TheProto-oncogenePKCiRegulatestheAlternativeSplicing of ... · located in exon 2 of Bcl-x pre-mRNA 277 to 295 bp upstreamfromintron2(30).Infurthermechanisticstudies, the involvement

Signaling and Regulation

TheProto-oncogene PKCiRegulates the Alternative Splicingof Bcl-x Pre-mRNA

Jacqueline C. Shultz1, Ngoc Vu1, Michael D. Shultz1, Mba-Uzoma U. Mba2, Brian A. Shapiro1, andCharles E. Chalfant1,3,4

AbstractTwo splice variants derived from the Bcl-x gene via alternative 50 splice site selection (50SS) are proapoptotic

Bcl-x(s) and antiapoptotic Bcl-x(L). Previously, our laboratory showed that apoptotic signaling pathways regulatedthe alternative 50SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusiveprosurvival signaling pathways that regulate the 50SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by using a number of small-molecule inhibitors of various mitogenic/survival pathways, we foundthat only treatment of non–small cell lung cancer (NSCLC) cell lines with the phosphoinositide 3-kinase (PI3K)inhibitor LY294002 (50 mmol/L) or the pan-protein kinase C (PKC) inhibitor G€o6983 (25 mmol/L) decreased theBcl-x(L)/(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCi and PKCz, had no effecton the Bcl-x(L)/(s) mRNA ratio. Additional studies showed that downregulation of the proto-oncogene, PKCi, incontrast to PKCz, also resulted in a decrease in the Bcl-x(L)/(s) mRNA ratio. Furthermore, downregulation ofPKCi correlatedwith a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the50SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss ofSAP155 complex formation at ceramide-responsive RNA cis-element 1. Finally, forced expression of Bcl-x(L)"rescued" the loss of cell survival induced by PKCi siRNA. In summary, the PI3K/PKCi regulates the alternativesplicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.Mol Cancer Res; 10(5); 660–9.�2012 AACR.

IntroductionNumerous studies have shown that overexpression of Bcl-

x(L) in cells confers resistance to many apoptotic stimuli andcooperates with oncogenic factors (e.g., c-Myc) in tumor-igenesis (1–10). Furthermore, many cell types spontaneous-ly resistant to chemotherapeutic agents also show increasedlevels of Bcl-x(L) (11–16). The regulation of Bcl-x(L)expression is a complex mechanism consisting of bothtranscriptional and posttranscriptional processes. In regardto posttranscriptional processing, the Bcl-x gene, via alter-native 50 splice site (SS) selection within exon 2, produceseither the proapoptotic Bcl-x(s) (upstream 50SS selection) orthe antiapoptotic Bcl-x(L) (downstream 50SS selection).

Several studies have shown that the Bcl-x splice variant,Bcl-x(s), in contrast to Bcl-x(L), promotes apoptosis (9, 17–20), and the mechanism of alternative 50SS selection of Bcl-xpre-mRNA has emerged as a potential target for anticancertherapeutics in non–small cell lung cancer (NSCLC). Forexample, Taylor and colleagues showed that Bcl-x alternativesplicing was specifically modulated using an antisense oli-gonucleotide targeting an RNA sequence surrounding theBcl-x(L) 50SS (21). Hybridization of this oligonucleotide toBcl-x pre-mRNA induced an increase in the expression ofBcl-x(s) mRNA with a concomitant decrease in the expres-sion of Bcl-x(L) mRNA resulting in sensitization of theNSCLC cells to cisplatin and eventually inducing apoptosisafter long-term exposure (>48 hours; ref. 21). These findingswere also shown byMercatante and colleagues who extendedthese findings to several different cancer types (22). Thus,regulation of the 50SS selection within the Bcl-x exon 2 is acritical factor in determining whether an NSCLC cell issusceptible or resistant to apoptosis in response to chemo-therapy (21–25).To this end, previous studies by our laboratory defined the

generation of de novo ceramide and the activation of proteinphosphatase-1 (PP1) as major components of an apoptoticsignaling pathway regulating the 50SS selection of Bcl-x exon2 in response to chemotherapeutic agents (26, 27). Recentstudies by Boon-Unge and colleagues (28) and Chang andcolleagues (29) verified these early findings and extended the

Authors' Affiliations: Departments of 1Biochemistry and Molecular Biol-ogy and 2Pharmacology & Toxicology, Virginia Commonwealth University-School of Medicine; 3Hunter Holmes McGuire Veterans AdministrationMedical Center; and 4The Massey Cancer Center, Richmond, Virginia

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

Corresponding Author: Charles E. Chalfant, Department of BiochemistryandMolecular Biology, Virginia Commonwealth University-School of Med-icine, Room 2-016, Sanger Hall, 1101 East Marshall Street, P.O. Box980614, Richmond, VA 23298. Phone: 804-828-9526; Fax: 804-828-1473; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0363

�2012 American Association for Cancer Research.

MolecularCancer

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list of chemotherapeutic agents to emetine, a potent proteinsynthesis inhibitor, and amiloride, a potassium-conservingdiuretic. Mechanistic studies from our laboratory identifiedthe ceramide-responsive RNA cis-element 1 (CRCE-1)located in exon 2 of Bcl-x pre-mRNA 277 to 295 bpupstream from intron 2 (30). In further mechanistic studies,the involvement of the splicing factor, SAP155, as an RNAtrans-factor that interacted with CRCE-1 and regulated the50SS selection of Bcl-x pre-mRNA was elucidated (31).Importantly, this RNA trans-factor was required for theeffect of ceramide on the alternative 50SS selection of Bcl-x,and downregulation of SAP155 by siRNA technology was aseffective as ceramide in reducing the Bcl-x(L)/(s) mRNAratio as well as the viability of NSCLC cells (31).This study sought to examine the hypothesis that Bcl-x

alternative splicing was dysregulated in certain cancer cellsand, thus linked to a specific oncogenic signaling pathway. Inthis regard, we show that the ratio of Bcl-x(L)/(s) mRNA issignificantly increased in a large number of NSCLC tumors.On the basis of these findings, we examined the survival/mitogenic pathways responsible for regulating the alternative50SS selection of Bcl-x pre-mRNA in malignant NSCLCcells. Whereas a classical protein kinase C (PKC) pathwayhas been implicated in nontransformed cells by Revil andcolleagues, the signaling pathways in transformed cells hasremained elusive. Indeed, classical PKC inhibitors had noeffect on Bcl-x RNA splicing in cancer cells in the Chabotstudy (32). In this study, we identify the phosphoinositide 3-kinase (PI3K) pathway as a key survival pathway regulatingthe alternative 50SS selection of Bcl-x pre-mRNA to producethe antiapoptotic Bcl-x(L) isoform. Furthermore, we showthat the atypical PKC (aPKC), PKCi, is downstream ofPI3K and regulates this alternative splicing mechanism andthe expression of SAP155. Finally, the presented studyshowed that Bcl-x(L) plays an important role in the survivalfunction of PKCi in NSCLC cells. Overall, this reportdefines a survival/oncogenic pathway regulating the alter-native splicing of Bcl-x pre-mRNA in NSCLC cells.

Materials and MethodsCell cultureThe NSCLC cell lines, A549, H292, H226, and H520

cells were grown in 50% RPMI-1640 (Invitrogen LifeTechnologies) and 50%Dulbecco's Modified Eagle's Medi-um (Invitrogen Life Technologies) supplemented with L-glutamine, 10% (v/v) FBS (Sigma), 100 units/mL penicillinG sodium (Invitrogen Life Technologies), and 100 mg/mLstreptomycin sulfate (Invitrogen Life Technologies). A549cells were purchased from American Type Culture Collec-tion. Cells were maintained at less than 80% confluencyunder standard incubator conditions (humidified atmo-sphere, 95% air, 5% CO2, 37�C).

Quantitative/competitive real-time PCRTotal RNA from cell lines was isolated using the RNeasy

Mini Kit (Qiagen Inc.) according to the manufacturer'sprotocol. Total RNA (1 mg) was reverse-transcribed using

Superscript III reverse transcriptase (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen) and PCRwas carried out for Bcl-x splice variants as previouslydescribed (31, 33). The final PCR products were resolvedon a 5% Tris-borate EDTA (TBE) acrylamide gel electro-phoresis, stained with SYBR Gold (Invitrogen), and visual-ized using a Molecular Imager FX (Bio-Rad) with a 488 nmexcision (530 nm BYPASS) laser.

Quantitative real-time PCRTotal RNA was reverse-transcribed using Superscript III

reverse transcriptase (SuperScript First-Strand SynthesisSystem for RT-PCR; Invitrogen) and quantitative real-timePCR (qRT-PCR) was carried out with SAP155 and 18S-specific primers (Applied Biosystems) using the TaqManPCR master mix in an Applied Biosystems 7500 Real-TimePCR System as previously described (34).

Western immunoblottingCells were lysed using CelLytic Lysis Buffer (Sigma-

Aldrich) supplemented with protease inhibitor cocktail(Sigma-Aldrich). Protein samples (5 mg) were subjected to10% SDS-PAGE, transferred to a polyvinylidene difluoride(PDVF) membrane (Bio-Rad), and blocked in 5%milk/1�PBS-0.1% Tween (M-PBS-T) for 2 hours. Primary anti-bodies were anti-PKCi (1:1,000, Santa Cruz), anti-PKCz(1:1,000, Santa Cruz), anti-SAP155 (1:1,000), anti-hnRNPK (1:2,000, Santa Cruz), anti-hnRNP L (1:2,000, SantaCruz), anti-hnRNP F/H (1:2,000, Santa Cruz), anti-Sam68(1:2,000, BD Biosciences), anti-Bcl-x(L) (1:2,000, CellSignaling), and anti-b-actin (1:5,000, Sigma-Aldrich). Sec-ondary antibodies were horseradish peroxidase–conjugatedgoat anti-mouse or anti-rabbit (1:5,000, Santa Cruz).Immunoblots were developed using Pierce enhanced chemi-luminescence (ECL) reagents and Bio-Max film.

siRNA transfectionFor inhibition of PKCi, PKCz, SAP155, or hnRNP K

expression, cell lines were transfected with PKCi SMART-pool multiplex, PKCz SMARTpool multiplex, SAP155SMARTpool multiplex, hnRNP K SMARTpool multiplex,or scrambled control siRNA (Dharmacon) using Dharma-FECT 1 transfection reagent (Dharmacon) as previouslydescribed by us (33, 35, 36). Briefly, cell lines were plated in6-well tissue culture dishes and allowed to rest overnight. At50% confluence, cells were transfected with siRNA (100nmol/L) using DharmaFECT 1 in Opti-Mem I–reducedserum medium. Forty-eight hours posttransfection, RNAand/or protein were isolated.

Inhibitor studiesFor Inhibitor studies, A549 cells (1.5 � 105) were plated

into 6-well tissue culture plates. The following day mediawere removed and replaced with the appropriate completegrowth media. Cells were subsequently treated with shamcontrol (1:1,000) or the appropriate concentration of activeinhibitor (1:1,000; Calbiochem). Twenty-four hours post-treatment, total RNA and/or protein was isolated.

The PI3K/PKCi Pathway Regulates Bcl-x pre-mRNA Splicing

www.aacrjournals.org Mol Cancer Res; 10(5) May 2012 661




Clonogenic assaysFor clonogenic assays, A549 cells were transfected with

control siRNA (100 nmol/L) or PKCi siRNA (100 nmol/L).After 24 hours, cells were then infected with either controladenovirus or Bcl-x(L) adenovirus (10 multiplicity of infec-tion; Applied Biological Materials Inc.). After an additional24 hours, 125 viable cells were seeded into 6-well tissueculture dishes with complete growth media. After 12 days,colonies were counted following fixation with methanol andstaining (0.1% crystal violet; colonies � 40 cells).

Electromobility shift assaysThe following sequence was used for the fluorescein

isothiocyanate (FITC)-tagged CRCE-1 RNA oligonucleo-tide (50FL-GAGGGAGGCAGGCGACGAC-30). RNA-binding reactions were conducted in a final volume of 25 mLcontaining: 600 ngCRCE-1 fluorescein-oligonucleotide, 40mg nuclear protein extract, 40 U RNasin, and 11.3 mgtRNAs in buffer composed of 10 mmol/L HEPES, 1mmol/L dithiothreitol (DTT), 120 mmol/L KCl, 3mmol/L MgCl2, and 5% glycerol as previously describedby us (31). The reaction mixtures were incubated at 4�C for25 minutes. Samples were loaded on a 5% TBE-PAGE(37.5:1 acrylamide/bis-acrylamide) for electrophoretic sep-aration of RNA-protein(s) complexes. The gel was thenscanned using Molecular Imager FX (Bio-Rad) with a488 nm excision (530 nm BP) laser.

Pathologist-verified tumor samplesTissueScanCancer andNormal Tissue cDNAArrays used

in this study were obtained fromOriGene (Lung cancer andnormal tissue samples, catalog no. HLRT102; Breast, Cer-vical and esophageal cancer and normal tissue samples,catalog no. CSRT103; see Supplementary Tables SI andSII).

Statistical analysisWhen appropriate, the data are presented as the mean �

SE or SD as indicated in the specific figure. Data points werecompared using a 2-tailed, independent samples t test, andthe P values calculated. P values less than 0.05 were con-sidered significant.

ResultsThe alternative splicing of Bcl-x is dysregulated inNSCLC tumorsPreviously, our laboratory showed that the alternative

splicing of Bcl-x pre-mRNA was regulated by apoptoticstimuli (27, 37). In this study, we expanded our investiga-tions to test the hypothesis that the alternative splicing ofBcl-x was dysregulated (in a prosurvival manner or increasedBcl-x(L)/(s) mRNA ratio) in cancer phenotypes, specificallyNSCLC tumors. Using total RNA samples from patholo-gist-verified humanNSCLC tumor samples (SupplementaryTable SI), quantitative/competitive RT-PCR analysis wasconducted to determine the degree of dysregulation in theBcl-x(L)/(s) ratio as compared with normal lung tissuecontrols. Tumor samples were categorized into 3 groups

respectively: normal, a Bcl-x(L)/(s) mRNA ratio of < 5.0;moderately dysregulated, a Bcl-x(L)/(s) mRNA ratiobetween 5.0 and 7.5; and highly dysregulated, a Bcl-x(L)/(s) mRNA ratio of > 7.5 (Fig. 1). These classifications aboutthe degree of dysregulation were due to the observation thatthe normal lung tissue samples had a Bcl-x(L)/(s) ratio of4.75 � 0.15 (N ¼ 4), which closely correlated with thereport from Minn and colleagues showing that Bcl-x(s)overcame Bcl-x(L) expression at a Bcl-x(L)/(s) ratio ofapproximately 4.0 (9). Furthermore, Bauman and colleaguesshowed using RNA oligonucleotides tomodulate the endog-enous ratio of Bcl-x(L)/(s) mRNA that a ratio less than 5.0for Bcl-x(L)/(s) mRNA [>20% Bcl-x(s)] led to a loss of cellviability for cancer cells (38). Furthermore, a ratio of about5.0 was induced in the lung tumors of mice in the samestudy, which corresponded to a 75% loss in tumormass (38).Hence, a Bcl-x(L)/(s) mRNA ratio > 5.0 indicates a pro-survival/dysregulated ratio compared with normal lungtissue. The classification for moderately dysregulated versushighly dysregulated was based on the findings of both Taylorand colleagues andMercatante and colleagues. These studiesshowed that cancer cells with a ratio of Bcl-x(L)/(s) mRNAof>7.5 correlated with strong resistance to various che-motherapies whereas cancer cells with a ratios between5.0 and 7.5 were partially resistant (e.g., PC-3 cells; refs. 21,22, 24).Using the aforementioned parameters, 78% (N ¼ 41) of

NSCLC tumors presented with amore than 25% increase inthe Bcl-x(L)/(s) mRNA ratio as compared with normal lungtissue controls. Even more dramatic, 32% of NSCLCtumors examined presented with a more than 50% increasein the Bcl-x(L)/(s) mRNA ratio (Fig. 1). Thus, the ratio ofBcl-x(L)/(s) mRNA is dramatically increased in a highpercentage of NSCLC tumors. Interestingly, a dysregulatedBcl-x(L)/(s)mRNA ratio correlated with tumor stage as 10 of18 tumors classified as stage II or higher presentedwith a Bcl-x(L)/(s) mRNA ratio more than 7.5 (Fig. 1C). Therefore,these data indicate that a significant portion of NSCLCtumors present with a dysregulated ratio of Bcl-x(L)/(s)mRNA favoring a prosurvival/pro-oncogenic phenotype.Next, our laboratory determined whether our findings in

NSCLC tumors on the Bcl-x(L)/(s) mRNA ratio translatedto other tumor types. Using total RNA extracted frompathologist-verified human breast, cervical, and esophagealtumors samples, quantitative/competitive RT-PCR analysiswas again conducted to determine the degree of dysregula-tion in the Bcl-x(L)/(s) ratio as compared with normal tissuecontrols. Of the samples examined, 43% of tumors of theesophagus (N ¼ 14) presented with a more than 25%increase in the Bcl-x(L)/(s) mRNA ratio as compared withnormal esophageal tissue controls (Supplementary TableSII). However, only 17% of breast tumor samples (N ¼23) and 20% of cervical tumor samples (N¼ 10) presentedwith a more than 25% increase in the Bcl-x(L)/(s) mRNAratio as compared with normal tissue controls (data notshown). Therefore, dysregulation in the alternative splicingof Bcl-x pre-mRNA translates tomultiple cancer types, albeitto a lesser extent in both breast and cervical tumors.

Shultz et al.

Mol Cancer Res; 10(5) May 2012 Molecular Cancer Research662




The PI3K pathway regulates the 50SS selection of Bcl-xpre-mRNAIn this study, we hypothesized that a major survival/

mitogenic pathway regulates the alternative processing ofBcl-x pre-mRNA to favor the production of antiapoptoticBcl-x(L), thereby increasing the Bcl-x(L)/(s) mRNA ratio.To investigate this hypothesis, these pathways were exam-ined for regulation of the alternative splicing of Bcl-x pre-mRNA using small-molecule inhibitors well characterized inthe scientific literature. Treatment of the NSCLC cell line,A549 cells, as previously described by us (39), with thefollowing inhibitors; the mitogen-activated protein (MAP)kinase inhibitor, PD98059 (10 mmol/L); the MAP/ERK(MEK)1/2 inhibitor, U0126 (10 mmol/L); the Rho-kinaseinhibitor, Y-27632 (10 mmol/L); the casein kinase II inhib-itor, DMAT (10 mmol/L); and an Src kinase inhibitor (25mmol/L), had no significant effect on the alternative splicingof Bcl-x pre-mRNA (Supplementary Table SIII). In con-trast, treatment of A549 cells with the PI3K inhibitor,LY294002 (50 mmol/L), resulted in a significant reductionin the ratio of Bcl-x(L)/(s) splice variants compared with theinactive, structurally related compound, LY303511 (50mmol/L; Fig. 2A). Specifically, the Bcl-x(L)/(s) mRNA ratiodecreased from 6.00 � 0.16 for LY303511 control-treatedsamples to 3.40� 0.19 for LY294002-treated samples (P <

0.01;N¼ 6). Therefore, these data show that PI3K regulatesthe alternative splicing of Bcl-x pre-mRNA in an antiapop-totic/prosurvival manner.To determine the translatability of the mechanism,

H226 and H292 were also treated with LY294002 (Fig.2B and C). As with the A549 cells, the Bcl-x(L)/(s) ratiowas dramatically decreased (H226 cells from 5.87 � 0.13to 3.15 � 0.12; H292 cells from 6.51 � 0.12 to 3.50 �0.19). Importantly, the effect of PI3K inhibition of Bcl-x50SS selection translated to the protein level as a decreasein the protein levels of Bcl-x(L) was observed in all 3 celllines after LY294002 treatment (Fig. 2D). To furtherconfirm the involvement of PI3K in regulating the alter-native splicing of Bcl-x, we used a cell-permeable Wort-mannin 17b-hydroxy analog (HWT) that acts as anirreversible PI3K inhibitor. Treatment of A549 cells withHWT (10 mmol/L) also resulted in a significant reductionin the ratio of Bcl-x(L)/(s) splice variants as compared withdimethyl sulfoxide (DMSO) controls (Fig. 2E). Specifi-cally, the Bcl-x(L)/(s) mRNA ratio decreased from 6.05 �0.18 for DMSO control–treated samples to 3.62 � 0.05for HWT-treated samples (P < 0.01; N ¼ 6). Therefore,the PI3K pathway regulates the alternative splicing of Bcl-x, and this signaling cascade translates to multiple NSCLCcell lines.

Figure 1. The Bcl-x(L)/(s) mRNAratio is dysregulated in NSCLCtumors. A population of cDNAs frompathologist-verified lungadenocarcinomas, squamous cellcarcinomas, and large cellcarcinomas (OriGene) underwentquantitative/competitive PCR forexpression of Bcl-x splice variants.A, a representation of the RT-PCRanalysis of matched normal andtumor samples used to determine thedegree of Bcl-x(L)/(s) dysregulation.N, normal lung tissue; T, tumortissue. B, quantitative/competitivePCR analysis of Bcl-x splice variantsshow that 46% of NSCLC tumorspresent a moderately dysregulatedBcl-x(L)/(s) mRNA ratio [Bcl-x(L)/(s)ratio of 5.0–7.5] and 32% of NSCLCtumors present a highly dysregulatedBcl-x(L)/(s) mRNA ratio [Bcl-x(L)/(s)ratio > 7.5; N¼ 41], as determined bydensitometric analysis of PCRproducts. C, the lung NSCLCsamples (N ¼ 41) used above andSCLC samples (N¼ 3) were groupedaccording to tumor stage to depictthe correlation between tumor stageand degree of Bcl-x(L)/(s) mRNAdysregulation.






PKCi regulates the activation of the Bcl-x(s) 50SS in A549cellsAkt/PKB, aPKCs (x and i), mTOR/S6 kinase, and PKCd

are downstream of PI3K. To investigate the downstreameffector of PI3K responsible for regulating the alternativesplicing of Bcl-x pre-mRNA, well-characterized small-mol-ecule inhibitors were again used. Treatment of A549 cellswith the small-molecule Akt1/2 inhibitor, AKT VIII (25mmol/L); the classical PKC (cPKC) inhibitor, GF109203X(10 mmol/L); the cPKC/novel PKC (nPKC) inhibitor,G€o6976 (10 mmol/L); or the mTOR/S6 kinase inhibitor,rapamycin (10 mmol/L), had no effect on the Bcl-x(L)/(s)mRNA ratio (Supplementary Table SIII). On the otherhand, treatment of A549 cells with the pan-PKC (cPKCs,nPKCs, and aPKCs) inhibitor, G€o6983 (10 mmol/L; Fig.3A), significantly decreased the Bcl-x(L)/(s) mRNA ratiofrom 6.04 � 0.18 for control samples to 3.16 � 0.29 (P <0.05; N¼ 6). The effects were specific to the 50SS selectionof Bcl-x pre-mRNA as the alternative splicing of caspase 9,another de novo ceramide target, was previously reported byus to be unaffected byG€o6983 inA549 cells (39). Therefore,these data, via the process of elimination, suggest that thePI3K survival pathway regulates the alternative splicing ofBcl-x via an aPKC.To confirm a role for an aPKC in this mechanism, siRNA

to both human aPKC isoforms, PKCi and PKCz, was used.In contrast to PKCz, downregulation of PKCi, a knowndownstream target of the PI3K, induced the activation of theBcl-x(s) 50SS, decreasing the Bcl-x(L)/(s) ratio from 6.12 �

0.12 for siControl-treated cells to 4.01 � 0.11 for siPKCi-treated cells (Fig. 3B and C), congruent with the inhibitionof aPKCs and PI3K by small-molecule inhibitors. Further-more, co-treatment of A549 cells with both siPKCi andLY294002 could not further decrease the Bcl-x(L)/(s) ratio,showing a linear pathway with PKCi as the downstreameffector of PI3K (Fig. 3D). Thus, the aPKC, PKCi, regulatesthe alternative splicing of Bcl-x pre-mRNA in a prosurvivalfashion.

The PI3K/PKCi pathway regulates SAP155 expressionOur laboratory has previously reported that the RNA

trans-factor, SAP155, regulates the 50SS selection of Bcl-xpre-mRNA. Here, we again show that downregulation ofSAP155 via siRNA decreased the Bcl-x(L)/(s) mRNA ratiofrom 6.10� 0.08 for siControl-treated cells to 3.21� 0.12for siSAP155-treated cells (Fig. 4A). To determine whetherthe expression of SAP155 was a distal mechanism regulatedby PI3K/PKCi, the LY294002 and G€o6983 inhibitors, andsiRNA to PKCi and PKCz were again used to examine theexpression of SAP155. Inhibition of PI3K, aPKCs andknockdown of PKCi by siRNA, but not knockdown ofPKCz by siRNA, induced a significant decrease in the levelsof SAP155 (Fig. 4B), analogous to a level of expressionshown to significantly lower the Bcl-x(L)/(s) ratio (31). Theobserved decrease in SAP155 protein levels induced byLY294002 treatment and PKCi downregulation was mim-icked at the RNA level (Fig. 4C and D). Specifically,treatment of A549 cells with LY294002 reduced the levels

Figure 2. The effect of PI3Kinhibiton on the alternativesplicing of Bcl-x pre-mRNA.Quantitative/competitive RT-PCRanalysis of Bcl-x splice variantsand the corresponding Bcl-x(L)/(s)mRNA ratios from (A) A549, (B)H226, and (C) H292 treated witheither structurally inactiveLY303511 (50 mmol/L) orLY294002 (50 mmol/L). D,Westernimmunoblot analysis of Bcl-x(L)expression from A549, H226, andH292 treated with LY303511control or LY294002. E,quantitative/competitive RT-PCRanalysis of Bcl-x splice variantsand the corresponding Bcl-x(L)/(s)mRNA ratios from A549s treatedwith 0.1% DMSO or the PI3Kinhibitor, HWT (10 mmol/L). Theratio of Bcl-x(L) to Bcl-x(s) mRNAwas determined by densitometricanalysis of RT-PCR fragments(P < 0.01, N ¼ 6). Data areexpressed as means � SD. Dataare representative of 3 separatedeterminations on 2 separateoccasions.

Shultz et al.





of SAP155 mRNA by 47% in comparison with LY303511control samples (Fig. 4C). Similarly, treatment of siPKCireduced the levels of SAP155mRNA by 25% in comparisonwith siControl samples (Fig. 4D). Interestingly, no changesin the expression of additional RNA trans-factors implicatedin regulating the 50SS selection of Bcl-x pre-mRNA (e.g.,hnRNP K, Sam68, hnRNP L, or hnRNP F/H) wereobserved following downregulation of PKCi (Fig. 4E;refs. 40–42). These data provide evidence for PKCi regu-lation of the 50SS selection of Bcl-x pre-mRNA via modu-lation of SAP155 levels.We next investigated that the role of hnRNP K on the

alternative splicing of Bcl-x in NSCLC cell lines as this RNAtrans-factor has been implicated in regulating Bcl-x 50SSselection in the prostate cancer cell line, PC-3 cells, and thecervical cancer cell line, HeLa cells (40). Downregulation ofhnRNPK resulted in no significant change in the ratio of Bcl-x (L)/(s) mRNA in A549 or H520 cells (Supplementary Fig.S1). These data show that the PI3K/PKCi pathway regulatesthe expression of SAP155 at themRNA level inNSCLC cellswith no effect on the expression of several RNA trans-factorsreported to regulate the 50SS selection of Bcl-x pre-mRNA.

The PI3K/aPKC pathway regulates the association ofSAP155 with CRCE-1Our laboratory has previously identified CRCE-1 located

277 to 295 bp upstream of intron 2, within exon 2 of Bcl-x

pre-mRNA (30). Furthermore, we have previously shownthat SAP155-deficient nuclear extracts resulted in the loss ofa specific protein complex to CRCE-1, showing thatSAP155 specifically interacts with CRCE-1 (31). Indeed,Supplementary Fig. S2 shows that addition of a SAP155antibody blocks the formation of the SAP155:CRCE-1complex in A549 nuclear extracts revalidating our previousreport (31). Importantly, A549 nuclear extracts fromLY294002-treated cells also resulted in the loss of theSAP155:CRCE-1 complex (Fig. 5A). Furthermore, inhibi-tion of aPKCs using G€o6983 also resulted in the same loss ofthe SAP155:CRCE-1 complex (Fig. 5B) in contrast to theclassical/novel PKC inhibitor, G€o6976 (data not shown).These data show that the PI3K/aPKC pathway regulates theformation of the SAP155:CRCE-1 complex analogous tothe observed loss of SAP155 expression.

PKCi requires Bcl-x(L) expression to sustain the survivalof NSCLC cellsPrevious studies have shown that the activation of the Bcl-

x(s) 50SS at the expense of Bcl-x(L) sensitized cells tochemotherapy and ultimately led to apoptosis (21, 26).Furthermore, the expression of PKCi was previously shownto have an important function in the survival ofNSCLC cells(43), and our results presented within this study link PKCito regulating both Bcl-x 50SS selection and Bcl-x(L) levels.Therefore, we hypothesized that Bcl-x(L) expression due to

Figure 3. Inhibition of PKCi decreases the Bcl-x(L)/(s) mRNA ratio in A549 cells. Quantitative/competitive RT-PCR analysis of Bcl-x splice variants and thecorresponding Bcl-x(L)/(s) mRNA ratios from A549s treated with either (A) 0.1% DMSO or G€o6983 (10 mmol/L), (B) PKCi siRNA (siPKCi), or (C) PKCz siRNA.The ratio of Bcl-x(L) to Bcl-x(s)mRNAwas determined by densitometric analysis of RT-PCR fragments (P < 0.01, N¼ 6). Data are expressed asmeans�SD. Band C, Western immunoblot analysis of PKCi and PKCz knockdown. Data are representative of 3 separate determinations on 2 separate occasions. D,quantitative/competitive RT-PCR analysis of Bcl-x splice variants and the corresponding Bcl-x(L)/(s) mRNA ratios from A549 treated with siCon, siPKCi,DMSO, or LY294002 (LY) as indicated. The ratio of Bcl-x(L) to Bcl-x(s)mRNAwasdetermined bydensitometric analysis of RT-PCR fragments (P <0.01, N¼6).Data are expressed as means � SD. Con, control.






the activation of the Bcl-x(L) 50SS by PKCi activation/expression was a key mechanism for the survival of NSCLCcells. Indeed, we examined the ability of forced expression ofBcl-x(L) to "rescue" the effects of downregulation of PKCion the survival of NSCLC cells. As shown by colonyformation assays, knockdown of PKCi resulted in decreasedclonogenic survival of A549 cells in agreement with previousreports (Fig. 6A and B; ref. 43). Importantly, forced expres-sion of Bcl-x(L) was able to completely "rescue" this effectdramatically inhibiting the ability of siPKCi to suppressclonogenic survival (Fig. 6A and B). These data support thehypothesis that the activation of PKCi enhances cell survivalof NSCLC cells via activation of the Bcl-x(L) 50SS at theexpense of the Bcl-x(s) 50SS use.

DiscussionThe presented study began from our first observation that

the alternative splicing of Bcl-x pre-mRNAwas dysregulatedin a large percentage of NSCLC cells. This led to the

hypothesis that a survival pathway of signal transductionregulated the alternative splicing of Bcl-x pre-mRNA.Indeed, we have previously shown that a contrasting path-way in apoptotic signaling existed for the activation of theBcl-x(s) 50SS. Specifically, our laboratory showed that theproduction of Bcl-x(s) via alternative splicing was dependenton the generation of de novo ceramide and the activation ofPP1 (26, 27). Therefore, a survival pathway regulating thiskey distal mechanism and balancing the cell between apo-ptosis and survival was likely to exist as well. The datapresented in this study show that the aPKC, PKCi, is amajor regulator of the alternative splicing of Bcl-x pre-mRNA in A549 cells, acting downstream of the majorsurvival/oncogenic enzyme, PI3K. Therefore, in contrast topublished reports showing a classical PKC responsible forregulating the 50SS selection of Bcl-x pre-mRNA in non-malignant/nontransformed cells basally and in response toDNA-damaging agents (32), an oncogenic aPKC regulatesthe 50SS selection in a prosurvival fashion in transformedphenotypes of lung cancer. Interestingly, the aPKC, PKCz,

Figure 4. Inhibition of thePI3K/PKCipathway decreases the levels of SAP155protein expression inA549 cells. A, quantitative/competitiveRT-PCRanalysis ofBcl-x splice variants and the correspondingBcl-x(L)/(s)mRNA ratios fromA549 treatedwith either control siRNAorSAP155 siRNA. The ratio of Bcl-x(L) to Bcl-x(s) mRNA was determined by densitometric analysis of RT-PCR fragments (P < 0.01, N ¼ 6). Data are expressed as means � SD. Western immunoblotanalysis of SAP155 knockdown. B, total proteins from A549 treated with LY303511 control (50 mmol/L), LY294002 (50 mmol/L), 0.1%DMSO control, G€o6983(10 mmol/L), G€o6976 (10 mmol/L), control siRNA, PKCi siRNA, or PKCi siRNA were subjected to Western blot analysis to determine expression levels ofSAP155andb-actin.Quantitative real-timePCRanalysis of SAP155mRNA inA549 following treatmentwith (C) LY303511control or LY294002 and (D) controlsiRNA or PKCi siRNA. Data are expressed as quantity of SAP155 mRNA over quantity of 18S rRNA. The columns represent the mean of 2 independentexperiments � SE. E, total proteins from A549 transfected with either control siRNA or PKCi siRNA were subjected to Western blot analysis to determineexpression levels of hnRNP K, Sam68, hnRNP L, hnRNP F/H, and b-actin. Con, control.

Shultz et al.





has been implicated in regulating the alternative splicing ofcaspase-2 (44). Although PKCz does not function inNSCLC cells to regulate the 50SS selection of Bcl-x pre-mRNA, this report coupled to the presented study suggestthat activating mechanisms for aPKCs can modulate thedistal mechanism of alternative splicing with high relevanceto the sensitivity of cancer cells to apoptotic stimuli (44).Once the upstream components in the survival signaling

pathway were determined, our laboratory focused on linkingthese upstream signals to downstream RNA trans-factorsknown to modulate the 50SS selection of Bcl-x pre-mRNA.For this purpose, we turned to the literature as well as our ownwork for these RNA trans-factors. In a collaboratory studywith Paronetto and colleagues, SAM68, a well-establishedRNA trans-factor with roles in cell signaling and transforma-tion, was shown to regulate the 50SS selection in HEK293cells. However, this finding did not completely translate toNSCLC cells, as siRNA directed against SAM68 only slightlyinhibited the use of the Bcl-x(s) 50SS (41). Other RNA trans-factors, hnRNP K and the hnRNPs F/H, have also beenimplicated in regulating the alternative splicing of Bcl-x(40, 42). Specifically, Revil and colleagues showed thatdownregulation of hnRNPKexpression byRNA interferenceresulted in an increase in the use of the Bcl-x(s) 50SS (40),whereas the contrasting effect was observed for downregula-tion of hnRNPs F/H. In this regard, downregulation ofhnRNP K had no effect on the alternative splicing of Bcl-xin A549 or H520 cells, suggesting the existence of cell type–specific mechanisms for NSCLC cells. Furthermore, theexpression of hnRNP K and the hnRNPs F/H was notaffected by PKCi downregulation, suggesting an alternativepathway for regulationof theBcl-x 50SS selection inNSCLCs.

In regard to an alternative pathway for the PI3K/PKCisignaling tomodulate the 50SS selection of Bcl-x pre-mRNA,we then examined the expression of the RNA trans-factor,SAP155. Our laboratory has previously reported this RNAtrans-factor functioning as a repressor of the Bcl-x(s) 50SS(31), which was later confirmed by Moore and colleaguesusing a high-throughput screening approach (45). Indeed,this study provides a link between the expression of SAP155to the activation/expression of the NSCLC proto-oncogene,PKCi. As to howPKCi regulates the expression of SAP155 iscurrently a conundrum, but PKCi has been implicated inregulating the transcription of genes activated by collagenasevia a STAT3- and c-fos–dependent mechanism (46). Cur-rently, there are no known regulatory sequences for STAT3or c-fos in the SAP155 promoter region; hence, furtherstudies are necessary to determine the signaling link betweenPKCi and SAP155. Also of note, our studies do not "rule-out" the possibility of alternative promoter choice or RNApolymerase II elongation in regulating the alternative splic-ing of Bcl-x pre-mRNA, although our data suggest a more"classical" regulation of 50SS selection by RNA trans-factors(47, 48). For example, inhibition of the PI3K/aPKC path-way reduces the levels of SAP155 with concomitant loss of

Figure 5. Inhibition of the PI3K/PKCi pathway decreases the formation ofthe SAP155:CRCE-1 complex in A549 cells. The effect of LY294002 andG€o6983 on the formation of the SAP155:CRCE-1 complex wasexamined. Specifically, nuclear extracts (NE) were prepared from A549cells after treatment with LY294002 (A) and G€o6983 (B) and subjected toelectromobility shift assay (EMSA)-binding conditions with labeledCRCE-1 as previously described. After a 25-minute reactionequilibration, samples were then subjected to electrophoretic separationusing a 5% TBE-PAGE (37.5:1 acrylamide/bis-acrylamide). The positionof the specific SAP155:CRCE-1 complex is indicated by the arrows. Dataare representative of N ¼ 3 reproduced on 2 separate occasions. Con,control.

Figure 6. Bcl-x(L) is required for PKCi to increase the clonogenic survivalof NSCLCcells. A, A549 cellswere transfectedwith the indicated siRNAs.Twenty-four hours posttransfection, cells were infected with control orBcl-x(L) adenovirus. Twenty-four hours postinfection, cells were platedas single cells (125 cells perwell) in 6-well dishes. Cells were then allowedto form colonies for 12 days after which colonies were fixed, stained withcrystal violet, and counted. Total protein lysates were isolated from cellsfollowing treatment with the indicated siRNAs and adenoviruses andsubjected to Western immunoblot analysis to determine PKCi and Bcl-x(L) expression. B, representative photographs of stained tissue cultureplates following treatment with the indicated siRNAs and adenoviruses.Data are expressed as mean � SE and are representative of 6 separatedeterminations on 2 separate occasions. Con, control.






the protein:RNA complex formation of SAP155 at CRCE-1. The loss of this complex has been previously reported byour laboratory to induce the activation of the Bcl-x(s) 50SS(31), which was confirmed again in this study. Hence, theregulation of the Bcl-x 50SS selection by the complex ofSAP155 with CRCE-1 is the more likely mechanism.The physiologic significance of the Bcl-x(L)/(s) mRNA

ratio has been documented by many reports in the literatureshowing that the fate of the cell can be determined by theproportion of antiapoptotic Bcl-x(L) to proapoptotic Bcl-x(s) (9, 21, 22). Furthermore, the induction of proapoptoticBcl-x(s) has also been shown to sensitize cells to apoptosisand loss of viability to chemotherapeutic agents (21, 22).Published findings from our laboratory corroborate thesefindings showing that treatment of A549 cells with con-centrations of ceramide known to activate the Bcl-x(s) 50SSalso lowered the IC50 of the chemotherapeutic agent, dau-norubicin (27). Taken together, these data suggest a linkbetween signal transduction pathways mediating the 50SSselection of Bcl-x pre-mRNA and the sensitivity of cells toapoptosis in response to chemotherapeutics. Specifically,SAP155 expression may be a key link, as we have previouslyshown that ceramide could not induce apoptosis or sensitizecells to daunorubicin in A549 cells above the extent ofSAP155 downregulation by siRNA. Unfortunately, little isknown about the PKCi signaling pathway to form a stronghypothesis as to how ceramide may be blocking signalsmediating SAP155 expression. The activation of the Bcl-x(s) 50SS is regulated by the serine/threonine protein phos-phatase, PP1. Therefore, it is conceivable that a ceramide-activated protein phosphatase such as PP1 may dephosphor-ylate PKCi effectively "shutting-down" the prosurvivalpathway and thus, SAP155 expression. Indeed, studies havelinked the inhibition of serine/threonine phosphatases toactivation of PKCi (49). In addition, Wang and colleagues

have recently shown that overexpression of PKCi resulted inenhanced survival of PC12 cells treated with ceramide (50).Outside of the realm of chemotherapy sensitivity, the

alternative splicing of Bcl-x pre-mRNA may also have rolesin oncogenesis. Recently, Finch and colleagues showed thatBcl-x(L) cooperated with c-Myc in oncogenic transforma-tion in vivo (6). These findings correlate well with theobservation that a large number of NSCLC tumors showeddysregulation of the alternative 50SS selection of Bcl-x exon 2to favor Bcl-x(L) expression. Furthermore, PKCi is a well-established oncogene for NSCLC as reported by Regala andcolleagues (51). Therefore, PKCimay in one aspect act as anoncogene via simple removal of Bcl-x(s) with concomitantincrease in Bcl-x(L) and promoting oncogenesis.In conclusion, this study shows that the PI3K/PKCi

pathway regulates the alternative 50SS selection of Bcl-xexon 2 in NSCLC cells. Second, this study provides datathat the proto-oncogene PKCi regulates this distal mecha-nism via the expression of SAP155, an RNA trans-factor thatregulates the 50SS selection of Bcl-x pre-mRNA.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.

Grant SupportThis work was supported by grants from the Veteran's Administration (VA Merit

Review I and a Research Career Scientist award to C.E. Chalfant); from the NIHHL072925, CA117950, CA154314 to C.E. Chalfant; NH1C06-RR17393 (toVirginia Commonwealth University for renovation), a T32 Post-Doctoral Fellowship(Postdoctoral Training Program in Cancer Biology, CA085159; to B.A. Shapiro); andfrom the Vietnam Education Foundation (VEF) as a predoctoral/postdoctoral fel-lowship (N. Vu).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received July 29, 2011; revised January 30, 2012; accepted March 7, 2012;published OnlineFirst April 20, 2012.

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2012;10:660-669. Published OnlineFirst April 20, 2012.Mol Cancer Res Jacqueline C. Shultz, Ngoc Vu, Michael D. Shultz, et al. Bcl-x Pre-mRNA

Regulates the Alternative Splicing ofιThe Proto-oncogene PKC

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TheProto-oncogenePKCiRegulatestheAlternativeSplicing of ... · located in exon 2 of Bcl-x pre-mRNA 277 to 295 bp upstreamfromintron2(30).Infurthermechanisticstudies, the involvement

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