Cohen-Eliav et al. Final PDF 2013

Journal of PathologyJ Pathol 2013; 229: 630–639Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.4129

ORIGINAL PAPER

The splicing factor SRSF6 is amplified and is an oncoproteinin lung and colon cancersMichal Cohen-Eliav,1 Regina Golan-Gerstl,1 Zahava Siegfried,1 Claus L Andersen,2 Kasper Thorsen,2Torben F Ørntoft,2 David Mu3 and Rotem Karni1*

1 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University - Hadassah MedicalSchool, Jerusalem, Israel2 Department of Molecular Medicine, Aarhus University Hospital, Denmark3 Department of Pathology, H083, Penn State University, Hershey Medical Center, PA, USA

*Correspondence to: Rotem Karni, Department of Biochemistry and Molecular Biology, Hebrew University - Hadassah Medical School, Jerusalem,91120, Israel. e-mail: [email protected]

AbstractAn increasing body of evidence connects alterations in the process of alternative splicing with cancer developmentand progression. However, a direct role of splicing factors as drivers of cancer development is mostly unknown.We analysed the gene copy number of several splicing factors in colon and lung tumours, and found that the geneencoding for the splicing factor SRSF6 is amplified and over-expressed in these cancers. Moreover, over-expressionof SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-inducedcell death and converted them to be tumourigenic in mice. In contrast, knock-down of SRSF6 in lung and coloncancer cell lines inhibited their tumourigenic abilities. SRSF6 up- or down-regulation altered the splicing ofseveral tumour suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumour-suppressiveisoforms. Our data suggest that the splicing factor SRSF6 is an oncoprotein that regulates the proliferation andsurvival of lung and colon cancer cells.Copyright 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: SRSF6; splicing; cancer; colon; lung; amplification; oncoprotein

Received 11 July 2012; Revised 3 October 2012; Accepted 9 October 2012

No conflicts of interest were declared.

Introduction

The process of alternative splicing is deregulated incancer and recently it has been shown that some splic-ing factors act as oncogenes and directly contribute tocancer development and progression [1–3]. Moreover,it has been demonstrated recently that in some pre-malignant conditions, such as myeloid haematopoieticmalignancies, there are somatic mutations in spliceo-somal components, indicating that splicing is also dys-regulated at the genetic level [4–6]. However, thecontribution of alternative splicing regulators to thedevelopment and progression of cancer and the mecha-nisms by which they are up- or down-regulated in can-cer are still largely unknown. SR proteins are importantregulators of RNA biogenesis, including constitutiveand alternative splicing, mRNA transport and transla-tion [7–10]. Over-expression of SR proteins in cancercells has been reported in several studies [1,11] butonly a few papers showed direct involvement of aSR protein in cancer development [1,12,13]. SRSF6is a prototypical SR protein that contains two RNA

recognition motifs (RRMs) and an RS domain, can reg-ulate alternative splicing and can shuttle between thenucleus and the cytoplasm [14–19]. The Drosophilahomologue of SRSF6, B52, is essential for properdevelopment and both its loss of function and over-expression abrogate proper development [18,20,21].B52 was also associated with cell cycle regulation byregulating the activity of the dE2F transcription fac-tors [22]. Another proposed activity of SRSF6 is toprotect the genome from the formation of DNA–RNAhybrids (R-loops) following transcription [19], a pro-cess which can cause DNA damage and accumulationof mutations [23]. Some reports implicated SRSF6 inalternative splicing regulation of the apoptic gene BIM ,suggesting that SRSF6 is important for the generationof the pro-Apoptotic isoform [24]. Other reports sug-gested that SRSF6 might play a role in the responseto DNA damage [25]. In addition, SRSF6 was shownto regulate the splicing of VEGF, inducing the anti-Angiogenic isoform of VEGF [26,27]. However, todate, SRSF6 has not been implicated in cancer devel-opment. Furthermore, the extent of SR protein geneamplification, an important genetic mechanism that

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SRSF6 is amplified and is an oncoprotein in lung and colon cancers 631

causes elevated expression of proto-oncogenes in can-cer, has never been thoroughly investigated. In thisstudy we examined the extent of SR protein gene copynumber changes in colon and lung cancers and foundthat SRSF6 is extensively amplified in colon, lungand breast cancers. We then investigated the oncogenicactivities of SRSF6 and found that slight elevation inSRSF6 levels can enhance proliferation, protect fromcell death and lead to transformation and tumourigenic-ity of immortal lung cells. Finally, we identified severaltargets that might explain some of SRSF6’s activities.

Materials and methods

Cells and viral transductionsMLE cells were generated as described previously[28,29]. MLE cells were grown in HITES medium;BEAS-2B cells were grown in BEB medium (Lonza);NCI-H460 and LcLc-103 H cells in RPMI; andRKO cells in Dulbecco’s modified Eagle’s medium(DMEM). All the media were supplemented with 10%fetal calf serum (FCS), 2 mM l-glutamine, 0.1 mg/mlpenicillin and 0.1 mg/ml streptomycin. For over-expression, cells were infected with pBABE-puroretroviral vector [30] expressing T7-tagged humanSRSF6 cDNA. The medium was replaced 24 h afterinfection, and 24 h later infected cells were selected forby the addition of puromycin (2 µg/ml) for 72–96 h.For knock-down experiments, cells were infected withMLP-puro-shRNAs vectors and cell transductants wereselected for with puromycin (2 µg/ml) for 96 h. shRNAsequences appear in Table S2 (see Supplementarymaterial).

Tumour samplesTumour samples were obtained from the Coopera-tive Human Tissue Network (CHTN; Philadelphia, PA,USA). The lung tumours were stage III adenocarci-nomas. The breast tumours were invasive ductal car-cinomas. The normal tissues were the histologicallynormal tissues adjacent to cancerous tissues. DNA copynumbers and expression of SRSF6 were determined byqPCR or qRT–PCR, as described below.

Gene copy number measurementsBlood and colorectal tumour samples were collectedat the Department of Surgery, Aarhus UniversityHospital, Aarhus, Denmark. All tissue samples werecollected immediately after surgery and snap-frozenin liquid nitrogen. Informed written consent wasobtained from all patients, and research protocols wereapproved by the Central Denmark Region Committeeson Biomedical Research Ethics.

Tumour and matched germline DNA (extracted fromblood and/or adjacent normal mucosa) from 37 adeno-mas and 28 carcinomas were labelled and hybridized

to the SNP 6.0 array, according to the manufacturer’sinstructions (Affymetrix, Santa Clara, CA, USA). Datapreprocessing, normalization, probe summarization andcalculation of raw total copy-number estimates weredone using the CRMAv2 method implemented in theAroma.affymetrix R package [31]. Segmentation ofthe .cn files produced by Aroma.affymetrix were doneusing the Rseg package [32]. This package allowssample-specific thresholds for calling gains and lossesto be defined and corrects for artifacts induced by nor-malization in case of unbalanced abnormalities. Gainor loss at the specified gene loci were called when thelog ratio copy number between tumour and germlinewas ≥ 0.17 or ≤ −0.17, respectively.

ImmunoblottingCells were lysed in Laemmli buffer and analysedfor total protein concentration as described previously[1]. 30 µg total protein from each cell lysate wasseparated by SDS–PAGE and transferred to a nitro-cellulose membrane. The membranes were blocked,probed with antibodies and detected using enhancedchemiluminescence. Primary antibodies were againstβ-catenin (1:2000; Sigma); SRp55 (SRSF6) (mAb 8-1-28 culture supernatant); T7 tag (1:5000; Novagen); andGAPDH (1:2000; Santa Cruz). Secondary antibodieswere HRP-conjugated goat anti-mouse, goat anti-rabbitor donkey anti-goat IgG (H + L) (1:10, 000; JacksonLaboratories).

Anchorage-independent growthColony formation in soft agar was assayed as describedpreviously [1]. Plates were incubated at 37 ◦C and5% CO2. After 10–18 days, colonies were countedfrom 10 different fields in each of two wells foreach transductant pool and the average number ofcolonies/well was calculated. The colonies were stainedand photographed under a light microscope at ×100magnification.

Growth curvesTransductant pools of MLE or BEAS-2B cells wereseeded at 2500 or 5000 cells/well in 96-well plates.Every 24 h, cells were fixed and stained with methyleneblue, as described previously [1], and the absorbanceat 650 nm of the acid-extracted stain was measured ona plate reader (BioRad).

Survival assaysMLE cells were transduced with the indicated retro-viruses. Following selection, 1 × 104 cells/well wereseeded in 96-well plates; 24 h later, the cells wereserum-starved for another 24 h. At 24 h before treat-ment, one 96-well plate was fixed and served as anormalizing control (‘time 0’). After starvation, themedium was replaced with starvation medium con-taining the indicated concentrations of CDDP (Sigma)and the cells were incubated for an additional 24 h.


632 M Cohen-Eliav et al

Cells were fixed and stained with methylene blue asdescribed previously [1] and the absorbance at 650 nmof the acid-extracted stain was measured on a platereader (BioRad) and normalized to cell absorbance at‘time 0’.

RT–PCRTotal RNA was extracted with Tri reagent (Sigma)and 2 µg total RNA was reverse-transcribed usingthe AffinityScript (Stratagene) reverse transcriptase.PCR was performed on 1/10 (2 µl) of the cDNA, in50 µl reactions containing 0.2 mM dNTP mix, 10×PCR buffer with 15 mM MgCl2 (ABI), 2.5 U TaqGold(ABI) and 0.2 mM of each primer; 5% v/v DMSOwas included in some reactions. PCR conditions were:95 ◦C for 5 min, then 33 cycles of 94 ◦C for 30 s, 57 ◦Cfor 30 s and 72 ◦C for 45 s, followed by 10 min at 72 ◦C.PCR products were separated on 1.5% or 2% agarosegels. Primers are listed in Table S2 (see Supplementarymaterial).

Tumour formation in micePools of MLE or BEAS-2B cells expressing SRSF6 orthe empty vector or NCI-H460 and RKO cells express-ing SRSF6 shRNAs, as indicated, were injected into therear flanks of NOD-SCID mice (2 × 106 cells/site in100 ml serum-free medium containing 0.25 v/v growthfactor-stripped Matrigel (BD Bioscience), using a26-gauge needle. Tumour growth was monitored asdescribed previously [2,30]. Veterinary care was pro-vided to all animals by the Hebrew University AnimalCare Facility staff, in accordance with AAALAC stan-dard procedures.

Statistical analysisFor growth curve comparisons between two cell lines,we used a within-and-between ANOVA with three timepoints as the within predictor, and two types of celllines as the between predictor. The interaction termbetween time points and cell line tests whether thegrowth rates differ. For all other statistical compar-isons, we used Student’s t-test (two-tailed) for inde-pendent samples where equal variances were assumed.p values associated with these tests are indicated in thefigure legends.

Results

SRSF6 is amplified and over-expressed in lung andcolon cancersWe analysed the DNA copy number and expres-sion levels of several splicing factors from theSR and hnRNP A/B protein families in colon andlung normal and tumour samples. We found thatSRSF6 (SRp55) was amplified in 12% of lung andbreast as well as in 37% of colon tumour samples

Figure 1. Elevated gene copy number and expression of SRSF6 inlung and breast tumours. (A, B) qPCR and qRT–PCR analyses ofSRSF6 DNA copy numbers and mRNA expression, respectively, fromfive normal lung or six lung tumour tissues (A) or breast normal ortumour tissues (B).

examined (Figure 1, Table 1). Notably, in colontumours, only hnRNP A2/B1 was amplified to asimilar extent and no other SR or hnRNP A/Bprotein had similar elevated gene copy numbers(Table 1). Interestingly, hnRNP A2/B1 has beenshown recently to be an oncogenic driver in glioblas-toma and other brain tumours [2]. SRSF6 was alsoover-expressed, as measured by its mRNA levels,in ∼50% of lung and breast tumours examined(Figure 1) [1]. Moreover, analyses of public databasesshow elevated gene copy numbers and significantover-expression of SRSF6 in colon tumours comparedto normal colon or rectum samples and in gastric andbladder tumours (see Supplementary material, FigureS1). These results suggest that SRSF6 is a marker forcolon and lung cancer development and may play arole in the development of these cancers.

SRSF6 enhances proliferation and inhibits celldeath of mouse lung epithelial cellsTo investigate whether SRSF6 promotes cellular trans-formation, we examined whether its over-expressioncan enhance proliferation or reduce cell death of nor-mal lung cells; and the reverse, whether knock-down ofSRSF6 can inhibit proliferation or enhance cell death



Table 1. SR proteins gene copy number in colon cancerAdenomas(n = 37)

Carcinomas(n = 28)

Gene ID Gain Loss Gain Loss Gain (% of total)SRSF1 6426 1 0 6 2 11SRSF2 6427 2 0 9 3 17SRSF3 6428 3 0 4 3 11SRSF4 6429 0 3 0 8 0SRSF5 6430 2 1 0 7 3SRSF6 6431 9 1 15 1 37SRSF7 6432 0 1 5 0 7SRSF9 8683 5 0 6 1 17TRA2B 6434 1 0 1 1 3SRSF10 10772 0 4 0 7 0SRSF11 9295 0 2 0 5 0SREK1 140890 0 1 5 1 7

of lung and colon cancer cells. Over-expression ofSRSF6 in immortal mouse lung epithelial (MLE) cells[28,29] or immortal human lung epithelial (BEAS-2B)cells enhanced their proliferation compared to cellsexpressing empty vector (Figure 2A–D). Knock-downof SRSF6 in MLE cells did not inhibit their pro-liferation, suggesting that SRSF6 is not required forproliferation of these cells but that its over-expressionmight trigger abnormal proliferation (Figure 2E, F).In addition, SRSF6 over-expression in MLE cells ren-dered them more resistant to chemotherapy-inducedcell death, as measured by their survival after treatmentwith cis-platinum (Figure 2G).These results suggestthat SRSF6 is both an anti-Apoptotic agent and canalso enhance proliferation.

SRSF6 cooperates with myc and its up-regulationtransforms mouse and human lung epithelial cellsin vitro and in vivoWe next examined whether SRSF6 can enhanceanchorage-independent growth of MLE and BEAS-2Bcells. Because MLE cells failed to form coloniesin soft agar in the absence or presence of SRSF6alone, we co-expressed the proto-oncogene c-myctogether with SRSF6 in MLE cells. c-myc expressionalone was not sufficient to induce colony formationin soft agar of MLE cells (Figure 3A, B). However,co-expression of SRSF6 and c-myc induced colonyformation in soft agar (Figure 3A, B). We nextexamined whether SRSF6 can transform immortalhuman bronchial epithelial cells (BEAS-2B). Wefound that expression of SRSF6 alone was sufficientto transform BEAS-2B cells, which formed colonies insoft agar (Figure 3C, D) To further investigate whetherSRSF6 can render cells tumourigenic in vivo, weinjected MLE and BEAS-2B cells expressing SRSF6or the empty vector subcutaneously into NOD-SCIDmice. We found that SRSF6 over-expression convertedMLE and BEAS-2B cells into tumourigenic cells thatformed tumours in mice (Figure 3E, F). These resultsindicate that SRSF6 is a cellular proto-oncogene inlung cancer which is not only up-regulated in lung

and colon cancers but may be a causative factor inlung tumour development.

Knock-down of SRp55 inhibits transformation andtumourigenesis of lung and colon cancer cellsTo examine whether SRSF6 is required for tumourmaintenance, we examined the effects of knock-downof SRSF6 on transformation of colon and lung tumourcell lines. We found that stable SRSF6 knock-down(Figure 4A, E) inhibited colony formation in soft agarof NCI-H460 lung cancer cells, as well as of RKOcolon cancer cells (Figure 4B, C, F, G). Furthermore,when injected into NOD-SCID mice, the ability ofthese cells to form tumours in vivo was stronglyinhibited, indicating that SRSF6 is required for bothtumour initiation and maintenance (Figure 4D, H).

SRp55 regulates the splicing of tumour suppressorsand oncogenesIn order to identify alternative splicing events thatare regulated by SRSF6 and might contribute to itsoncogenic activity, we examined the effects of SRSF6up- or down-regulation on splicing events reported tobe altered in cancer and that contribute to the trans-formed phenotype (Figure 5; see also Supplementarymaterial, Figure S2, Table S1). We found that splicingof the insulin receptor (INSR) was changed upon up-or down-regulation of SRSF6. INSR exon 11 alterna-tive splicing was shown to be regulated by some SRproteins and hnRNP A/B proteins [33,34], and modu-lates the affinity of the insulin receptor to its ligands– insulin or IGF-II [35,36]. A switch in INSR splic-ing in cancer which leads to elevated skipping of exon11 has been reported in several cancers [35,37]. Wefound that SRSF6 up-regulation increases the skip-ping of INSR exon 11, while its knock-down elevatedexon 11 inclusion (Figure 5A, B). These results sug-gest that SRSF6 regulates INSR alternative splicingand leads to the production of the more mitogenicisoform of the insulin receptor [35,36]. Another splic-ing target of SRSF6 we identified is the kinase Mnk2(MKNK2) [1,38]. We recently found that MKNK2alternative splicing generates a tumour-suppressive iso-form (Mnk2a), which activates the p38–MAPK stressresponse and can induce apoptosis, while the Mnk2bisoform is pro-oncogenic [38–39]. We found that whileSRSF6 up-regulation did not change MKNK2 splicingin BEAS-2B cells, its knock-down in NCI-H460 cellsreduced the formation of the pro-oncogenic Mnk2b iso-form, and increased levels of the tumour-suppressiveMnk2a isoform (Figure 5A, B). DLG-1 is a putativetumour suppressor which has been implicated in cellpolarity and tissue organization and its expression isdown-regulated in cancer and contributes to enhancedinvasion [40–42]. We found that while SRSF6 over-expression did not affect DLG-1 splicing in BEAS-2Bcells, its knock-down induced inclusion of exons 3–4and the production of the full-length tumour suppressorDLG-1 (Figure 5A, B).



Figure 2. SRSF6 enhances proliferation of mouse and human lung epithelial cells and protects them from cell death. (A) Pools of mouselung epithelial cells (MLE) (A, B) or BEAS-2B human lung bronchial cells (C, D) were transduced with the indicated retroviruses encodingSRSF6 or an empty vector. After puromycin selection cells were lysed, and after western blotting membranes were probed with theindicated antibodies against SRSF6 or T7. β-Catenin served as loading control (A, C). To measure proliferation (B, D), cells mentioned in(A, C) were seeded on 96-well plates and proliferation was measured as described in Materials and methods; * p ≤ 0.001 ** p ≤ 0.001.(E) MLE cells were transduced with the indicated retroviruses encoding shRNAs against SRSF6 or an empty vector. After selection, levelsof SRSF6 knock-down were measured by western blot analysis, as in (A). (F) Cells mentioned in (E) were seeded on 96-well plates andproliferation was measured as described in Materials and methods. (G) MLE cells described in (A) were seeded on 96-well plates and 24 hlater were treated with cis-platinum (CDDP) and cell survival was measured as described in Materials and methods; *p ≤ 1.80018E-06; **p≤ 2.78911E-05; ***p ≤ 3.12537E-11.

Discussion

Increasing evidence suggests that the process of alter-native splicing is not only deregulated in cancer, butalso directly contributes to cancer development andprogression [4,5,43–46]. In recent years, several alter-native splicing regulators have been shown to act asoncogenes in some cancers [1–3]. However, the role ofmost of the splicing factors in cancer is unknown. Oneof the hallmarks of a putative oncogene is its geneticalteration in tumours, including mutations and translo-cations (which alter the activity of the gene product),or gene amplifications (which cause up-regulation ofthe gene product) [46,47]. Here we compared the genecopy numbers of several splicing factors from the SRprotein family in normal or tumour tissues of colon andlung cancers.

We found that some SR proteins have elevated genecopy numbers in these cancers (Table 1). Among theSR proteins, SRSF6 was most commonly amplified(37% in colon and 12% in lung and breast tumours;Figure 1, Table 1). Previously, it has been shown thatSRSF1 is up-regulated in several cancers, and its geneis amplified in breast cancer. Moreover, SRSF1 actsas a potent oncogene in breast cancer when over-expressed [1,13]. Although SRSF1 was amplified in∼11% of colon tumours (Table 1), SRSF6 amplifica-tion was more pronounced in colon and lung cancersand it was also amplified in breast tumours (Figure 1,Table 1). Since the genetic evidence suggested thatSRSF6 is amplified and up-regulated (see Supplemen-tary material, Figure S1) in colon and lung tumours, wesought to examine whether SRSF6 possesses oncogenicactivity and whether it contributes to lung and coloncancer development. We moderately over-expressed



Figure 3. SRSF6 expression induced transformation of mouse and human lung epithelial cells and cooperated with c-myc. Pools ofMLE cells were co-transduced with the indicated retroviruses encoding SRSF6 or empty vector, and c-myc. Pools of BEAS-2B cells weretransduced with the indicated retroviruses encoding SRSF6 or an empty vector. After puromycin selection, cells were seeded into softagar, as indicated in Materials and methods. (A) Graph represents the average and SD of number of colonies/plate of MLE cells; n = 2,*p = 1.6787E-09. (B) Representative fields of colonies were photographed 14 days after the cells were seeded. (C) Graph representsthe average and SD of number of colonies/plate of BEAS-2B cells; n = 2; **p = 2.9926E-14. (D) Representative fields of colonies werephotographed 14 days after cells were seeded. (E) MLE cells described in (A) were injected (2 × 106 cells/site) into the rear flanks ofNOD-SCID mice (n = 8) and tumour volume was measured and calculated as described in Materials and methods. (F) BEAS-2B cellsdescribed in (C) were injected and measured as in (E); (n = 8).

SRSF6 in mouse and human immortal lung epithe-lial cells (MLE and BEAS-2B, respectively) and foundthat SRSF6 increased the proliferation of these cells(Figure 2A–D). Knock-down of SRSF6 did not inhibitproliferation (Figure 2E, F), suggesting that SRSF6 isnot required for proper growth or survival of thesecells, but that over-expression may enhance prolifer-ation by gain of function. SRSF6 also increased thesurvival of MLE cells treated with cis-platinum,suggesting that it might reduce cell death as well(Figure 2G). In order to examine whether SRSF6 caninduce transformation and tumourigenicity of immor-tal mouse and human lung cells, MLE and BEAS-2B cells were transduced with SRSF6 and seededinto soft agar. MLE cells with or without SRSF6did not form any colonies in soft agar (data notshown). However, when co-transduced with c-myc andSRSF6, MLE cells formed large colonies in soft agar,while MLE cells transduced with c-myc alone hardlyformed colonies (Figure 3A, B). These results indicatethat SRSF6 cooperates with the proto-oncogene c-mycto induce transformation. Many genes that cooperate

with c-myc in transformation possess anti-Apoptoticactivity, as myc strongly activates an apoptotic armsimultaneously with its proliferative activity [48,49].Thus, it is possible that the anti-Apoptotic activity ofSRSF6 (Figure 2G) contributes to this cooperation.In BEAS-2B cells, SRSF6 expression was sufficientto induce colony formation in soft agar (Figure 3C,D). In both cell lines, SRSF6 over-expression wassufficient to induce tumour formation in NOD-SCIDmice (Figure 3E, F). These results strongly suggestthat SRSF6 acts as a potent proto-oncogene in lungcells and is capable of converting immortal epithe-lial lung cells to be tumourigenic. In order to exam-ine whether SRSF6 is required for maintenance ofthe transformed phenotype of lung and colon cancercells, we knocked-down SRSF6 in lung (NCI-H460)and colon (RKO) cancer cell lines and examined theironcogenic properties in vitro and in vivo. We foundthat knock-down of SRSF6 inhibited colony formationof both lung and colon cancer cells (Figure 4A–C,E–G). Furthermore, SRSF6 inhibited tumour formationin NOD-SCID mice, indicating that SRSF6 is required



Figure 4. SRSF6 knock-down inhibits transformation and tumourigenesis of lung and colon cancer cells. (A) Pools of NCI-H460 lungcancer cells were transduced with the indicated retroviruses encoding shRNAs or the empty vector; after selection cells were lysed, andafter western blotting membranes were probed with the indicated antibodies against SRSF6 or β-catenin as loading control. (B) Cellsdescribed in (A) were seeded into soft agar, as indicated in Materials and methods. Graph represents the average and SD of number ofcolonies/plate of MLE cells; n = 2; *p = 3.9721E-19; **p = 6.3129E-21. (C) Representative fields of colonies were photographed 14 daysafter cells were seeded. (D) Cells described in (A) were injected (2 × 106 cells/site; n = 8) into the rear flanks of Nude mice and tumourvolume was measured and calculated as described in Materials and methods. (E) Pools of RKO colon carcinoma cells were transduced withthe indicated retroviruses encoding an shRNA against SRSF6 or the empty vector; after selection cells were lysed, and following Westernblotting membranes were probed with the indicated antibodies against SRSF6 or β-Actin as loading control. (F) Cells described in (E) wereseeded into soft agar, as indicated in Materials and methods. Graph represents the average and SD of number of colonies/plate of RKOcells; n = 2; *p = 1.5293E-14. (G) Representative fields of colonies were photographed 14 days after cells were seeded. (H) Cells describedin (E) were injected (2 × 106 cells/site; n = 8) into the rear flanks of NOD-SCID mice and tumour volume was measured and calculated, asdescribed in Materials and methods.

for tumour maintenance in addition to its role in tumourinitiation (Figure 4D, H).

Because SRSF6 is a splicing factor, we hypothe-size that its oncogenic activity might be mediated byits activity as a splicing factor. We examined alter-native splicing of many genes known to be alterna-tively spliced in cancer and found three alternativesplicing events which might contribute to the onco-genic activity of SRSF6. The insulin receptor (INSR) isexpressed in almost every cell in the body and increases

glucose uptake and metabolism upon insulin binding[50,51]. INSR is alternatively spliced and generatestwo splicing isoforms: INSR-A and INSR-B, whereexon 11 is skipped or included, respectively [35].Biochemical analysis of INSR isoforms shows thatthe receptor lacking exon 11 (INSR-A) has higheraffinity to insulin and can also bind IGF-II, whileINSR-B binds only insulin [35,36]. INSR-A levels areup-regulated, while INSR-B is down-regulated in manytumours, suggesting it might play an important role



Figure 5. SRSF6 regulates the splicing of tumour suppressors and oncogenes. (A) Pools of BEAS-2B cells were transduced with theindicated retroviruses encoding SRSF6 or an empty vector. After puromycin selection, cells were lysed and total RNA was extracted. Afterreverse transcription, cDNA was subjected to PCR, using primers that detect the indicated alternative splicing events. (B) Pools of NCI-H460lung cancer cells were transduced with the indicated retroviruses encoding shRNAs or the empty vector; after selection cells were lysedand total RNA was extracted. After reverse transcription, cDNA was subjected to PCR, using primers that detect the indicated alternativesplicing events.

in enhanced insulin/IGF-II signalling in tumour cells[37]. We found that SRSF6 over-expression stronglyinduced skipping of exon 11, while its knock-downenhanced inclusion (Figure 5). These results suggestthat SRSF6 regulates exon 11 alternative splicing andinduced the more active form of INSR. Although SRproteins usually enhance inclusion of exons, there areseveral examples of SR proteins enhancing skippingof exons [52,53]. DLG-1 is a putative tumour suppres-sor which is important for proper tissue organizationand cell polarity [41,42]. Proper tissue organization isdisrupted in cellular transformation and in the EMTprocess, and thus loss of DLG-1 expression contributesto motility and invasion [40,41]. We found that whileSRSF6 over-expression does not enhance skipping ofexons 3–4 of DLG-1, its knock-down enhances inclu-sion of these exons (Figure 5). These results suggestthat SRSF6 over-expression might reduce the levelsof the full transcript of DLG-1, while its knock-downup-regulates the tumour-suppressive full-length tran-script. MKNK2 (Mnk2) is a kinase that phosphorylateseIF4E on serine 209 and is downstream to the MAPKsignalling pathway [38]. We found recently that theMnk2a isoform is tumour-suppressive, down-regulatedin many tumours and activates the p38–MAPK stressresponse [Maimon et al . (submitted)]. Mnk2b, on theother hand, is pro-oncogenic and does not activate thisstress response. We found previously that SRSF6 ele-vates Mnk2b and reduces Mnk2a in human fibroblasts[1]. Here we show that knock-down of SRSF6 ele-vates Mnk2a, suggesting that SRSF6 down-regulatesthe tumour suppressive isoform Mnk2a (Figure 5).SRSF6 affects the splicing of a subset of genes, asmany alternative splicing events are not affected bysilencing or over-expression of SRSF6 (see Supple-mentary material, Table S1, Figure S2).

Taken together, SRSF6 is amplified and up-regulatedin lung and colon cancers and acts as a potent onco-protein able to transform immortal lung epithelial cells.

SRSF6 is also important for tumour maintenance, asits knock-down inhibits transformation and tumourige-nesis of lung and colon cancer cells. Finally, SRSF6regulates alternative splicing to down-regulate tumoursuppressors and activate oncogenic isoforms thatcontribute to the cancerous phenotype. Further studiesof the targets of SRSF6 will determine which pathwaysare directly responsible for the tumourigenic activity ofSRSF6.

Acknowledgments

The authors wish to thank members of the Karnilaboratory for helpful discussions and technical help,and Professor Avi Kluger for help with the statisticalanalysis. This study was partially supported by theIsrael Science Foundation (ISF; Grant No. 780/08, toRK).

Author contributions

MC-E and RK designed the experiments; MC-E, RGand ZS performed the experiments; CA, KT andTØ analysed DNA copy numbers from tumour sam-ples; DM analysed DNA copy numbers and expres-sion of normal tumour samples; and RK wrote themanuscript.

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SUPPORTING INFORMATION ON THE INTERNETThe following supporting information may be found in the online version of this article.

Figure S1. Elevated gene copy number and expression of SRSF6 in colon, bladder and gastric tumours compared to normal tissues. (A, B)Analysis of SRSF6 DNA copy numbers from normal blood (351 patients), colon (79 patients), rectum (15 patients), rectal adenocarcinoma (90patients) and rectosigmoid adenocarcinoma (24 patients). Analyses was done using the Oncomine database (www.oncomine.org). (C) mRNAexpression levels of SRSF6 in gastric mucosa (31 patients) compared to gastric intestinal-type adenocarcinoma (26 patients). Analyses was doneusing the Oncomine database (www.oncomine.org). (D) mRNA expression levels of SRSF6 in bladder (48 patients) compared to infiltrating bladderurothelial carcinoma (81 patients) Analyses was done using the Oncomine database (www.oncomine.org).

Figure S2. Alternative splicing changes affected by SRSF6 over-expression or knock-down. Total RNA from BEAS-2B transduced with emptyvector (pBABE ) or SRSF6 and from NCI-H460 and RKO cells transduced with the indicated shRNA against SRSF6 was extracted and subjectedto reverse transcription. cDNA was subjected to PCR, using the indicated primers, and the products were separated and visualized using agarosegels. Alternative splicing products are indicated on the right.

Table S1. Changes in alternative splicing and expression upon SRSF6 down- or up-regulation.

Table S2. Primers and shRNAs.


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