Splicing Factor hnRNP A2/B1 Regulates Tumor Suppressor Gene Splicing...

Molecular and Cellular Pathobiology

Splicing Factor hnRNP A2/B1 Regulates Tumor SuppressorGene Splicing and Is an Oncogenic Driver in Glioblastoma

Regina Golan-Gerstl1, Michal Cohen1, Asaf Shilo1, Sung-Suk Suh2, Arianna Bak�acs3,Luigi Coppola3, and Rotem Karni1

AbstractThe process of alternative splicing is widely misregulated in cancer, but the contribution of splicing regulators

to cancer development is largely unknown. In this study, we found that the splicing factor hnRNP A2/B1 isoverexpressed in glioblastomas and is correlated with poor prognosis. Conversely, patients who harbor deletionsof the HNRNPA2B1 gene show better prognosis than average. Knockdown of hnRNP A2/B1 in glioblastoma cellsinhibited tumor formation in mice. In contrast, overexpression of hnRNP A2/B1 in immortal cells led tomalignant transformation, suggesting that HNRNPA2B1 is a putative proto-oncogene. We then identified severaltumor suppressors and oncogenes that are regulated by HNRNPA2B1, among them are c-FLIP, BIN1, andWWOX,and the proto-oncogene RON. Knockdown of RON inhibited hnRNP A2/B1 mediated transformation, whichimplied that RON is one of the mediators of HNRNPA2B1 oncogenic activity. Together, our results indicate thatHNRNPA2B1 is a novel oncogene in glioblastoma and a potential new target for glioblastoma therapy. Cancer Res;71(13); 4464–72. �2011 AACR.

Introduction

The process of alternative splicing is widely misregulated incancer, andmany tumors express newsplicing isoforms that areabsent in the corresponding normal tissue (1–3). Many onco-genes and tumor suppressors are differentially spliced in cancercells, and it has been shown that many of these cancer-specificisoforms contribute to the transformed phenotype of cancercells (4–6). However, the contribution of alternative splicingregulators to cancer development has been largely unknown.Only recently the first functional evidence showed that somesplicing factors can act as potent oncogenes and are upregu-lated in human cancers (7, 8). hnRNP proteins are abundantRNA-binding proteins expressed in most human tissues (9, 10).The hnRNP A/B family is a subset of hnRNP proteins withclosely related sequences and a conserved modular structure(10). They can affect alternative splicing, frequently by antag-onizing SR proteins, in part, through the recognition of exonicsplicing silencers elements (11–13). Additional functions ofthese proteins in postsplicing events, such asmRNA trafficking,

and replication and transcription of cytoplasmic RNA viruses,have also been reported (10). Recent studies have shown thathnRNP A1 also affects the maturation of specific miRNAsamong them pre-miR-18a, which is part of a cluster of miRNAswith oncogenic activity (onco-mIRs; refs. 14–16). Previousstudies showed the overexpression of hnRNP A1 and hnRNPA2/B1 in lung and breast cancer (17, 18). Moreover, knockdownof hnRNPA1andA2/B1 inbreast cancer cells induced apoptosisthat was specific for cancer cells (19). However, a direct role forHNRNPA2B1 as an oncogene in human cancer has not yet beenshown. Recent studies found that hnRNP A1 and A2/B1 mod-ulate alternative splicing of the glycolytic PKM2 enzyme incancer cells, suggesting a possible role for hnRNP A1 andA2/B1in the regulation of tumor metabolism (20, 21).

Another unsolved question is the biochemical and biologicaldifference betweenmembers of the hnRNP A/B protein family.To date, their splicing activities, both in vitro and in knockdownor transfection assays, showed similar splicing effects onseveral substrates (11, 20–22). Thus, it is not clear to whatextent there is redundancy in their splicing targets and biolo-gical activities. We found that hnRNP A2/B1, but not any otherhnRNP A/B or SR protein, is a prognostic marker for glioblas-toma patient survival and a potent oncogene in glioblastomadevelopment and tumor maintenance (Figs. 1–3; Supplemen-tary Figs. S1–S3). In a search for its alternative splicing targets,we found that hnRNP A2/B1 modulates alternative splicing ofthe tumor suppressors BIN1, WWOX, the antiapoptotic pro-teins c-FLIP and caspase-9B, the insulin receptor (IR), and theRON proto-oncogene among others (Fig. 4; SupplementaryTable S1). In all of these splicing events, hnRNP A2/B1enhanced the expression of the oncogenic isoforms of thesegenes (Fig. 4). We further show that RON knockdown inhibitstransformation of glioma cells overexpressing exogenous

Authors' Affiliations: 1Department of Biochemistry and Molecular Biol-ogy, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel; 2Department of MolecularVirology, Immunology and Medical Genetics, Comprehensive CancerCenter, The Ohio State University, Columbus, Ohio; and 3UOC AnatomiaPatologica, Azienda-Complesso Ospedaliero San Filippo Neri, Rome, Italy

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Rotem Karni, Hebrew University, Ein Karem,Jerusalem 91120, Israel. Phone: 972-2-6758289; Fax: 972-2-6757379;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4410

�2011 American Association for Cancer Research.

CancerResearch

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hnRNP A2/B2, suggesting that RON is an important target inhnRNP A2/B1–mediated transformation (Fig. 5).

Materials and Methods

CellsU87MG and T98G cells were grown in Dulbeco's modified

Eagle's medium (DMEM), supplemented with 10% fetal

calf serum (FCS), penicillin and streptomycin. NIH 3T3 cellswere grown in DMEM supplemented with 10% calf serum(CS), penicillin, and streptomycin. To generate stable trans-ductant pools, NIH 3T3 and U87MG cells were infected withpBABE-puro retroviral vectors expressing T7-tagged humanhnRNP A2 cDNA. At 24 hours after infection, the medium wasreplaced, and 24 hours later, infected cells were selected withpuromycin (2 mg/mL) for 72 to 96 hours. In the case of

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Figure 1. hnRNP A2/B1 is upregulated in brain tumors, and the expression and gene copy number of hnRNP A2/B1 are inversely correlated with glioma patientsurvival. A, hnRNP A2/B1 expression in samples of glioblastoma, oligodendrogioma, and astrocytoma patients (red) compared with normal brainsamples (blue). Data taken from Oncomine (http://www.oncomine.org). B, qRT-PCR quantitation of hnRNP A2 expression was done on total RNA extractedfrom normal brain (blue) and glioblastoma WHO grade IV tumors (gray and red columns). Red columns represent tumor samples in which hnRNP A2expression is more than 2-fold over the average expression of the normal brain samples (normalized as 1 and represented as black line). C, Kaplan–Meiersurvival plots show the survival of glioma patients with differential expression of hnRNP A2/B1: samples with upregulation (red line; n ¼ 28). Samples withintermediate expression (yellow; n ¼ 316). The blue line represents the survival of all glioma patients. Log-rank P value (upregulated vs. intermediate):7.89948E-5. Data based on the National Cancer Institute REMBRANDT data set (http://rembrandt.mci.nih/gov). D, Kaplan–Meier plots show the correlationbetween hnRNP A2/B1 gene copy number and glioma patient survival. Samples with gene amplification (red line; n ¼ 179). Samples with gene deletion(green line; n ¼ 179). Blue line represents the survival of all glioma patients. Log-rank P value (upregulated vs. intermediate): 1.54771E-5. Data basedon the National Cancer Institute REMBRANDT data set (http://rembrandt.mci.nih/gov).

HNRNPA2B1 Is a Driving Oncogene in Glioblastoma

www.aacrjournals.org Cancer Res; 71(13) July 1, 2011 4465




infection with MLP-puro short hairpin RNA (shRNA) vectors,U87MG, T98G cells transductants were selected with puro-mycin (2 mg/mL) for 96 hours. For RON knockdown we usedHuman pLKO.1 lentiviral MST1R (RON) shRNA (Open Biosys-tems) sh RON-1 clone ID TRCN0000121161; sh RON-2 clone IDTRCN0000121151.

Anchorage-independent growthColony formation in soft agar was assayed as described (23).

Plates were incubated at 37�C and 5% CO2. After 14 to 21 days,colonies from 10 different fields in each of 2 wells werecounted for each treatment and the average number ofcolonies per well was calculated. The colonies were stainedas described (23) and photographed under a light microscopeat �100 magnification.

Tumorigenesis assays in nude miceU87MG cells expressing MLP-puro or MLP-puro contain-

ing hnRNP A2 shRNAs or NIH 3T3 cells overexpressinghnRNP A2 or an empty vector (pBABE) were injected(2 � 106 cells per site in 200 mL of PBS) subcutaneouslyinto each rear flank of (Atimic-Nu/Nu) nude mice by using a26-gauge needle. Tumor growth was monitored twice a weekas described (7).

ImmunoblottingCells were lysed in SDS and analyzed for total protein

concentration as described (7). A total of 30 or 20 mg of totalprotein from each cell lysate was separated by SDS-PAGE andtransferred onto a nitrocellulose membrane. The membraneswere blocked, probed with antibodies, and detected usingenhanced chemiluminescence detection. Primary antibodieswere as follows: anti-b-catenin (1:2,000; Sigma); anti-b-actin(1:2,000; Santa Cruz Biotechnology); anti-RON (1:1,000 SantaCruz); anti-hnRNAP A2/B1 (1:1,000; Santa Cruz); and T7 tag(1:5,000; Novagen). Secondary antibody was horseradish per-oxidase (HRP)-conjugated goat anti-mouse or anti-rabbit(1:10,000; Jackson Laboratories).

Growth curvesU87MG or NIH 3T3 cells were infected with the indicated

retroviruses. After selection, 5,000 cells per well were seeded in96-well plates. Cells were fixed and stained with methyleneblue as described (7), and the A650 of the acid-extracted stainwas measured on a plate reader (Bio-Rad).

Reverse transcriptase-PCRTotal RNA was extracted with TRIzol reagent (Sigma) and 2

mg of total RNA was reverse transcribed with AffinityScript II

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Figure 2. hnRNP A2/B1 is required for glioblastoma transformation.A, U87MG cells were transduced with retroviruses encoding hnRNPA2/B1–specific shRNA or empty vector [MLP(�)] and after selection cellswere analyzed by Western blotting for hnRNP A2/B1 protein expression.b-Catenin was used as a loading control. B, quantification of colonyformation in soft agar of cells described in A. Error bars represent SD(n ¼ 2). C, representative fields of colonies in soft agar described in B. D,cell proliferation of cells described in A was examined by methylene bluestaining. Error bars represent SD (n ¼ 6). E, U87MG cells transduced withempty vector (pBABE) or hnRNP A2/B1 (A2) were analyzed by Westernblotting for hnRNP A2/B1 protein expression, and b-catenin was used as aloading control. F, quantification of colony formation in soft agar of thecells described in E. Colonies were considered "Big colonies" if they

contained approximately more than 100 cells or were more than 1 mmin diameter. Error bars indicate SD (n ¼ 2). G, representative fields of softagar colonies described in F. H, cell proliferation of cells describedin E was examined by methylene blue staining. Error bars representSD (n ¼ 6). I, cells described in A were injected (106 cells/site)subcutaneously near both rear flanks of nude/nude mice, and tumorvolume was measured biweekly. Error bars indicate SD (n ¼ 8).J, representative mice described in I are shown.

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Cancer Res; 71(13) July 1, 2011 Cancer Research4466




(Stratagene). Reverse transcriptase-PCR (RT-PCR) was con-ducted on one tenth (1–2 mL) of the cDNA, in 25 to 50-mLreactions containing 0.2mmol/L deoxynucleotide (dNTP)mix,10� PCR buffer with 15 mmol/L MgCl2 (Invitrogen), 2.5 unitsof TaqGold (Invitrogen), and 0.2 mmol/L of each primer; 5%(v/v) DMSO was included in some reactions. PCR conditionswere 95�C for 5 minutes, then 35 cycles of 94�C for 30 seconds,60�C for 30 seconds, and 72�C for 45 seconds, followed by 10minutes at 72�C. PCR products were separated on 1.5% or 2%agarose gels or on 6% nondenaturing polyacrylamide gels.Primers are described in Supplementary Table S2.

Quantitative RT-PCRTotal RNA was extracted with Tri reagent (Sigma) and 2 mg

of total RNA was reverse transcribed using the AffinityScript

(Stratagene) reverse transcriptase. We determined the mRNAlevels of hnRNP A2/B1 in normal brain and glioblastomatumors by performing quantitative PCR with SYBER Green(SYBR Premix Ex Taq # RR041A) using the CFX96 (Bio-Rad)machine. Unknown samples were compared to a standardcurve, which is established by serial dilutions of a knownconcentration of cDNA. The standard that was used is b-actin.Threshold cycle from b-actin and unknown samples wereinserted to the standard curve formula and the final valuewas the ratio between the unknown sample divided by theb-actin standard gene. Primers: hnRNP A2/B1: For: TTTGAT-GACCATGATCCTGT; Rev: CTCTGAACTTCCTGCATTTC.b-actin (NM_001101) sense primer: GGCACCCAGCACAAT-GAAGA; antisense primer: AGGATGGAGCCGCCGATC. ThePCR reaction steps were 1 cycle at 95�C for 10 seconds, 40cycles of 95�C for 10 seconds, and 58�C for 20 seconds.

Results

The splicing factor hnRNP A2/B1 is upregulated inbrain cancer

We analyzed the expression of hnRNP A2/B1 inseveral types of brain cancers, using the Oncomine database(http://www.oncomine.org), and found hnRNP A2/B1 over-expression in tumor samples from glioblastoma, oligoden-drogioma, and astrocytoma patients compared with normalbrain tissues (Fig. 1A). These results support a recent findingthat hnRNP A2/B1, hnRNP A1, and polypyrimidine tract-binding protein (PTB) are overexpressed in glioblastoma(20). We further analyzed 3 normal brain (blue bars) and 22glioblastoma grade IV tumor samples by quantitative RT-PCR (qRT-PCR) and found that, in 19 of the 22 tumors (redbars), hnRNP A2/B1 was overexpressed more than 2-foldthan the average expression in the normal brain samples(Fig. 1B).

The expression and gene copy number of the splicingfactor hnRNP A2/B1 are inversely correlated withglioma patient survival

Using the REMBRANDT database of the National CancerInstitute (https://caintegrator.nci.nih.gov), we analyzed thecorrelation of hnRNP A2/B1 expression and gene copynumber and patient survival. We found a very significantinverse correlation (P ¼ 2.62E-5) between patient survivaland elevated expression or gene copy number ofHNRNPA2B1 (Fig. 1C and D). These results indicate thatpatients with tumors harboring elevated copy number ofHNRNPA2B1 have poor prognosis of survival. Analysis of thesame samples for overexpression of known oncogenes (MYCand EGFR) or downregulation of know tumor suppressors(TP53 and TP73) did not show significant correlation withpatients survival (Supplementary Figs. S1 and S2). Genecopy number of the tumor suppressor PTEN, however,showed significant correlation with patient survival, andAkt2 expression and copy numbers were also inverselycorrelated with patient survival (Supplementary Fig. S3)corroborating previous findings (24). It is importantto mention that hnRNP A2/B1 prognostic value was

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Figure 3. hnRNP A2/B1 can transform NIH 3T3 cells in vitro and in vivo. A,NIH 3T3 cells transduced with empty vector (pBABE) or hnRNP A2/B1 (A2)were analyzed by Western blotting for hnRNP A2/B1 protein expressionand b-catenin was used as a loading control. B, quantification of colonyformation in soft agar of cells described in A. Error bars represent SD(n ¼ 2). C, representative fields of colonies in soft agar described in B. D,proliferation of cells described in A was examined by methylene bluestaining. Error bars indicate the SD (n ¼ 6). E, cells described in A wereinjected (106 cells/site) subcutaneously near both rear flanks of nude/nudemice, and tumor volume was measured biweekly. Error bars indicate SD(n ¼ 8). F, representative mice described in E are shown. G, lightmicrographs of formalin-fixed, paraffin-embedded tissue sections fromtumors derived from NIH 3T3 cells overexpressing hnRNP A2/B1, stainedwith hematoxylin and eosin.






statistically better than AKT2 (Fig. 1C and D) and thus itmight be a better biomarker for glioma prognosis. Interest-ingly, neither gene copy number nor expression of the othersplicing factors from the hnRNP A/B protein family (hnRNP

A1, hnRNP A0, and hnRNP A3) or SR protein family showedsignificant inverse correlation with patient survival (Supple-mentary Fig. S4).

hnRNP A2/B1 is required for glioblastomatransformation and tumorigenicity

To examine the importance of hnRNP A2/B1 to gliomatumor maintenance, hnRNP A2/B1 was knocked down inU87MG and T98G cells or overexpressed in the U87MGglioblastoma cell lines and anchorage-independent growth

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Figure 4. hnRNP A2/B1 regulates the alternative splicing of tumorsuppressors and oncogenes. U87MG glioblastoma cancer cells weretransduced with retroviruses encoding shRNA empty vector (MLP), orhnRNP A2/B1–specific shRNA, empty pBABE vector, or pBABEcontaining hnRNP A2/B1. After selection cells were lysed, RNA isolated,and the alternative splicing patterns of RON, INSR, ENAH, CFLAR, BIN,CASP9, and WWOX were examined by RT-PCR, using the indicatedisoform-specific primers (arrowheads). The splice variants are indicated byboxes at the right side of each gel. A, RON exon 11 skipping/inclusion.B, INSR exon 11 skipping/inclusion. C, ENAH exon 11a skipping/inclusion.D, CFLAR exon 7 skipping/inclusion. E, BIN1 exons 12a and 13 skipping/inclusion. F, CASP9 exons 3 to 6 skipping/inclusion. G,WWOX exons 6 to8 skipping/inclusion. H, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) control. Numbers under each panel represent the average andSD of fold change in the inclusion of the indicated exon compared with theempty vectors (normalized as 1) in 2 to 4 independent experiments.

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was analyzed by colony formation in soft agar. Colony for-mation of U87MG and T98G cells with hnRNP A2/B1 knock-down was reduced in contrast to increased colony numberand size in glioblastoma cells overexpressing hnRNP A2/B1(Fig. 2A–C and E–G; Supplementary Fig. S5). One of thefeatures that can contribute to colony formation is the pro-liferation rate of the cells. Notably, for tumor development andprogression, other aspects such as motility, invasiveness, theability to grow in an anchorage-independent manner and toresist apoptotic cues can be more prominent. To examinewhether hnRNP A2/B1 plays a role in proliferation, wemeasured proliferation rates of U87MG glioblastoma cellswith up- and downregulation of hnRNP A2/B1. The knock-down of hnRNP A2/B1 did not reduce the viability of theglioblastoma cells and slightly inhibited their ability to pro-liferate on plastic in 10% serum but inhibited their prolifera-tion in lower serum concentrations (Fig. 2D; SupplementaryFig. S6), suggesting that hnRNP A2/B1 contributes to cellproliferation in low growth factor conditions. Importantly,in hnRNP A2/B1 knockdown and overexpression cell pools,hnRNP A2/B1 silencing and overexpression were sustainedover the growth curve period, eliminating the possibility thatcell density or an aberrant clone could overcome HNRNPA2B1silencing (Supplementary Fig. S6G–H). However, overexpres-sion of hnRNP A2/B1 increased the proliferation rate ofU87MG and NIH 3T3 cells in all growth factor concentrations(Figs. 2H and 3D; Supplementary Fig. S6A–C), indicating that itcan contribute to enhanced proliferation at both low and highgrowth factor conditions when overexpressed. To determinethe importance of hnRNP A2/B1 for glioma tumor formationin vivo, nude mice were injected with U87MG cells with andwithout knockdown of hnRNP A2/B1. Cells with empty vectorgave rise to fast-growing large tumors whereas mice injectedwith U87MG cells, in which hnRNP A2/B1 was knocked down,developed either small tumors in some of the injection sites orno tumors at all (Fig. 2I and J). The importance of hnRNP A2/B1 to brain cancer maintenance was also confirmed in theT98G glioma cell line (Supplementary Fig. S5). T98G cells withhnRNP A2/B1 knockdown showed reduced colony formationin soft agar, similar to results in U87MG cells (SupplementaryFig. S5).

hnRNP A2/B1 overexpression transformed NIH 3T3 andconverted them to be tumorigenicOn the basis of our results that hnRNP A2/B1 is required

for the glioblastoma-transformed phenotype, we examinedthe ability of this factor to transform normal immortal cells.Upregulation of hnRNP A2/B1 in NIH 3T3 cells inducedcolony formation in soft agar in contrast to control cellsthat did not form colonies (Fig. 3A–C). Moreover, these cellsformed fast-growing tumors when injected subcutaneouslyinto nude mice (Fig. 3E and F). Pathologic analysis of thetumors formed by cells overexpressing hnRNP A2/B1showed that these tumors look like high-grade invasivesarcoma with high mitotic index (Fig. 3G). These resultssuggest that HNRNPA2B1 is a driving oncogene on itsown and probably directly contributes to glioblastomadevelopment.

hnRNP A2/B1 levels affect the alternative splicing ofseveral tumor suppressors and oncogenes

To examine the effect ofHNRNPA2B1 on alternative splicingin glioma tumor cells, we analyzed the alternative splicingpattern of several genes for which alternatively spliced iso-forms have been characterized and for some of them shown tocontribute to transformation, invasion, and apoptosis or to beaffected by hnRNP A2/B1 (Fig. 4; Supplementary Fig. S7 andTable S1). Knockdown of hnRNP A2/B1 in U87MG cellsincreased the inclusion, whereas upregulation of hnRNPA2/B1 increased the skipping of exon 11 of RON, a tyrosinekinase receptor involved in the invasiveness and motility oftumor cells (refs. 25, 26; Fig. 4A). These results suggest thatsimilar to the splicing factor oncoprotein SRSF1 (SF2/ASF),hnRNP A2/B1 upregulation contributes to cellular transfor-mation by increased skipping and upregulation of the DRONoncogenic splicing isoform (25). Importantly, the levels ofSRSF1 (SF2/ASF) or SRSF6 (SRp55), another SR protein,and the levels of hnRNP A1, another hnRNP A/B familymember, did not change upon hnRNP A2/B1 down- or upre-gulation (Supplementary Fig. S8). In NIH 3T3 cells overexpres-sing hnRNP A2, we could detect only one isoform of RON,suggesting that either this splicing event occurs only inhumans or that it hardly occurs in fibroblasts. The IR is themain mediator of insulin metabolism and glucose levels in thebody and is expressed in most tissues. Skipping of exon 11from the INSR transcript generates the splicing variant IR-Athat binds the growth factor insulin-like growth factor II (IGF-II), in addition to insulin. This splicing variant is overproducedin many cancers and has been implicated in an autocrine loopin cancer cells (27, 28). hnRNP A2/B1 knockdown increasedthe inclusion of exon 11 whereas its overexpression increasedexon 11 skipping generating the mitogenic isoform (Fig. 4B).The ENAH gene has been shown to play a role in the epithelial-to-mesenchymal transition process and to affect cellularmotility and invasion. Overexpression did not affect theinclusion of exon 11a, suggesting that it is not a splicingtarget of hnRNP A2/B1 in U87MG cells (Fig. 4C). CFLAR (c-FLIP) is an antiapoptotic protein that is alternatively splicedand has been shown not only to inhibit TNF and TNF–relatedapoptosis-inducing ligand (TRAIL)-induced apoptosis (29, 30)but also to enhance motility and invasion through activationof the mitogen-activated protein kinase-extracellular signalregulated kinase (MAPK-ERK) pathway (31, 32). We found thathnRNP A2/B1 downregulation decreased the levels of the longisoform of c-FLIP whereas overexpression of hnRNP A2/B1increased the level of the long isoform, raising the possibilitythat upon hnRNP A2/B1 knockdown, glioma cells mightbecome more sensitive to apoptotic stimuli and at the sametime be less motile and less invasive (ref. 31; Fig. 4D). Thetumor suppressor BIN1 has been shown to be regulated byalternative splicing and inclusion of exon 12a of BIN1 inacti-vates its tumor suppressor activity (33, 34). BIN1 exon 12a wasalso identified as a target of the SR protein SRSF1 (SF2/ASF;ref. 7). We found that similar to the effect of SRSF1 over-expression, hnRNP A2/B1 overexpression in both U87MG andNIH 3T3 cells enhanced exon 12a inclusion generating theantiapoptotic isoform of BIN1 whereas its knockdown






enhanced exon 12a skipping (Fig. 4E; Supplementary Fig. S7).CASP9, the gene coding for caspase-9 has been previouslyshown to be alternatively spliced by skipping of exons 3 to 6 togenerate a truncated dominant negative isoform that inhibitsapoptosis and is overexpressed in several cancers (35, 36). Wefound that hnRNP A2/B1 knockdown enhanced the produc-tion of full-length caspase-9 whereas its overexpressionenhanced skipping of exons 3 to 6 generating the antiapop-totic isoform caspase-9B (Fig. 4F). WWOX is a known tumorsuppressor that resides at a common fragile site (37) and isfrequently inactivated in several types of cancer includingglioblastoma (38). Skipping of exons 6 to 8 ofWWOX has beenreported in breast cancer (39). We found that hnRNP A2/B1knockdown enhanced inclusion of alternatively spliced exons6 to 8 whereas its overexpression induced skipping of theseexons (Fig. 4G). Skipping of these exons causes deletion of 180amino acids including its substrate binding domain and itsalcohol dehydrogenase domain probably inactivating its cat-alytic activity (39). However, the functional role of theseskipped isoforms requires further examination. WWOXtumor-suppressive activity is related to its anti-invasive andantiapoptotic functions (37, 39), which are a common themeshared with the other targets of hnRNP A2/B1 we identified.hnRNP A2/B1 also affected the alternative splicing of exon 7Bof hnRNP A1, another member of the hnRNP splicing factorfamily (Supplementary Figs. S7 and S8A). Skipping of this exonhas been shown to be affected by hnRNP A1 and hnRNP A2/B1levels in an autoregulatory fashion and thus our resultscorroborate these previous findings (40). The levels of hnRNPA2/B1 also affected the alternative splicing of other genes,including known HNRNPA2B1 targets such as exon 7 of theSMN gene and other splicing events (Supplementary Fig. S7and Table S1). Importantly, many splicing events we examinedwere not affected by HNRNPA2B1 depletion or overexpression(Supplementary Table S1).

Knockdown of RON reverses transformation ofglioblastoma cells overexpressing hnRNP A2/B1

To examine whether RON contributes to hnRNP A2/B1–mediated transformation, we knocked down the expression ofthe RON proto-oncogene in U87MG cells overexpressing ecto-pic hnRNP A2. (Fig. 5A). We found that stable knockdown by 2different shRNAs reduced RON levels and inhibited colonyformation in soft agar of U87MG cells (Fig. 5B and C). Theseresults indicate that RON is one of the important mediators ofhnRNP A2/B1 oncogenic activity in glioblastoma cells, eventhough other targets can probably contribute as well.

Discussion

An emerging body of data shows that alternative splicingmisregulation plays an important role in cancer developmentand tumor maintenance (5–8). Alternative splicing regulatorsfrom the SR and hnRNP A/B protein families are overex-pressed or downregulated in various cancers and can probablyaccount for many of the cancer-specific alternative splicingchanges. Some alternative splicing regulators such as the SRprotein SRSF1 (SF2/ASF) have been shown to be upregulated

in many cancers and act as potent oncogenes when slightlyoverexpressed (7).

hnRNP A/B proteins are important regulators of alternativesplicing and in several examples of in vitro and in vivo splicingassays seemed to act antagonistically to SR proteins (11–13).Even though hnRNP A/B proteins can alter alternative splicingin an opposite manner to oncogenic splicing factors such asSRSF1 (SF2/ASF), there is no evidence that they act as tumorsuppressors, and, on the contrary, members of the hnRNP A/Bprotein family are overexpressed in some cancers (7, 17–20)and change the splicing pattern of genes that contribute to thetransformed phenotype (20, 21, 41). Moreover, recently, it hasbeen shown that the splicing factors hnRNP A1 and A2/B1 aswell as PTB are under the transcriptional control of the mycproto-oncogene and can modulate the splicing of PKM2,activating a metabolic switch to aerobic glycolysis that is ahallmark of cancer cells (the "Warburg effect"; refs. 20, 21).However, to date, there is no direct evidence that any memberof the hnRNP A/B family is an oncogene that can transformcells and is genetically upregulated (amplified, translocated, ormutated) in cancer. We show here that the splicing factorhnRNP A2/B1 is overexpressed in gliomas compared withnormal brain samples in many cases because of the amplifica-tion of the HNRNPA2B1 gene (Fig. 1A–D). Moreover, over-expression and amplification of hnRNP A2/B1 correlate withpoor prognosis of glioma patients whereas deletion of theHNRNPA2B1 gene correlates with better prognosis thanaverage (Fig. 1C and D). Importantly, none of the other hnRNPA/B proteins, as well as the SR proteins or some other knownoncogenes, showed significant inverse correlation with survi-val of glioma patients (Supplementary Figs. S1 and S2). Inaccordance with previous findings, deletion of the tumorsuppressor PTEN and overexpression of the proto-oncogeneAkt2 showed significant inverse correlation with gliomapatient survival (24; Supplementary Fig. S3). Taken together,these findings suggest that hnRNP A2/B1 is a valuable prog-nostic marker for glioblastoma development and patientsurvival. To examine whether hnRNP A2/B1 is causativelyinvolved in glioblastoma tumor development, we downregu-lated the expression of hnRNP A2/B1. We found that knock-down of hnRNP A2/B1 in U87MG glioblastoma cells partiallyinhibited their proliferation, especially in low-serum condition(Fig. 2D; Supplementary Fig. S6). This result suggests that thecell cycle and viability may not be the major antioncogeniceffectors in cells with hnRNP A2/B1 knockdown. However,knockdown of hnRNP A2/B1 inhibited colony formation insoft agar as well as tumor formation in nude mice of glio-blastoma cells (Fig. 2A–C, I, and J; Supplementary Fig. S5),suggesting that hnRNP A2/B1 is important for glioblastomatumor development and maintenance. Moreover, overexpres-sion of hnRNP A2/B1 in the U87MG glioblastoma cellsenhanced their proliferation in all serum concentrationsand increased colony size and number in soft agar(Fig. 2E–H; Supplementary Fig. S6), indicating that hnRNPA2/B1 may not be a limiting factor when growth factors arepresent but plays a positive role in glioblastoma transforma-tion when upregulated. To examine whether HNRNPA2B1 actsas an oncogene, we slightly overexpressed (2-fold) hnRNP A2/

Golan-Gerstl et al.





B1 in immortal mouse fibroblasts (NIH 3T3 cells) and exam-ined their oncogenic properties. NIH 3T3 cells overexpressinghnRNP A2/B1 became transformed, formed colonies in softagar, and were tumorigenic in nude mice forming tumors withhallmarks of high-grade sarcomas (Fig. 3). These data suggestthat HNRNPA2B1 acts as a proto-oncogene when slightlyupregulated and thus it is not only a marker but also a drivingoncogene in glioblastoma development. To identify possiblealternative splicing targets of hnRNPA2/B1 thatmightmediateits oncogenic activity, we examined the alternative splicingpattern of several genes known to undergo alternative splicingin cancer and to contribute to the transformed phenotype. Weidentified several alternative splicing events modulated byhnRNP A2/B1, among them the tumor suppressor BIN1, theantiapoptotic gene CFLAR (c-FLIP), and CASP9 all regulators ofthe apoptotic process (29–36). The tumor suppressor geneWWOX, the INSR gene, the long isoform of CFLAR, and the RONtyrosine kinase receptor are all involved in motility and inva-sion (25–28, 31, 32, 37–39). Our results suggest that hnRNP A2/B1 activates an alternative splicing program that enhances theproduction of antiapoptotic isoforms of genes such as BIN1,CASP9, and CFLAR, as well as invasion-promoting isoformssuch as DRON, CFLAR-long, and IR-A, which contribute totransformation and invasion.Surprisingly, in several cases of alternative spliced exons

shown in this article (BIN1 exon 12a and RON exon 11),hnRNP A2/B1 showed similar splicing effects to the onco-genic SR protein SRSF1 (SF2/ASF), raising the possibilitythat in many cases in vivo, these splicing factor are notantagonistic as previously thought from in vitro and tran-sient-transfection splicing assays (11–13). hnRNP A2/B1 is ageneral splicing factor and it is expected to change thesplicing of many (currently unknown) transcripts. We knowthat the alternative splicing of several transcripts did notchange upon up- or downregulation of hnRNP A2/B1 (Fig. 4;Supplementary Table S1). We are limited by the smallnumber of transcripts tested, and only a genome-wideanalysis will reveal how many transcripts are regulateddirectly and indirectly by hnRNP A2/B1.To examine whether RON is an important target that

mediated hnRNP A2–induced transformation, we stablyknocked down RON in U87MG glioblastoma cells overexpres-sing exogenous hnRNP A2 cDNA and found that RON knock-down significantly inhibited transformation of these cells,similar to the effect of hnRNP A2/B1 knockdown (Fig. 5).We conclude that RON is one of the important mediators ofhnRNP A2–induced transformation and contributes to thetransformed phenotype of glioblastoma cells.

Because hnRNP A2/B1 is a general alternative splicingfactor and participates in RNA processing steps other thansplicing (10, 12), we assume that it has many splicingtargets other than RON and that it targets other RNAprocessing steps (such as miRNA maturation; refs. 14, 15)that contribute to its transforming activity. For example,hnRNP A1 has been shown to promote the maturation ofonco-miRNAs such as miR-18a and to downregulate tumor-suppressive miRNAs such as Let-7a (14, 42). hnRNP A2/B1has also been linked to tumor metabolism (20, 21, 43). It hasbeen shown that hnRNP A2/B1 is downregulated posttran-scriptionally by the tumor suppressor gene VHL, which islost in many tumors and induces aerobic glycolysis (the"Warburg effect") through hypoxia-inducible factor 1-alpha(HIF-1a) stabilization (43). It will be intriguing to examinewhether hnRNP A2/B1 can affect HIF-1a stabilization bycompetitive binding to VHL when overexpressed. In recentstudies, hnRNP A2/B1 has been shown to be transcription-ally controlled by the myc proto-oncogene and to regulatethe splicing of the glycolytic enzyme PKM2 contributing tothe induction of aerobic glycolysis by another mechanism(20, 21).

Taken together, our data suggest that HNRNPA2B1 is a newbiomarker for glioblastoma patient survival and a new proto-oncogene that regulates the splicing and other RNA proces-sing steps of several tumor suppressors and oncogenes.Furthermore, downregulating hnRNP A2/B1 levels in glioblas-toma cells should be considered as a new strategy for glio-blastoma therapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Prof. Oded Meyuhas and Dr. Zahava Kluger for commentson the manuscript.

Grant Support

This study was partially supported by grants from the Israel Cancer Associa-tion; Israel Science Foundation; Marie Curie IRG re-integration grant (FP7); andthe Concern Foundation (Los Angeles, CA).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received December 7, 2010; revised April 14, 2011; accepted May 5, 2011;published OnlineFirst May 17, 2011.

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2011;71:4464-4472. Published OnlineFirst May 17, 2011.Cancer Res Regina Golan-Gerstl, Michal Cohen, Asaf Shilo, et al. Gene Splicing and Is an Oncogenic Driver in GlioblastomaSplicing Factor hnRNP A2/B1 Regulates Tumor Suppressor

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