceof this distal mechanism in the treatment of NSCLC.Specifically, we identify a novel and strong intronic splicingenhancer (C9-I6/ISE) for caspase 9 pre-mRNA and showthat SRSF1 specifically interacts with this ISE. Further-more, SRSF1 was shown to regulate the alternative splicingof caspase 9 via this novel RNA cis-element. Finally, weshowed that modulation of the alternative splicing ofcaspase 9 regulates the synergistic effects of daunorubicin(DNR) and erlotinib, suggesting that this molecular splicingmechanism is a relevant therapeutic target for NSCLC.

Material and Methods

Cell cultureA549 cells were grown in 50%RPMI 1640 (BioWhittaker

Cambrex Bio Science Walkersville, Inc.) and 50% DMEMsupplemented with L-glutamine (Invitrogen), 10% (v/v)FBS (Invitrogen), 100 units/mL penicillin, and 100 mg/mL streptomycin (BioWhittaker Penicillin/Streptomycin,Cambrex Bio Science Walkersville, Inc.). H2030, H838,and HeLa cell lines were cultured with 100% RPMI 1640supplemented with L-glutamine, 10% (v/v) FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin. HBEC-3KTcells were cultured with keratinocyte serum-free mediumcontaining bovine pituitary extract and recombinant epider-mal growth factor (Life Technologies). Cells were main-tained at less than 80% confluence under standard incubatorconditions (humidified atmosphere, 95% air, 5% CO2,37�C). For transfection, cells were plated at 2.5 � 105

cells/35-mm plate in appropriate culture media. The che-motherapeutic drugs; DNR, cisplatinum (Cp), and pacli-taxel (Pac) were purchased from Sigma-Aldrich, Inc.(Sigma). Erlotinib was obtained from Genetech, Inc.

RNA transfectionssiRNA. A549, H838, H2030, HeLa, and HBEC-3-

KT cell lines were transfected with the SRSF1 SMART-pool multiplex designed RNA (siRNA; Dharmacon)using DharmaFECT 1 transfection reagent (Dharmacon)following the manufacturer's protocol. A549 and H2030cells were also transfected with caspase 9b siRNA desi-gned against following sequence; GATTTGGTGATG-TCGAGCATT. The siRNA for caspase 9b (9b-si) andnontargeting control siRNA using DharmaFECT 1 trans-fection reagent (Dharmacon) following the man-ufacturer's protocol. Briefly, cell lines were plated inregular growth medium at 40% to 50% confluence ina 6-well tissue culture dish 24 hours before transfection.Cells were placed in Opti-Mem I medium without anti-biotics/FBS and transfected with 100 nmol/L (dilution in1� siRNA buffer) of the siRNA of choice (DharmaFECT1/Opti-MEM I reduced serum medium and incubatedfor 4 hours at standard incubator conditions). After in-cubation, 0.5 mL Opti-MEM I reduced serum mediumcontaining 3 times the normal concentration of antibio-tics/FBS was added to the transfected cells lines withoutremoving the transfection mixture. After 48 hours, totalRNA isolation or total protein lysates were collected aspreviously described in our laboratory (15) for reversetranscription PCR (RT-PCR) or Western immunoblotanalysis.ASROs. 20-O-(2-methoxy) ethyl (MOE) 23-mer anti-

sense RNA oligonucleotides (ASRO) were purchased fromDharmacon. This study focuses onE4ASRO (2-O-methoxyderivative; 50- GAGTGTACCTTGGCAGTCAGGTC-30)that is targeted to the 50splice site of caspase 9 exon 4 (20).A549 cells were transfected with ASRO (400 nmol/L) usingDharmaFECT I reagent (Dharmacon) and following thesame protocol as the siRNA transfection.

Shultz et al.

Mol Cancer Res; 9(7) July 2011 Molecular Cancer Research890




Western immunoblottingTotal protein was extracted by direct lysis with Laemmli

buffer. Cells were lysed with 0.1 mL of 2� Laemmli buffer(50 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,0.04% bromophenol blue, and 250 mmol/L b-mercap-toethanol) after resuspension in 0.1 mL of ice-cold PBS.Samples were boiled for 10 minutes and either examineddirectly by SDS-PAGE or stored at �20�C. Proteinsamples (10 mg) were subjected to 10% SDS-PAGEand transferred to polyvinylidene difluoride membrane(PDVF; Bio-Rad) and blocked in 5%milk/1� PBS/0.1%Tween (M-PBS-T) for 2 hours. The membrane wasincubated with one of the following primary antibodies,anti-SRSF1 (1:1,000; Zymed), anti-T7 tag (1:10,000;Novagen), anti-Caspase 9 (1:1,000; Cell Signaling Tech-nology), or anti-a-tubulin (1:5,000; Sigma), or anti-b-actin (1:5,000; Sigma) for 2 hours in M-PBS-T fol-lowed by 3 washed with PBS-T. The membrane was thenincubated with the appropriate secondary antibody(horseradish peroxidase-conjugated), either goat anti-mouse IgG antibody (Sigma), goat anti-rabbit IgG anti-body (1:10,000; Pierce), or goat anti-mouse IgM(1:10,000; Calbiochem) for 1 hour in M-PBS-T followedby 3 washes with PBS-T. Immunoblots were developedusing Pierce enhanced chemiluminescence (ECL) reagentsand Bio-max film.

Quantitative and competitive RT-PCRTotal RNA from A549 cells was isolated using the

RNeasy Mini Kit (Qiagen Inc.) according to the man-ufacturer's protocol. Total RNA (1 mg) was reverse-tran-scribed using Superscript III reverse transcriptase(SuperScript First-Strand Synthesis System for RT-

PCR, Invitrogen) and oligo (dT) as the priming agent.After 50 minutes of incubation at 42�C, the reactions werestopped by heating at 70�C for 15 minutes. TemplateRNA was then removed using RNase H (Invitrogen). Toevaluate the expression of endogenous caspase 9 splicevariants, an upstream 50 primer to caspase 9 (50-GCTCTT CCT TTG TTC ATC TCC-30) and a 30 primer (50-CAT CTG GCT CGG GGT TAC TGC-30; IntegratedDNA Technologies, Inc.) were used. Using these primers,20% of the reverse transcriptase reaction was amplified for25 cycles (94�C, 30 seconds melt; 58�C, 30 secondsanneal; 72�C, 1 minutes extension) using Platinum TaqDNA polymerase (Invitrogen Life Technologies). Geneproducts produced from endogenous caspase 9 PCRresulted in a 1,248-bp caspase 9a splice variant and798-bp caspase 9b splice variant. To specifically evaluatethe expression of the splice variant products of the caspase9 minigene (Fig. 1), 50 primer to caspase 9 minigene (50-CAT GCT GGC TTC GTT TCT G-30) and a 30 primer(50-AGG GGC AAA CAA CAG ATG G-30) were used.Gene products produced from caspase 9 minigene PCRresulted in a 889-bp caspase 9a splice variant and a 443-bpcaspase 9b splice variant. Using these primers, 20% of thereverse transcriptase reaction was amplified for 20 cycles(94�C, 30 seconds denaturation; 57�C, 30 secondsanneal; 72�C, 1 minutes extension) using PlatinumTaq DNA polymerase (Invitrogen). The final PCR pro-ducts were resolved on a 5% TBE acrylamide gel electro-phoresis and visualized by staining with SYBR Gold(Invitrogen) and scanned using a Molecular Imager FX(Bio-Rad) with a 488 nm EX (530-nm BYPASS) laser.These RT-PCR based assays are quantitative as to the ratioof caspase 9a/caspase 9b mRNA between analyzed samples

Figure 1. Identification of novel RNA cis-elementsthat regulate the inclusion of exon 3,4,5,6 cassetteof caspase 9. A–D, RT-PCR analysis of caspase 9minigene-derived transcripts from A549stransfected with the WT caspase 9 minigene and(A) C9-I6/ISE, (B) C9-E6/ESE, (C) C9-E4/ESS-1, or(D) C9-E4/ESS-2. The caspase 9a/9b mRNA ratiowas determined by densitometric analysis of RT-PCR fragments. Data represent 3 separatedeterminations on 3 separate occasions.

Caspase 9a MG Caspase 9a MG

BA

C D

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T MG

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/ISE M

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ESE MG

Caspase 9b MG Caspase 9b MG

C9a/C9b ratio

Caspase 9a MG

Caspase 9b MG

C9a/C9b ratio

Caspase 9a MG

Caspase 9b MG

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C9a/C9b ratio2.8 0.1

2.9 4.9 2.9 4.8

2.8 1.9

Regulation of Caspase 9 Splicing

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as confirmed previously by our laboratory (14, 19, 20) andthe initial reports by Siebert and Larrick (21).

Mutation of the caspase 9 minigeneTo mutate the 13 purine-rich sequences in the caspase 9

minigene (20) selected for replacement mutagenesis (fol-lowing manufacturers protocol), the QuikChange II XLSite-Directed Mutagenesis Kit (Stratagene) was used. Allmutations were verified by DNA sequencing.

Plasmid transfectionCell lines were transfected with caspase 9 minigene

constructs and additional plasmid vectors using EffecteneTransfection Reagent (Qiagen Inc.) following the manu-facturers protocol. Briefly, cell lines were plated at �70%confluency in 6-well tissue culture dishes. Cells were trans-fected with plasmid constructs (2 mg total) followed by 24hours standard incubation. After 24 hours standard incuba-tion, the cells were examined for minigene expression byquantitative/competitive real-time RT-PCR. This proce-dure routinely gave more than 50% transfection efficiencyfor A549 cells. The generation of A549 cell lines stablyexpressing either caspase 9b cDNA or caspase 9b shRNAwas previously described (19, 20).

Affinity purification assayThe following sequences were created for affinity pur-

ification assays: 50-biotinylated-tagged RNA oligonucleo-tide for C9-I6/ISE: (50Bio-CUC UGGGUGGGUCUG-30); 50-biotinylated-tagged RNA oligonucleotide forC9-I6/ISE MUT: (50Bio-CUC UCG CUG CGUCUG-30); 50-biotinylated-tagged RNA oligonucleotidefor nonspecific competitor (SC): Bio-NSC-2: (50Bio-AGA GCU AGU CCU GU-30); nonlabeled SC oligonu-cleotide: SC: (50-CUG GGU GGG UC-30); and nonla-beled nonspecific competitor oligonucleotide: NSC-1:(50-GAA UUC GCA CGU UA-30), (Dharmacon). Topreclear, streptavidin-agarose beads (Invitrogen) in theamount of 20 mL were placed in buffer containing 50mg A549 nuclear extract, 40 U RNASIN, 11.3 mg tRNAs,10 mmol/L HEPES, 5 mmol/L DTT, 120 mmol/L KCl,3 mmol/L MgCl2, and 5% glycerol. Binding reactionswere placed in 4�C for 2 hours with gentle agitation, andreaction mixtures were collected by centrifugation.Reaction mixtures were then incubated with 400 ngBio-C9-I6/ISE, Bio-C9-I6/ISE MUT, or Bio-NSC-2for 5 minutes following an addition of 100-fold molarexcess of NSC or SC oligos and incubated on ice for 30minutes. New streptavidin–agarose beads were then addedto RNA-binding reactions and incubated at 4�C for 2hours with gentle agitation. RNA–protein-binding reac-tions were washed with a buffer containing 100 mmol/LKCl, 20 mmol/L Tris-HCl at pH 7.5, and 0.2 mmol/LEDTA. After washing, the complex was pelleted bycentrifugation. The pellet was resuspended in Laemmlibuffer, dry boiled for 10 minutes, and subjected to SDS-PAGE/Western immunoblotting (anti-SRSF1 antibody)analysis.

Immunodepletion assayFor immunodepletion, Lamin-B2 (Abcam), SRSF1 (Invi-

trogen), or mouse IgG (Sigma) was first coupled to protein-G-sepharose beads in lysis buffer (150 mmol/L NaCl, 2mmol/L EDTA, 0.05% NP-40, 20 mmol/L HEPES, and 5mg/mL BSA) overnight at 4�C with little agitation. Theprotein-G-coupled antibody beads were washed 3 timeswith lysis buffer and subsequently incubated with 100 mgA549 nuclear extract in lysis buffer overnight at 4�C. Aftercentrifugation, supernatants were either resolved by SDS-PAGE and analyzed by Western blot analysis or utilized inEMSAs.WST assay. A549 cells (1.0 � 105) were plated into

each well of a 24-well plate in a 1.0 mL media. After 24hours at standard incubator conditions (humidified atmo-sphere, 95% air, 5% CO2, 37�C), the cells were transfectedwith ASRO/siRNA as previously described (20). After 24hours of ASRO/siRNA treatment, the cells were treatedwith DNR at its IC50 concentration (750 nmol/L) for 48hours. After 48 hours of incubation, WST-1 reagent (RocheApplied Science, Indianapolis, IN) was added to these cells(final 1:10 dilution). The cells were then incubated alongwith the reagent for 30 minutes. The plates were then readagainst blank using a microplate (ELISA) reader at 420 to480 nm with the reference wavelength more than 600 nm.Clonogenic assay. Clonogenic assays were conducted

with the indicated chemotherapeutic agents as previouslydescribed (19).Apoptosis assay. A549 cells (2.0 � 105) were plated

into each well of a 6-well plate in 3.0 mL media. After 24hours at standard incubator conditions (humidified atmo-sphere, 95% air, 5% CO2, 37�C), the cells were transfectedwith ASRO following the manufacturer's (Invitrogen LifeTechnologies) protocol (briefly described above). The cellswere treated with the appropriate concentration of DNR(150 nmol/L; Sigma; 9 hours posttransfection) for 48 hours.After the drug treatment cells were stained with Hoechststain (10 mg/mL; Sigma) at room temperature for 10 min-utes. The cells were later observed under fluorescent micro-scope (20�). Five fields were observed and in each fieldtotal numbers of cells and apoptotic cells were counted.Percentage apoptotic cells were counted over total numberof cells.

Data analysisComparison of the effects of various treatments was done

using a 2-tailed, unpaired Student's t test when appropriate.Differences with a P < 0.05 were considered statisticallysignificant. Experiments shown are the means of multipleindividual points (�SEM). Median dose–effect isobolo-gram analyses to determine the synergism of drug interac-tion were done according to the methods of Chou andTalalay using the CalcuSyn program for Windows (Biosoft;ref. 22). Cells were treated with agents at a fixed concen-tration dose. A combination index value of less than 1.00indicates synergy of interaction between the drugs, a valueof 1.00 indicates additivity, and a value of more than 1.00indicates antagonism of action between the agents.

Shultz et al.





Results

Identification of enhancing RNA cis-elements for theinclusion of the exon 3,4,5,6 cassette into caspase 9mRNAPreviously, our laboratory reported that SRSF1 was a

required enhancer for the inclusion of the exon 3,4,5,6cassette of caspase 9 pre-mRNA (15). Many studies over2 decades have shown that SRSF1 interacts with specificRNA cis-elements to mediate enhanced inclusion of exons.Therefore, the sequence (exonic and intronic) of the exon3,4,5,6 cassette of caspase 9 was examined for purine-richsequences, repeating purine-rich sequences, and consensusSRSF1 binding sites from the literature and those identifiedby ESE Finder 2.0 (23) and Splicing Rainbow (24). Analysisof the sequence revealed 13 possible interaction sites forSRSF1 (Table 1). To determine whether these RNA cis-elements played a role in regulating the alternative splicing ofcaspase 9 pre-mRNA, a functional minigene for caspase 9splicing was utilized as previously described (20). Thepossible RNA cis-elements were modified in the minigeneconstruct by replacement mutagenesis (Table 1). Impor-tantly, these mutations did not affect the distances betweenthe possible RNA cis-elements and exonic splice sites, thusreducing the possibility of artifactual results. Of the 13possible RNA cis-elements, 5 mutations affected the caspase9a/9b ratio (Table 1 and Fig. 1).We have previously reportedthat mutation of the RNA cis-element, C9-E3/ESS, resultedin a dramatic increase in the caspase 9a/9b mRNA ratio andwas shown to be an exonic splicing silencer in exon 3 ofcaspase 9 pre-mRNA (Table 1; ref. 20). Therefore, disre-garding C9-E3/ESS, 4 additional RNA cis-elements were

identified. Specifically, 2 enhancer elements, one located inintron 6 termed C9-I6/ISE (Table 1 and Fig. 1A) and theother located in exon 6 termed C9-E6/ESE (Table 1 andFig. 1B), and 2 additional silencer elements, both located inexon 4 and termed C9-E4/ESS-1 (Table 1 and Fig. 1C) andC9-E4/ESS-2 (Table 1 and Fig. 1D), were identified.Importantly, mutation of C9-I6/ISE induced a completeinversion of the caspase 9a/9b ratio from 2.8� 0.1 to 0.1�0.0 (n¼ 6; P < 0.01; Fig. 1A), whereas mutation of C9-E6/ESE decreased the caspase 9a/9b ratio from 2.8 � 0.1 to1.9� 0.1 (n¼ 4; P < 0.01; Fig. 1B). Furthermore, mutationof C9-E4/ESS-1 induced an increase in the caspase 9a/9bratio from 2.9� 0.1 to 4.9� 0.2 (n¼ 4; P < 0.01; Fig. 1C)in a similar manner as mutation of C9-E4/ESS-2, whichincreased the caspase 9a/9b ratio from 2.9 � 0.1 to 4.8 �0.1 (n ¼ 4; P < 0.01; Fig. 1D). The caspase 9 minigenemutants showing no effect on the caspase 9a/9b ratio are alsorepresented (Supplementary Fig. S1). Therefore, these datashow novel RNA cis-elements located in caspase 9 pre-mRNA required for the inclusion/exclusion of the exon3,4,5,6 cassette. As the goal of this study was to defineenhancing RNA cis-elements, the remainder of this reportfocuses on these RNA cis-elements.

SRSF1 specifically interacts with C9-I6/ISEPreviously, our laboratory reported that downregulation

of SRSF1 by siRNA induced a dramatic reduction in theendogenous caspase 9a/9b mRNA ratio (1.67 � 0.11 to0.56 � 0.08 (*P < 0.005); ref. 15). Because mutation ofthe C9-I6/ISE enhancer element resulted in the moredramatic decrease in the caspase 9a/9b ratio and produceda comparable effect as siRNA downregulation of SRSF1 in

Table 1. Schematic representation of potential regulatory cis-elements involved in regulating the alter-native splicing of caspase 9

Site Location Designation WT sequence Mutated sequence

1 Exon 3 C9-E3/ESS GAGAGTTTGAGGGGAAATa GAGAGTTTGCTACTAAAT2 Exon 4 TGGTGGAGGT TGCTCGACGT3 Intron 4 TGGAGGGAGAC TCGACGCACAC4 Intron 4 AGGGTGGGGGG ACGCTCGCGCG5 Intron 4 CAGTGGGTGGGGAAG CAGTCGCTGCGCAAC6 Intron 4 CATGGGAGGTAGGAC CATCCGCTTTAG G AC7 Intron 5 TGGGAGAGGGAGGGCAG TGGGAGACGCTTTGCAG8 Exon 6 CAGCCTGGGAGGG CACCCTCGCAGCG9 Intron 6 C9-I6/ISE TGGGTGGGT TCGCTGCGT10 Intron 6 CTGGTGGGGAGGGA CTCGTCGCCAGCGA11 Exon 4 C9-E4/ESS-1 CCTGAGCATGGAGC CCTCACCCTCTCGC12 Exon 4 C9-E4/ESS-2 TGAAGGGCG TCCACCTCG13 Exon 6 C9-E6/ESE AAGCCCAAGC CCTCCCATCC

NOTE: Depicted are the location, wild-type sequences, and corresponding mutagenic sequences (Sites 1–13) in the caspase 9minigene construct of each of the potential regulatory cis-elements. Sites boxed represent RNA cis-elements that, when mutated,affected the caspase 9a/9b ratio.aThe sequence termed as C9-E3/ESS also affected the caspase 9a/9b ratio and was previously shown to be an exonic splicingsilencer in exon 3 of caspase 9 pre-mRNA (21).






A549 cells, we hypothesized that this C9-I6/ISE specificallyinteracted with SRSF1. To explore this hypothesis, C9-I6/ISE was subjected to streptavidin-agarose affinity purifica-tion with A549 nuclear extract. Biotinylated RNA oligo-nucleotide corresponding to C9-I6/ISE (Bio-I6/ISE),biotinylated RNA oligonucleotide corresponding to amutant C9-I6/ISE (Bio-I6/ISE MUT), biotinylated RNAoligonucleotide corresponding to a NSC sequence (Bio-NSC-2), nonlabeled SC and nonlabeled NSC-1 were uti-lized for this affinity assay. Of note, the biotinylated RNAoligonucleotide corresponding to mutant C9-I6/ISE pos-sessed the same mutations as the mutant C9-I6/ISE mini-gene utilized in this study. As predicted, SRSF1 specificallyinteracted with the Bio-ISE-1 cis-element, whereas nointeraction of SRSF1 was observed with Bio-NSC-2.Importantly, the mutant C9-I6/ISE (Bio-I6/ISE) RNA

oligonucleotide (Fig. 2A) showed a 89% reduction in theinteraction with SRSF1. Furthermore, the addition ofunlabeled SC completely abolished the observed SRSF1/ISE-1 interaction. Thus, SRSF1 specifically interacts withC9-I6/ISE in vitro.The specificity of this interaction was further shown by

immunodepletion (ID) of SRSF1 from A549 nuclearextracts (Fig. 2B). Immunodepletion analysis of SRSF1showed a loss of a specific C9-I6/ISE protein complex incontrast to ID-nuclear lamin B2 extract (Fig. 2C). Theculmination of these data (e.g., competition/affinity pur-ification and immunodepletion/affinity purification)showed that SRSF1 specifically binds to C9-I6/ISE.

The regulation of the alternative splicing of caspase 9pre-mRNA by SRSF1 translates to multiple cell linesand is via C9-I6/ISEBased on previous findings from our laboratory, down-

regulation of SRSF1 in A549 cells induced an increase in thecaspase 9b splice variant at the expense of caspase 9a, therebyinducing a decrease in the caspase 9a/9b mRNA ratio (15).We now show that the change in the caspase 9a/9b ratiotranslates to the protein level (Fig. 3B). Additionally, weshowed translatability of this mechanism into multiple celllines (Fig. 3A and B). Specifically, HeLa cells, H838 cells,and the nontransformed cell line, HBEC-3KT cells, weresubjected to downregulation of SRSF1 utilizing siRNA. Aswith the A549 cells, downregulation of SRSF1 resulted in adramatic decrease in the caspase 9a/b ratio in all cell linesexamined [HeLa cells from 3.1� 0.12 to 0.6� 0.08 (n¼ 4;P < 0.01); H838 cells from 2.2� 0.11 to 1.2� 0.09 (n¼ 4;P < 0.01); and HBECK-3KT cells from 3.5� 0.07 to 1.1�0.09 (n¼ 4; P < 0.01); Fig. 3A]. As with our previous data,this effect on the caspase 9a/9bmRNA ratio translated to theprotein level (Fig. 3B). Therefore, the regulation of thealternative splicing of caspase 9 by SRSF1 is a specificmechanism that translates to multiple cell lines. Of note,we have previously shown that the effect of SRSF1 on thismechanism was not due to a generalized effect on cellularRNA splicing (15).Previous findings from our laboratory have found that

ceramide treatment led to the dephosphorylation of SRproteins, and SRSF1 was required for ceramide to affect thealternative splicing of caspase 9 pre-mRNA (15). Further-more, our laboratory has recently shown that the phosphor-ylation status of SRSF1 on serine199, 201, 227, and 234

regulates the alternative splicing of caspase 9 pre-mRNAin A549 cells downstream of Akt (19). Specifically, ectopicexpression of the SRSF1 quadruple (SRSF1-QD) mutant,which harbors serine to aspartic acid mutations at serineresidues 199, 201, 227, and 234, induced a significantdecrease in the caspase 9a/9b ratio (19). Conversely, ectopicexpression of the SRSF1 quadruple (SRSF1-QA) mutant,which harbors serine to alanine mutations at serine residues199, 201, 227, and 234, induced a significant increase inthe caspase 9a/9b ratio (19). Based on these results, wehypothesized that mutated C9-I6/ISE co-transfected witheither wild-type SRSF1 (SRSF1-WT) or SRSF1-QD would

A

B

CHigh Low

SRSF1 (30kDa)

Lamin B2 (70 kDa)

β-Actin

A549 NE –

–

–

–

+

+

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Figure 2. SRSF1 specifically interacts with the C9-I6/ISE element. A, A50-biotinylated C9-I6/ISE oligonucleotide (Bio-C9-I6/ISE), a 50-biotinylatedC9-I6/ISE mutant oligonucleotide (Bio-C9-I6/ISE MUT) or 50- biotinylatednonspecific competitor oligonucleotide (Bio-NSC-2; negative control) wereincubated in the presence of nuclear extract fromA549 cells or IgG (control)and subjected to Western immunoblot (anti-SRSF1 monoclonal antibody)analysis. Unlabeled competitor RNA oligonucleotides (e.g., NSC-1, SC)were also added to the reactions as indicated. The data are representativeof 4 separate determinations on 2 separate occasions. B, Western blotanalysis of immunodepleted A549 nuclear extracts (supernatants) toconfirm Lamin B2 and SRSF1-immunodepletions. C, Antibodies to SRSF1and Lamin B2 were coupled to protein-G-sepharose beads (see Materialsand Methods). Protein-G-coupled antibody beads were added to A549nuclear extract to allow immunodepletion. Fluorescein-tagged C9-I6/ISEoligonucleotide was incubated in the presence of SRSF1-immunodepletednuclear extracts or LaminB2-immunodepletednuclear extracts followedbyelectromobility shift assays. High, high-intensity laser scan and Low,low-intensity laser scan. The data are representative of 7 separatedeterminations on 3 separate occasions.

Shultz et al.





abolish any observed effect on the caspase 9a/9b mRNAratio. To test this hypothesis, A549 cells were transfectedwith the mutated C9-I6/ISE minigene in conjunction withSRSF1-WT, SRSF1-QD, or SRSF1-QA. Neither the wild-type nor mutated SRSF1 were able to affect the minigene-derived caspase 9a/9b mRNA ratio when cotransfected withthe mutated C9-I6/ISE, in contrast to cotransfection withwild-type caspase 9 miniene (Fig. 3C). These data supportthe hypothesis that SRSF1 regulates the alternative splicingof caspase 9 via the strong, intronic splicing enhancer,C9/I6-ISE.

Clonogenic survival and chemotherapy sensitivity isaltered in cells by direct manipulation of the alternativesplicing of caspase 9NSCLC cells in general show high resistance to che-

motherapy (25) due to a lack of caspase activation (26, 27),and we have reported that the ratio of caspase 9 splicevariants (C9a/C9b) is dysregulated in NSCLC tumors andlung cancer cell lines (also see Supplementary Fig. S2; refs.19, 20). Furthermore, our laboratory reported that theclinically relevant therapeutic, erlotinib, and the lipid sec-ond messenger, ceramide, induced an increase in the caspase9a/9b mRNA ratio at concentrations below the IC50 ofeither agent via a pathway involving the phospho-state ofSRSF1 and likely via the RNA splicing enhancers reportedhere (15, 19). As erlotinib and de novo ceramidewill synergizewith various chemotherapies such as adriamycin-derivatives

in vitro (14, 28), we hypothesized that the caspase 9a/9bratio may be significant in chemotherapeutic sensitivityinduced by erlotinib toward chemotherapies that do notinduce de novo ceramide (e.g., DNR). To study the role ofalternative splicing of caspase 9 in chemotherapy sensitivity oflung carcinoma cells, ASROs and siRNA were utilized todirectly manipulate the alternative splicing of caspase 9 as wehave previously described (20). Specifically, an ASROdesigned to hybridize to 50SS of exon 4 (E4 ASRO) waseffective in reducing the caspase 9a/9b splice ratio (Fig. 4Aand B). Specifically, E4 ASRO shifts the caspase 9 spliceratio to favor caspase 9b with a 57% reduction in the caspase9a/9b mRNA ratio (Fig. 4B). To achieve an increase in theratio of C9a/C9b mRNA, siRNA to specifically down-regulate caspase 9b (9b-si) was developed (Fig. 4C). RT-PCR analysis revealed that transfection of A549s with 9b-siRNA resulted in an increase in the caspase 9a/9b ratio(Fig. 4C).To examine the role that direct manipulation of the

alternative splicing of caspase 9 on the sensitivity ofchemothereutic agents for NSCLC cells that do notgenerate appreciable amounts of de novo ceramide, 3clinically relevant agents used in clinical combinationstherapies for cancer were chosen, DNR, Cp and Pac.These chemotherapies were also chosen because none ofthese agents had an appreciable effect on the caspase 9a/9bmRNA ratio individually (Supplementary Fig. S3). Both,DNR and Cp, are similar in that they are DNA damaging

Caspase 9a MGEV SRSF1-

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HeLa H838 HBEC-3KTsiCon siSRSF1 siCon siSRSF1 siCon siSRSF1

3.0 0.6 0.6

Figure 3. Downregulation of SRSF1 decreases the caspase 9a/9b splice ratio in multiple cell lines. A, the indicated cell lines were transfected with 100 nmol/Lcontrol siRNA or 100 nmol/L SRSF1 SMARTpool siRNA for 48 hours. Total RNA was isolated and analyzed by competitive/quantitative RT-PCR for caspase 9splice variants (15). The C9a/C9b mRNA ratio was determined by densitometric analysis of RT-PCR fragments. B, simultaneously, total protein lysates werealso produced, subjected to SDS-PAGE analysis, and immunoblotted for SRSF1, caspase 9a, caspase 9b, and b-actin. C, RT-PCR analysis of Casp9minigene-derived transcripts from A549s transfected with the wild-type caspase 9 minigene (WT MG) or mutant C9-I6/ISE caspase 9 minigene (I6/ISEMUT MG; 1 mg) in conjunction with either SRSF1-WT (1 mg), mutant SRSF1-QD (SRSF1-QD; 1 mg), or mutant SRSF1-QA (SRSF1-QA; 1 mg). All ratiosof caspase 9a/9b were determined by densitometric analysis of RT-PCR fragments. Data are expressed as means � SE. The protein expression levelsof SRSF1-WT, SRSF1-QD, and SRSF1-QA were also assayed by Western immunoblot analysis using an anti-T7 tag antibody.






agents with DNR being an intercalating agent versus Cpbeing an alkylating agent. Pac is the traditional cytotoxicagent targeting spindle fibers, and thus blocking cell

division. Lowering the caspase 9a/9b ratio using theE4, ASRO induced significant resistance to DNR-inducedapoptosis (n ¼ 6; P < 0.05; Fig. 4D) as well as suppression

F G

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Caspase 9aCaspase 9b

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E4 ASRO*

Figure 4. The E4-ASRO treatment increases the survival and chemotherapy resistance of A549 cells. A, schematic representation of the alternativesplicing mechanism of caspase 9 and binding sites for the E4 ASRO and caspase 9b siRNA utilized in these studies. *, binding site for E4 ASRO and **, bindingsite for caspase 9b siRNA. B, RT-PCR analysis of caspase 9 splice variants from A549s transfected with control ASRO (CON) or E4 ASRO (E4; 400 nmol/L)and (C) control siRNA or caspase 9b siRNA. All ratios of caspase 9a/9b were determined by densitometric analysis of RT-PCR fragments. Data areexpressed as means � SE. D, A549 cells were transfected with the indicated ASROs (400 nmol/L). Twenty-four hours following ASRO transfection, cellswere treated with or without DNR (150 nmol/L) for 48 hours. After drug treatment, apoptosis assays were conducted as described in the Materials andMethods section. The graph depicts percentage apoptosis for cells treated� DNR. Data are expressed as mean� SE; *, P < 0.05. E, the viability of A549 cellstransfected with the indicated ASROs was monitored as mitochondrial function using WST assays as described in Materials and Methods. A549 cellswere treated with DNR (150 nmol/L) or sham control for 48 hours. Data are expressed as mean � SE and are representative of 4 separate determinations on2 separate occasions. F and G, A549 cells were transfected with the indicated ASROs or siRNAs. Twenty-four hours posttransfection, cells were replated(500 cells/well of a 6-well dish) and treated with DNR (0.0–20.0 nmol/L) for 48 hours. Cells were then allowed to form colonies for 10 days. The colonies werescored and percentage survival was calculatedwith respect to the control treated cells *,P < 0.05. F, also includes representative photographs of stained tissueculture plates following treatment with 3 nmol/L DNR to show the increased survival imparted by the E4 ASRO in the presence of DNR. Data are expressedas mean � SE and are representative of 3 separate determinations on 4 separate occasions.

Shultz et al.





of cell viability (n ¼ 6; P < 0.05; Fig. 4E) and cell survival(increased the IC50 from 6.0 to 13.0 nmol/L; Fig. 4F).This effect was also observed for Cp (SupplementaryFig. S4A) and to a lesser, but significant extent, for Pactreatment (Supplementary Fig. S4B).Based on the above findings, we also hypothesized that an

increase in the caspase 9a/9b ratio would produce the oppo-site effect and sensitize NSCLC cells to the same chemother-apeutic agents. More importantly, 9b-siRNA treatment ofA549 cells showed a reducedDNR IC50 of 6.0� 0.7 nmol/Lcompared with cells transfected with control siRNA (12.5nmol/L) as measured by the clonogenic survival assays(Fig. 4G). In addition, 9b-siRNA treatment also reducedthe IC50 for Cp (from 0.25 mg/mL to 0.08 mg/mL; Supple-mentary Fig. S5A). A similar reduction in the IC50 of Pac wasalso observed inA549 cells, although to a slightly lesser extentas the DNA-damaging agents (Supplementary Fig. S5B).Importantly, these effects translated to more than 1 NSCLCcell line as similar increases in the drug de-sensitization wereobserved inH2030cellswhen transfectedwithE4ASROandtreated with DNR and Pac (Supplementary Fig. S6). Simi-larly, H2030 cells transfected with 9b-siRNA also resulted ina reductionof the IC50 as comparedwithCon-siRNA-treatedcells (Supplementary Fig. S7). Overall, these data show thatthe ratio of caspase 9a/9b mRNA is an indicator of theresistance/sensitivity of NSCLC cells to broad-types of che-motherapeutic agents.

Modulation of the alternative splicing of caspase 9regulates the synergistic effects of daunorubicin anderlotinibBecause modulation of the caspase 9a/9b mRNA ratio

sensitized NSCLC cells to DNA damaging agents cur-rently utilized in combination therapies for NSCLCpatients, we next tested our overall hypothesis that erlo-tinib sensitized NSCLC cells to adriamycin-derivatives viamanipulation of the caspase 9a/9b ratio. Specifically, weexamined if genetic manipulation of the alternative spli-cing of caspase 9 would modulate the synergistic effects oferlotinib and DNR in low and high dose combinations. Asshown by colony formation assays, erlotinib and DNRinteracted in a synergistic manner to inhibit the colonyformation of A549 cell as indicated by CI values of lessthan 1.00 (Table 2) and a further reduction of cell survivalwas observed when the NSCLC cells were treated withsub-IC50 concentrations of these drugs (Fig. 5A and B).Importantly and validating our hypothesis, the synergismof erlotinib and DNR in combination at low concentra-tions (sub-IC50 values) was dramatically diminished inNSCLC cells expressing C9b-shRNA and presenting witha low caspase 9a/9b ratio (Table 2 and Fig. 5A). Lowectopic expression of caspase 9b (a 2-fold decrease in thecaspase 9a/9b ratio) also dramatically inhibited the syner-gism of DNR and erlotinib irrespective of dose (Table 2and Fig. 5B). Therefore, these data support the hypothesisthat erlotinib acts to synergize with DNA-damagingagents, such as DNR, through its ability to modulatethe alternative splicing of caspase 9.

Discussion

The importance of the presented study lies in several keyfindings. First, our mechanistic knowledge of how thealternative splicing of caspase 9 is regulated has beenexpanded by our identification of a novel RNA cis-elementvia which SRSF1 (ASF/SF2) enhances the inclusion of theexon 3,4,5,6 cassette. Furthermore, SRSF1 was shown tospecifically interact with this RNA cis-element, and regulatethe alternative splicing of caspase 9 via this novel RNA cis-element. Second, we showed that the alternative splicingof caspase 9 is a relevant therapeutic target as shown bydirect manipulation of this splicing cascade having signifi-cant effect on the sensitivity NSCLC cells to clinicallyrelevant chemotherapeutics. Finally, one of the major find-ings of the report is our data showing that the synergism oferlotinib combination therapy is in part via modulation ofthe alternative splicing of caspase 9.In regard to the RNA cis-element that specifically inter-

acts with SRSF1, a purine-rich RNA cis-element was iden-tified in intron 6, 24 bp downstream of the 50 splice site ofexon 6. The position of this RNA cis-element makes logicalsense as it is localized near a juxta-exon for the large exon3,4,5,6 cassette. We had initially anticipated that regulatoryelements for the exon 3,4,5,6 cassette would be positionedin or near exon 3 and 6, and indeed, this study showedthat an intronic splicing enhancer (now termed C9-I6/ISE)was immediately downstream of exon 6. There is still a

Table 2. Synergistic effects of DNR and erloti-nib in A549 cells

Cell line DNR(nmol/L)

Erlotinib(mmol/L)

CI

A549 Con vector 1.5 1.5 0.425 5 0.2810 10 0.2720 20 –

A549 C9b shRNA 1.5 1.5 0.915 5 0.29

A549 C9b ectopic 10 10 0.5720 20 0.50

NOTE: A549cells expressingvector control, ectopicCasp9bcDNA, or Casp9b shRNA were treated 2 hours after platingas single cells (150–400cells/well) with vehicle (DMSO), DNR(1.5–20 nmol/L), erlotinib (1.5–20 mmol/L), or with both drugscombined, as indicated at a fixed concentration ratio, toconduct median dose–effect analyses for the determinationof synergy. After drug exposure, the medium was changed,and cells were cultured in drug-free media for an additional10 to 14 days. Cells were fixed, stained with crystal violet,and counted. Colony formation data were entered into theCalcuSyn program, and CI values were determined. A CIvalueof less than1.00 indicatessynergy; aCI valueofgreaterthan 1.00 indicates antagonism.






possibility that C9-I6/ISE is only one of several requiredsplicing enhancers for SRSF1 within the exon 3,4,5,6cassette, and indeed, mutation of 1 other possible RNAcis-element for SRSF1 interaction in exon 6 had a signifi-cant, albeit smaller effect on the caspase 9a/9b mRNA ratio.Still, C9-I6/ISE is the strongest enhancer element identifiedto date, and mutation of this RNA cis-element produced thesame results as downregulation of SRSF1 by siRNA (15).This is another key indicator that C9-I6/ISE is the majorenhancer element for inclusion of the 3,4,5,6 cassette.Furthermore, our studies show that SRSF1 specificallybinds C9-I6/ISE, and SRSF1-WT as well as the phos-pho-mutants of SRSF1 could not affect the ratio of mini-gene caspase 9a/9b mRNA when C9-I6/ISE was mutated.Therefore, the culmination of these date shows that C9-I6/ISE is the major splicing enhancer for the inclusion of theexon 3,4,5,6 cassette via specific interaction with SRSF1.As more data are accumulated on the regulation of the

exon 3,4,5,6 cassette of caspase 9, a complex and novelmechanism is beginning to emerge. For example, werecently showed that an exonic splicing silencer (ESS)was located in exon 3 termed C9/E3-ESS. Indeed, wefurther showed that the RNA trans-factor, hnRNP L,bound this sequence in NSCLC cells to repress theinclusion of the exon 3,4,5,6 cassette into the maturetranscript. In this same report, we addressed the possibilitythat this was an oversimplification, and indeed, we iden-

tified 2 additional ESS sequences in the exonic cassette,both in exon 4. These sequence share homology to theC9/E3-ESS sequence suggesting that hnRNP L may alsoassociate with these sequences to repress the entirety of thecassette. This repression by hnRNP L activated by phos-phorylation along with the inactivation of SRp30a byphosphorylation on ser199, 201, 227, 234 bound to exon 6is a logical mechanism to rapidly regulate the alternativesplicing of caspase 9. Overall, the additional regulatoryRNA cis-elements identified in these studies suggest acomplex mechanism involving a number of RNA cis-elements along the exon 3,4,5,6 cassette required for bothinclusion and exclusion (repression). For example, repres-sion of the exonic cassette requires not one, but a numberof exonic splicing silencers bound to inhibitory RNAtrans-factors (e.g., phosphorylated hnRNP L). Hence,the default splicing paradigm is inclusion of the exoniccassette as long as SRSF1 is expressed and not phosphory-lated on ser199, 201, 227, 234, peripheral phosphorylationsites to the standard RS domain of the RNA trans-factor.Taken together with our recent reports on SRSF1 andhnRNP L in this alternative splicing mechanism, thesenew results suggest that RNA trans-factors not onlyplay constitutive roles in RNA splicing, but also activatedroles in modulating alternative splicing induced byposttranslational modifications in contrast to simpleexpression gradients.

An = 6; P < 0.01

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Figure 5. The alternative splicing of caspase 9 regulatesthe synergistic effects of erlotinib and DNR incombination. A and B, clonogenic survival of A549 cellsexpressing vector control, ectopic caspase 9b cDNA, orCasp9b shRNA. Depicted are representativephotographs of stained tissue culture plates of theindicated cells treated with DMS vehicle control or DNRand Erlotinib in combination. Bar graphs represent thepercent death of the indicated cell line followingtreatment with DNR and erlotinib in combination asdetermined by the number of colonies counted incomparison to DMSO control treated (n ¼ 6; *, P < 0.01).Data are mean � SE represented as percent control.

Shultz et al.





In regard to biology, the role of SRSF1 in this mechanism istranslatable to more than 1 NSCLC cell line as well as othercell types. Our laboratory previously published that down-regulation of SRSF1 by siRNA induced a dramatic decrease inthe caspase 9a/9bmRNA ratio of A549 cells (15). Essentially,the inclusion of the exon 3,4,5,6 cassette was abolished intothemature transcript. Here, we expanded this early study andshowed that downregulation of SRSF1 in the NSCLC celllines, H2030 and H838 cells, the cervical cancer cell line,HeLa cells, and in the nontransformed immortalized humanbronchial epithelial cell line, HBEC-3 cells, also lowered thecaspase 9a/9b mRNA ratio. Whereas the effect of SRSF1 inthe NSCLC cell lines and HBEC-3 cells was not unexpected,the result inHeLa cells was surprising due to a previous reportby Karni and colleagues (29). Specifically, downregulation ofSRSF1 was reported to have no effect on the alternativesplicing of caspase 9 (29). Unfortunately, our findings are notin agreement. This may be due to the extent of downregulat-ing SRSF1 inHeLa cells or other technical differences.Duringthe preparation of this report, an additional study publishedbyMoore and colleagues, which also reported that the down-regulation of SRSF1 affected the alternative splicing of caspase9 inHeLa cells, but in this case, the conclusionwas an increasein the caspase 9a/9bmRNAratio (30). This difference is likelydue to this report examining only the levels of caspase 9a fortheir conclusions (30). Regardless of these contrasting find-ings between research groups, all of the data reported in thismanuscript show that SRSF1 is a required RNA trans-factorfor the inclusion of the 3,4,5,6 exon cassette of caspase 9 pre-mRNA in all cell lines examined to date, which is furthersupported by the corroborating effect observedbymutationofthe SRSF1 binding site in the caspase 9 minigene (e.g., alsolowered the caspase 9a/9b ratio).Also in this study, we continued to address the biological

consequences of a dysregulated caspase 9a/9b ratio, and showthat this distal splicing mechanism was related to the sensi-tivity of NSCLC cells to additional types of chemotherapiesbeyond EGFR inhibitors like erlotinib. For example, C9bsiRNA induced a 2.7-fold increase in the caspase 9a/9bmRNA ratio, which translated to a significant decrease in theIC50 ofDNA-damaging agents. Thus, the ratio of caspase 9a/9b mRNA may be a prognostic/predictive indicator ofchemotherapy response to DNA-damaging agents. On theother hand, the ratio of caspase 9a/9b mRNA may notfunction as an indicator of response to classical cytotoxicagents such as Pac. As this study shows, the shift in IC50 aftereither E4 treatment or 9b-si treatment in the case of Pac wassignificant, but not as nearly as dramatic when comparedwith the effects on the IC50 of DNR and Cp. This could beattributed to the mode of action for Pac versus the DNAdamaging agents. For example, DNR sensitivity is highlylinked to apoptosis capacity, thus manipulation of caspase 9activity dramatically affecting the IC50 of this agent is logical.Regardless of the limited effect of the manipulating thecaspase 9a/9b ratio on Pac, the effect on the IC50 of thesechemotherapeutic agents translated to more than 1 NSCLCcell line suggesting a broad-based mechanism. Therefore,this study shows that a single mechanism in these cells (e.g.,

the alternative splicing of caspase 9) has a large impact on thesensitivity of NSCLC cells to several chemotherapeuticagents.These findings have direct implications to the resistance

observed in NSCLC patients to these compounds in theclinic. Specifically, our results suggest that direct manip-ulation of the caspase 9a/9b ratio would sensitize NSCLCtumors to already available chemotherapies. Furthermore,direct manipulation of this splicing event may allow forthe use of 1 anti-cancer agent limiting side-effects andpossibly toxic deaths as synergism between erlotinib andthe DNA-damaging agent, DNR, was lost by directmanipulation of the caspase 9a/b ratio. This is furtherhighlighted as caspase 9b is only appreciably expressed intransformed cells, and hence, should have essentially nounwanted side-effects in patients. Currently, preclinicalanimal studies are being undertaken to assess the efficacyof this treatment regime.In conclusion, this study links our previous findings on

the phospho-status of SR proteins and the alternativesplicing of caspase 9 during apoptotic signaling (15) byestablishing a mechanistic link between the SR proteins,SRSF1, and the alternative splicing of caspase 9 via a novelISE in intron 6 that regulates the inclusion of the exon3,4,5,6 cassette. These current studies also strongly suggestthat the alternative splicing of caspase 9 is an importantregulatory mechanism that influences the chemotherapysensitivity of NSCLC cells. The direct manipulation of thismechanism with the help of ASROs, siRNA, and RNAscavengers gives us the promise of a new generation ofmolecular-therapeutic agents for NSCLC that sensitizesNSCLCs to a variety of chemotherapeutic agents with littleor no unwanted side-effects.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Drs. John D. Minna and Jerry Shay of the University ofTexas Southwestern Medical Center, Dallas, Texas, for their gracious gift of non-transformed immortalized HBEC-3KT cells, which were created using funds from theNational Institutes of Health via an NCI Lung Cancer SPORE P50CA70907(J.D. Minna) and from the National Aeronautics and Space Agency NNJO5HD36G(J.D. Minna). We also thank Dr. Paul Dent and Clint Mitchell of the Department ofNeurosurgery, Virginia Commonwealth University for their help with clonogenicsurvival assays. Finally, we would like to thank Dr. Javier Cacares for the gracious giftof the SRSF1 wild-type and phospho-mutant constructs used in this study.

Grant Support

This work was supported by grants from the Veteran's Administration (VA MeritReview I and a Research Career Scientist Award to C.E. Chalfant), from the NationalInstitutes of Health via HL072925 (C.E. Chalfant), CA117950 (C.E. Chalfant),NH1C06-RR17393 (to Virginia Commonwealth University for renovation), and aNational Research Service Award-T32 Post Doctoral Fellowship in Wound Healing(D.S. Wijesinghe).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 1, 2011; revised April 1, 2011; accepted April 14, 2011;published OnlineFirst May 26, 2011.






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2011;9:889-900. Published OnlineFirst May 26, 2011.Mol Cancer Res Jacqueline C. Shultz, Rachel W. Goehe, Charuta S. Murudkar, et al.

Small Cell Lung Cancer Cells−Sensitivity of Non Intronic Splicing Enhancer Affecting the Chemotherapeutic

SRSF1 Regulates the Alternative Splicing of Caspase 9 Via A Novel

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