A novel human heparanase splice variant, T5, endowed with protumorigenic characteristics

The FASEB Journal • Research Communication

A novel human heparanase splice variant, T5, endowedwith protumorigenic characteristics

Uri Barash,* Victoria Cohen-Kaplan,* Gil Arvatz,* Svetlana Gingis-Velitski,*Flonia Levy-Adam,* Ofer Nativ,† Ronen Shemesh,‡ Michal Ayalon-Sofer,‡ Neta Ilan,*and Israel Vlodavsky*,1

*Cancer and Vascular Biology Research Center, Bruce Rappaport Faculty of Medicine, Technion,Haifa, Israel; †Department of Urology, Bnai-Zion Medical Center, Haifa, Israel; and ‡Compugen Ltd.,Tel Aviv, Israel

ABSTRACT Heparanase is a mammalian endo-�-D-glucuronidase that can cleave heparan sulfate sidechains, an activity strongly implicated in tumor celldissemination. The current study aimed to identify andcharacterize heparanase splice variants. LEADS, Com-pugen’s alternative splicing modeling platform (Com-pugen, Tel Aviv, Israel), was used to search for splicevariants in silico; tumor-derived cell lines (i.e., CAGmyeloma) and tumor biopsies were utilized to validateT5 expression in vivo; signaling (i.e., Src phosphoryla-tion) was evaluated following T5 gene silencing oroverexpression and correlated with cell proliferation,colony formation, and tumor xenograft development.A novel spliced form of human heparanase, termed T5,was identified. In this splice variant, 144 bp of intron 5are joined with exon 4, which results in a truncated,enzymatically inactive protein. T5 overexpression re-sulted in increased cell proliferation and larger colo-nies in soft agar, mediated by Src activation. Further-more, T5 overexpression markedly enhanced tumorxenograft development. T5 expression is up-regulatedin 75% of human renal cell carcinoma biopsies exam-ined, which suggests that this splice variant is clinicallyrelevant. Controls included cells overexpressing wild-typeheparanase or an empty plasmid and normal-lookingtissue adjacent the carcinoma lesion. T5 is a novel func-tional splice variant of human heparanase endowed withprotumorigenic characteristics.—Barash, U., Cohen-Kaplan, V., Arvatz, G., Gingis-Velitski, S., Levy-Adam, F.,Nativ, O., Shemesh, R., Ayalon-Sofer, M., Ilan, N., Vlo-davsky, I. A novel human heparanase splice variant, T5,endowed with protumorigenic characteristics. FASEB J.24, 1239–1248 (2010). www.fasebj.org

Key Words: cell proliferation � colony formation � xenograft� Src � phosphorylation

Heparanase is an endo-�-d-glucuronidase that cancleave heparan sulfate (HS) side chains at a limitednumber of sites. The consequence of this activity com-bines structural alteration of the extracellular matrix(ECM) underlying epithelial and endothelial cells,which makes it more susceptible to cellular invasion,and liberation of a multitude of biological mediatorssequestered in the ECM or tethered to HS on the cell

membrane. Heparanase activity has long been associ-ated with metastatic potential of tumor-derived cells (1,2), a notion that has been established by using specificanti-heparanase siRNA and ribozyme methodologies(3). Although heparanase in cellular invasion andtumor metastasis plays a decisive role (4–7), its functionin primary tumor progression remains largely unknownbut likely involves tumor angiogenesis. Several in vitroand in vivo model systems, including wound healing (8,9), tumor xenografts (10), Matrigel plug assay (8) andtube-like structure formation (11), have confirmed theangiogenic potency of heparanase. Moreover, microvesseldensity was significantly reduced in tumor xenograftsdeveloped by T-lymphoma cells transfected with anti-heparanase ribozyme (3), and clinical findings con-cluded with a proangiogenic feature of heparanase (7,12). These observations, the anticancerous effect ofheparanase gene silencing (3) and of heparanase-inhibiting molecules (4, 13), as well as the unexpectedidentification of a single functional heparanase (6, 14),suggest that the enzyme is a valid target for anticancerdrug development and a promising tumor marker (4,13, 15–17).

Apart from the well-studied enzymatic feature of theenzyme, findings indicated that heparanase exerts biolog-ical functions apparently independent of its enzymaticactivity. Inactive heparanase was noted to enhance adhe-sion and migration of normal and tumor-derived cells(10, 11, 18, 19) and to promote the phosphorylation ofsignaling molecules such as Akt (10, 11, 20, 21), whichlikely support cell survival (22). Similarly, enzymaticallyinactive heparanase was noted to enhance the phosphor-ylation of p38 and Src, associated with induced tissuefactor (TF) and VEGF expression (23, 24). Heparanasealso augmented the phosphorylation of EGFR in a Src-dependent manner (25). EGFR activation by heparanasewas associated with enhanced cell proliferation and col-ony formation in soft agar (25). Furthermore, head andneck carcinoma showed a correlation between hepara-nase and EGFR phosphorylation levels (25), with the

1 Correspondence: Cancer and Vascular Biology ResearchCenter, Faculty of Medicine, Technion, P.O. Box 9649, Haifa31096, Israel. E-mail: [email protected]

doi: 10.1096/fj.09-147074

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heparanase-Src-EGFR axis playing an important route intumor progression (26). A splice variant of humanheparanase was described (27), yet its function remainsobscure. Here, we describe a novel splice form of humanheparanase termed T5, in which 144 bp of intron 5 arejoined with exon 4, which results in a truncated 169-aaprotein. We provide evidence that T5 augments Srcphosphorylation to levels comparable with the full-lengthheparanase. Moreover, T5 overexpression was associ-ated with increased cell proliferation, larger coloniesin soft agar, and markedly enhanced tumor xeno-graft development. T5 is overexpressed in the major-ity (75%) of human renal cell carcinoma biopsiesexamined, which suggests that this splice variant isclinically relevant.

MATERIALS AND METHODS

Discovery of heparanase splice variants

The discovery of T4 and T5 heparanase splice variants wascarried out using LEADS, Compugen’s alternative splicingmodeling platform (Compugen, Tel Aviv, Israel) (28–31).Briefly, human ESTs and cDNAs were obtained from the U.S.National Center for Biotechnology Information GenBank(www.ncbi.nlm.nih.gov/Genebank) and aligned to the hu-man genome build (www.ncbi.nlm.nih.gov/genome/guide/human) using the LEADS clustering and assembly algo-rithms. The platform cleans the expressed sequences fromvectors and immunoglobulins, masks them for repeats andlow-complexity regions, and aligns the expressed sequencesto the genome while modeling alternative splicing. Thediscovery of the skip 10 splice variant was carried out using a“non-EST-based method for exon-skipping prediction” (30).In principle, this method relies on a gene’s exon structure/size and the human/mouse homology of the exon and itssurrounding sequences.

T5 Cloning and gene constructs

3�-Rapid amplification of cDNA ends (RACE) analysis (Am-bion, Austin, TX, USA) was performed using the forwardheparanase primers 5�-GAGAATTCAGGTGAGCCCAAGAT-GCTGCTG-3�, 5�-GGAATTCATGCTGCTGCGCTCG-3�, ac-cording to the manufacturer’s instructions. The PCR productwas purified by Wizard SV Gel PCR Clean-Up System (Pro-mega, Madison, WI, USA), and sequenced. The T5 cDNA wassubcloned into eukaryotic expression plasmids pcDNA3,pSecTag2A, or pTK208 lentivirus vector, essentially as de-scribed (32).

Antibodies and reagents

Anti-Myc-tag (sc-40), anti-Src (sc-18), and anti-calnexin (sc-11397) antibodies were purchased from Santa Cruz Biotech-nology (Santa Cruz, CA, USA). Anti-phospho-Src (Tyr416)antibody was purchased from Cell Signaling Technologies(Beverly, MA, USA). Anti-heparanase 1453 and 733 havepreviously been characterized (33). Anti-mouse platelet en-dothelial cell adhesion molecule (PECAM)-1 (CD31) poly-clonal antibody was kindly provided by Dr. Joseph A. Madri(Yale University, New Haven, CT, USA) (23). ConcanavalinA-Sepharose and bromodeoxyuridine (BrdU) were pur-chased from GE Healthcare (Little Chalfont, UK) and anti-BrdU monoclonal antibody-HRP conjugated was purchased

from Roche (Mannheim, Germany). Fluorescein wheat germagglutinin was purchased from Vector Laboratories (Burlin-game, CA, USA). Src inhibitors PP1, PP2, and Src inhibitor I(Merck Biosciences, GmbH, Germany) were dissolved inDMSO as stock solution. DMSO was added to the cell cultureas a control.

Cells and cell culture

HEK 293, human choriocarcinoma JAR, U87-MG glioma,Colo205 and HT29 colon carcinoma, PC-3 prostate carci-noma, MDA-MB-231 breast carcinoma, and PANC-1 pancre-atic carcinoma cells were purchased from the American TypeCulture Collection (ATCC; Manassas, VA, USA). FaDu phar-ynx carcinoma cells were kindly provided by Dr. Eben L.Rosenthal (University of Alabama at Birmingham, Birming-ham, AL, USA) (34) and CAG myeloma cells were kindlyprovided by Dr. Ben-Zion Katz (Tel Aviv Sourasky MedicalCenter, Tel Aviv, Israel) (35). Cells were grown in Dulbec-co’s modified Eagle’s medium (Biological Industries, BeitHaemek, Israel) supplemented with 10% fetal calf serum(FCS) and antibiotics. Human umbilical vein endothelialcells (HUVECs) were isolated and grown essentially asdescribed previously (11).

Gene silencing and PCR analysis

Anti-heparanase and anti-T5 siRNA oligonucleotides (siG-enome On-Target plus SMART pool duplex) were pur-chased from Dharmacon (Thermo Fisher Scientific Inc,Waltham, MA, USA), and transfection was carried out withDharmaFECT reagent (Dharmacon), according to themanufacturer’s instructions. PCR was preformed with thefollowing primer sets: heparanase, F 5�-ACAGTTCTAAT-GCTCAGTTGCTC-3� and R 5�-TTGCCTCATCACCACTTC-TATT-3�; T5, F 5�-TTGGCCAGAGGCTTGTCTCC-3� and R5�-CCCATTTGGGAAATGCTTCAG-3�; GAPDH, F 5�-CCAGCCGAGCCACATCGCTC-3� and R 5�-ATGAGC-CCCAGCCTTCTCCAT-3�.

Immunocytochemistry

Immunofluorescent staining was performed essentially asdescribed previously (34).

Cell lysates, activity assay, and protein blotting

Preparation of cell lysates, protein blotting, and measurementof heparanase enzymatic activity were carried out as describedpreviously (24, 25, 34).

Cell proliferation

Cell proliferation was analyzed by BrdU incorporation usingcell proliferation labeling reagent (1:1000, GE Healthcare),as described previously (10). At least 1000 cells were countedfor each cell type.

Colony formation in soft agar

DMEM (3 ml) containing 0.5% low-melt agarose (Bio-Rad,Hercules, CA, USA) and 10% FCS was poured into 60-mmPetri dishes. The layer was covered with cell suspension(0.5�104 cells) in 1.5 ml DMEM containing 0.3% low-meltagarose and 10% FCS, followed by addition of 2 ml DMEMcontaining 10% FCS. Medium was exchanged every 3 d.

1240 Vol. 24 April 2010 BARASH ET AL.The FASEB Journal � www.fasebj.org

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Colonies were visualized and counted under a microscopeafter 2–5 wk, as described previously (25).

Tumorigenicity and immunohistochemistry

Cells from exponential cultures of control-, heparanase-,and T5-infected CAG myeloma cells were detached withtrypsin/EDTA, washed with PBS, and brought to a concen-tration of 1 � 107 cells/ml. Cell suspension (1�106/0.1ml) was inoculated subcutaneously at the right flank of5-wk-old female athymic nude mice (n�7). Xenograft sizewas determined 2�/wk by externally measuring tumors in2 dimensions using a caliper. At the end of the experiment,mice were sacrificed, and xenografts were removed,weighed, and fixed in formalin. Paraffin-embedded 5-�msections were subjected to immunostaining with anti-PECAM-1(CD31) or anti-smooth muscle actin (1A4; Sigma) antibodies,using the Envision kit (Dako, Glostrup, Denmark) accordingto the manufacturer’s instructions, as described previously(25, 34). The Animal Care Committee of the Technion(Haifa, Israel), approved all animal experiments.

Statistics

Data are presented as means � se. Statistical significance wasanalyzed by 2-tailed Student’s t test. Values of P � 0.05 wereconsidered significant. All experiments were repeated �3times with similar results.

RESULTS

Discovery, cloning, and expression of heparanasesplice variant T5

We have computationally discovered three different spliceforms of heparanase (T4, T5, and skip 10; Fig. 1A) usingCompugen’s alternative splicing modeling platformLEADS (T4 and T5), and a non-EST-based method forexon-skipping prediction (skip 10). 3�-RACE analysisyielded a product of 687 bp that was highly enriched inRNA extracted from lung carcinoma tissue and chronicmyeloid leukemia (CML) cells compared with controls[adjacent normal lung tissue and white blood cells(WBCs) collected from healthy donors, respectively;Fig. 1B]. Sequencing of the 3�-RACE product matchedthe expected T5 sequence (Fig. 1C), indicating expres-sion of the T5 splice variant. The expression of T4 andskip 10 splice variants could not be detected underthese experimental settings. T5 expression was subse-quently confirmed by RT-PCR analysis of total RNAextracted from several cancer-derived cell lines apply-ing T5-specific primers (Fig. 2A, top panel). T5 expres-sion is detected, along with heparanase, to variouslevels in essentially all cell lines examined. JAR cellsreported to be negative for heparanase (36) (Fig. 2A,

Figure 1. Expression of T5 splice variant. A) Schematic structure of heparanase splice variants emerging from Compugen’s LEADSsearch. B) 3�-RACE analysis of RNA extracted from white blood cells collected from healthy donor (WBC), patient with chronicmyeloid leukemia (CML), lung carcinoma, and adjacent normal lung tissue. C) Sequence alignment indicating that the 3�-RACEproduct corresponds to the predicted T5 splice variant sequence. D) Schematic presentation of heparanase (WT) and T5 exoncomposition.

1241T5, A NOVEL HEPARANASE SPLICE VARIANT

middle panel) were also negative for T5 expression(Fig. 2A, top panel). To verify that T5 encodes for aprotein, the T5 sequence was cloned into a mammalianexpression vector, and lysate samples prepared fromstably transfected 293 cells were subjected to immuno-blotting. Anti-heparanase antibody clearly detected an�15-kDa protein band, a molecular mass slightlyhigher than that anticipated from the deduced aminoacid sequence (14,207; Fig. 2B, 0), whereas no reactivitywas detected in control cells transfected with an emptyplasmid (Fig. 2B, Vo). In addition, an �17-kDa proteinband was also seen (Fig. 2B, 0). We considered twopossibilities for the appearance of two T5 protein bands:different glycosylation pattern or proteolytic cleavage ofthe high-molecular-mass protein. We examined the firstpossibility by incubating T5-overexpressing cells with in-creasing concentrations of tunicamycin, an inhibitor ofprotein N-glycosylation. Indeed, tunicamycin treatment

markedly reduced the amount of the �17-kDa band,while levels of the �15-kDa protein were increased (Fig.2B, 10, 20). In contrast, chloroquine, reported to inhibitproteolytic processing of heparanase within lysosomes(33), did not significantly affect the ratio between the twoT5 forms (Fig. 2, Chl). These results indicate that T5 isbeing expressed as an �15-kDa protein and a moreglycosylated 17-kDa form and that T5 is not subjected toprocessing within lysosomes. As would be expected fromthe presence of a signal peptide, T5 is secreted and foundin the cell conditioned medium (Fig. 2B, right). Thisfinding is in agreement with its localization to the ER andGolgi apparatus (Fig. 2C), as demonstrated previously forheparanase (34). Subjecting lysates of stably transfectedcells to heparanase activity assay indicated that T5 lacksheparanase enzymatic activity (Fig. 2D, T5 vs. Hepa) aswould be expected from a heparanase variant comprisingonly a small portion of the 50-kDa subunit (Fig. 1A, T5).

Figure 2. Cloning and expression of T5. A) RT-PCR analysis. Total RNA was extracted from the indicated cell line and subjectedto RT-PCR analysis applying T5 (top panel), heparanase (middle panel), and GAPDH (bottom panel) specific primers, specifiedin the Materials and Methods section. B) T5 transfection. T5 was cloned into mammalian expression vector (pcDNA3), andtransfected HEK 293 cells were left untreated (0) or incubated with tunicamycin (10, 20 �g/ml) or chloroquine (Chl; 100 �M).Control cells were transfected with an empty vector (Vo). Cell lysates (left panel) and cell conditioned medium (right panel)were blotted with anti-heparanase 1453 antibody. C) Cellular localization. Stably transfected HEK 293 cells were triple stainedfor Myc-tag (T5; red), the ER marker calnexin (ER; top panels, green), and merged with cell nuclei labeled with TO-PRO (toppanels, blue). Cells were similarly stained with anti-Myc-tag (T5; red), the Golgi marker wheat germ agglutinin-FITC (Golgi;bottom panels, green) and merged with cell nuclei labeled with TO-PRO (bottom panels, blue). Shown is one image in whichthe two colors are of equal intensities, representative of many images, all exhibiting similar localization patterns. D) Enzymaticactivity. HEK 293 cells transfected with heparanase (Hepa), T5, or control empty vector (Vo) were subjected to 3 freeze-thawcycles and applied onto culture dishes coated with 35S-labeled ECM. Release of sulfate-labeled material eluted in fractions 15–30was evaluated as measure of heparanase activity, as described in Materials and Methods.


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Moreover, T5 expression did not seem to impinge signif-icantly on the activity of endogenous heparanase (Fig. 2D,T5 vs. Vo), which suggests that T5 does not function as amodulator of heparanase enzymatic activity.

T5 activates Src and facilitates cell proliferation

Since T5 does not appear to modulate heparanase enzy-matic activity (Fig. 2D), we sought to determine analternative function of the splice variant and examinedwhether T5 facilitates the phosphorylation of Src, shownpreviously to be induced by heparanase (24–26). Srcphosphorylation was increased nearly 3-fold in CAG my-eloma cells infected with heparanase (Fig. 3A, left; thirdand bottom panels, Hepa), in agreement with a similareffect noted in diverse cell systems (24, 25). Notably, CAGcells infected with T5 exhibited comparable induction ofSrc phosphorylation (Fig. 3A, left; third panel, T5), asdetermined by densitometry analysis (Fig. 3A, left; bottompanel). Even higher enhancement of Src phosphorylation

(�4-fold) was found in 293 cells overexpressing hepara-nase or T5 proteins (Fig. 3A, right panels). Src phosphor-ylation enhanced by heparanase or T5 was not affected byheparin (Fig. 3A, ), which suggests that the observedstimulation is not mediated by cell surface HS (26, 37). Incontrast, Erk phosphorylation was not affected by hepara-nase or T5 overexpression (Fig. 3A, fifth and sixth pan-els), suggesting a specific trait.

We next utilized the opposite approach and designedsiRNA specific for the inhibition of endogenousheparanase or T5 expression by 293 cells (Fig. 3B).Anti-T5 siRNA significantly and specifically decreasedT5 expression while heparanase expression was notinhibited (Fig. 3B, top and second panels, T5). Incontrast, anti-heparanase siRNA inhibited both hepara-nase and T5 expression (Fig. 3B, top and secondpanels). Heparanase gene silencing was associated withreduced Src phosphorylation (Fig. 3B, fourth panel,Hepa), which is in agreement with the notion thatendogenous heparanase is engaged in Src regulation

Figure 3. T5 augments Src phosphorylation. A) Overexpression. CAG myeloma (left) and 293 (right) cells infected with heparanase(Hepa), T5, or control empty vector (Vo) were grown in the absence (–) or presence () of heparin (50 �g/ml) under serum-freeconditions. Total cell lysates were subjected to immunoblotting, applying anti-heparanase 1453 (top and second panels),anti-phospho-Src (p-Src, third panels), anti-Src (fourth panels) anti-phospho-Erk (p-Erk, fifth panels), and anti-Erk2 antibodies (sixthpanels). Bottom panels: Src phosphorylation index was calculated by densitometry analysis of phosphorylated Src levels divided bytotal Src values. Data are presented as average � se fold increase of Src phosphorylation vs. control Vo cells (set to arbitrary value of1) of 5 independent experiments. B) Gene silencing. Parental 293 cells were transfected with anti-heparanase, anti-T5, or controlsiRNAs. Total RNA was extracted, and RT-PCR analysis was performed, applying T5 (top panel), heparanase (Hepa; second panel),and GAPDH (third panel) specific primers. Corresponding cell lysates were subjected to immunoblotting, applying phospho-Src(p-Src; fourth panel), Src (fifth panel), phospho-Erk (p-Erk; sixth panel) and Erk2 (seventh panel) antibodies. Bottom panel: Srcphosphorylation index, calculated as in A. Note decreased Src phosphorylation levels in response to T5 down-regulation.


(25). Notably, not only heparanase but also T5 silenc-ing resulted in reduced Src phosphorylation (Fig. 3B,fourth panel, T5). Densitometry analysis revealed 2-fold decrease in Src phosphorylation levels followingheparanase and T5 silencing (Fig. 3B, bottom panel).In contrast, the phosphorylation levels of Erk were notaffected (Fig. 3C, sixth panel), which suggests a specificeffect.

We have reported previously that interaction ofheparanase with cell membrane HS results in clusteringof syndecans (37). Syndecan clustering by heparanaseor by a peptide (termed KKDC) corresponding to theheparin-binding domain of heparanase, stimulated theadhesion and spreading of various cell types, involvingPKC, Src, and Rac1 activity (37). The heparin-bindingsequence Lys158-Asn162 (38) is retained in the T5 splicevariant and is followed by the addition of three uniqueamino acids, SKK, thought to comprise additionalbinding sites for heparin (Fig. 1A). Src activation by T5,noted above, may therefore result from clustering andactivation of cell membrane HSPG. Our findings argue,however, against this possibility. First, inclusion ofheparin did not inhibit Src activation by T5 (Fig. 3A,). In fact, T5 failed to bind heparin (SupplementalFig. 1A, left; bottom panel), while interaction with thelectin concanavalin A was retained (Supplemental Fig.1A, bottom panel, ConA), in agreement with T5 beingglycosylated (Fig. 2B). Similarly, T5 levels in the cellculture medium were not affected by the addition ofheparin, whereas heparanase levels are markedly in-creased (Supplemental Fig. 1A, right). Second, a pep-

tide harboring the heparin binding domain, includingthe unique SKK residues (CKKFKNSTYSSKK), failed tointeract with heparin (Supplemental Fig. 1B, CKKK) orto inhibit heparanase enzymatic activity (SupplementalFig. 1C), both accomplished by the KKDC peptide(Supplemental Fig. 1B, C). We, therefore, concludedthat Src activation by T5 is HS-independent but ratherlikely involves interaction with other cell surface mole-cules.

The intimate involvement of Src in different aspectsof tumor progression, including cell proliferation (39),led us to examine this aspect in heparanase- andT5-overexpressing cells (Fig. 4). Control CAG cellsmainly grow while adhering to the culture dish as amonolayer (Fig. 4A, top panel, Vo) (35). In contrast,heparanase- and T5-infected CAG cells were noted toform large foci (Fig. 4A, top panel; Hepa, T5; arrows),indicating their overgrowth. Increased cell prolifera-tion was revealed further by BrdU incorporation (Fig.4A, middle panel). Thus, while 24 � 3% of controlCAG cells incorporated BrdU (Fig. 4A, middle panel,Vo), heparanase- and T5-infected cells exhibited 49 �6% and 52 � 5% BrdU incorporation, respectively, anincrease that is statistically highly significant (P�0.0002and 0.00001 for heparanase vs. Vo and T5 vs. Vo,respectively), reflecting 2-fold increase in cell prolif-eration (Fig. 4A, middle panel, right). Similar resultsalso were observed in heparanase- and T5-overexpress-ing 293 cells (data not shown). Likewise, heparanase-and T5-silencing was associated with a comparable

Figure 4. T5 augments cell proliferation. A) BrdU incorporation. Top panel: morphology of control (Vo)-, heparanase (Hepa)-, andT5-infected CAG myeloma cell cultures. Middle panel: direct evaluation of DNA synthesis is demonstrated by BrdU incorporation.Subconfluent cultures of Vo-, Hepa-, and T5-infected CAG cells were grown in serum-free medium for 20 h, followed by incubationwith BrdU (1:1000) for 2 h. Cells were then fixed and immunostained with anti-BrdU monoclonal antibody. Positively stained,red-brown nuclei were counted vs. blue, hematoxilin counterstained nuclei. At least 1000 cells were counted for each cell type;percentage of positively stained cells is noted in each panel. Fold increase in BrdU incorporation is shown graphically at right. Bottompanel: gene silencing. HEK 293 cells were transfected with anti-GFP (si-GFP), anti-heparanase (si-Hepa), or anti-T5 siRNAoligonucleotides, and BrdU incorporation was evaluated as above, except that cells were kept in serum-containing medium. Note2-fold decrease in BrdU incorporation following heparanase or T5 gene silencing (right panel). B) Colony formation in soft agar. Vo-,Hepa-, and T5-infected CAG (top panel), Fadu (second panel) and 293 (third panel) cells (5�103 cells/dish) were mixed with softagar and cultured for 3–5 wk. CAG cells were similarly grown in the absence (DMSO; fourth panels) or presence of Src inhibitor (PP2,0.4 nM; bottom panels). Photomicrographs of colonies are representative (�100 view).


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�50% decrease in BrdU incorporation (Fig. 4A, bot-tom panel).

The effect of heparanase and T5 on cell proliferationwas evaluated further by monitoring cell growth andcolony formation in soft agar (Fig. 4B and Supplemen-tal Fig. 2). Colony number (Supplemental Fig. 2), size,and cell density were increased markedly followingheparanase- or T5-overexpression in CAG myeloma(Fig. 4B, top panel), FaDu pharynx carcinoma (Fig. 4B,second panel) and 293 cells (Fig. 4B, third panel).Enhanced colony formation on heparanase or T5-overexpression appeared to be mediated by Src activa-tion, because colonies formed in the presence of theSrc inhibitor PP2 (Fig. 4B, fourth and bottom panels)as well as two additional Src inhibitors (PP1, Src inhib-itor I; data not shown) were similar in shape and size tocontrol colonies. Collectively, these findings stronglyimply that heparanase and the T5 splice variant en-

hance cell proliferation and anchorage-independentcell growth by activating Src.

T5 enhances myeloma xenograft development

Having demonstrated a role for T5 in cell proliferationand colony formation, we next examined whether T5enhances tumor development. Until d 21, xenograftsproduced by control (Vo)-, heparanase-, and T5-in-fected CAG myeloma cells exhibited similar growthrates on subcutaneous inoculation into athymic nudemice (Fig. 5A). Thereafter, however, the developmentof tumor xenografts produced by heparanase- andT5-infected cells was enhanced markedly comparedwith xenografts generated by control cells (Fig. 5A). Ontermination of the experiment on d 37, noticeabledifferences in tumor xenograft development were ob-served (Fig. 5A, inset). Thus, average weight of control

Figure 5. T5 enhances tumor xenograft development. A, B) Control (Vo)-, heparanase-, and T5-infected CAG myeloma cellswere injected subcutaneously (1�106/ 0.1 ml) and tumor volume was calculated (A). At the end of the experiment on d 37,tumors were resected, photographed (A, inset) and weighed (B). C) Immunohistochemical analysis. Paraffin-embedded 5-�msections were stained with hematoxilin and eosin (H&E; left column), anti-CD31 (left center column), and anti-smooth muscleactin antibodies (SMA; right center and right columns) antibodies. Note increased blood vessel density and maturation(recruitment of SMA-positive cells) in xenografts produced by heparanase- and T5-infected cells.


xenografts was 58 � 11 mg compared with 648 � 69and 738 � 114 mg for heparanase and T5 xenografts,respectively, differences that are statistically highly sig-nificant (P�0.00007 and 0.0005 for heparanase vs. Voand T5 vs. Vo, respectively). We next evaluated mi-crovessel density in the resulting CAG xenografts byimmunohistochemical analysis of the endothelial cellmarker, CD31. A 2-fold increase in vessel density wasfound following heparanase overexpression (Fig. 5C;CD31, Hepa), in agreement with previous studies uti-lizing this cell system (40), and a similar elevation ofvessel density was observed on T5 overexpression (Fig.5C; CD31, T5). Not only vessel density but also vesselmaturation was affected by heparanase and T5 overex-pression. Staining for smooth muscle actin, whichlabels vessel-associated pericytes, showed a significantlyincreased number of pericytes associated with bloodvessels in xenografts produced by heparanase- andT5-infected CAG cells (Fig. 5C, SMA low), in agreementwith previous findings (9). Closer examination revealedthat while the pericyte coverage appeared discontinu-ous in xenografts produced by heparanase-overexpress-ing CAG cells (Fig. 5C; SMA high, Hepa), a densepericyte layer surrounded even small capillaries follow-ing T5 overexpression (Fig. 5C, SMA high, T5). Theproangiogenic feature of T5 was further concludedfrom endothelial cell organization (tube formation) onreconstituted basement membrane (Matrigel) that wasmaintained for a relatively long period of time vs.control (Vo) cells (Supplemental Fig. 3A). These re-sults suggest that enhanced xenograft progression isdue, in part, to augmented tumor angiogenesis andblood vessel maturation.

T5 is abundantly expressed in renal cell carcinoma

To investigate the clinical significance of T5, we examinedits expression in renal cell carcinoma biopsies by applyingRT-PCR analysis (Fig. 6). T5 expression was observedreadily in the carcinoma (Fig. 6, C) samples and appearedelevated in 75% of the cases available for us (8/12)compared to the adjacent, normal-looking tissue (Fig. 6,N). Moreover, renal cell carcinomas appeared to containsimilar levels of heparanase and T5, suggesting that bothwild-type and spliced heparanase T5 contribute to theprogression of this and likely other carcinomas.

DISCUSSION

Almost all protein-coding genes contain introns that areremoved in the nucleus by RNA splicing and are oftenalternatively spliced. Alternative splicing increases thecoding capacity of the genome, generating multiple pro-teins from a single gene. The resulting protein isoformsfrequently exhibit different biological properties that mayplay an essential role in tumorigenesis (41–43). T5 ap-pears to be such a protumorigenic splice variant. Splicevariant of human heparanase has been recognized previ-ously, yet no biological function was demonstrated (27).Clearly, the T5 splice variant is endowed with protumori-genic properties, enhancing cell proliferation, anchorageindependent growth, and tumor xenograft development(Figs. 4 and 5). These features were observed in severaltumor-derived cell lines overexpressing T5, or followingT5 gene silencing (Figs. 3 and 4), suggesting that itsfunction is relevant to multiple tumor types. The clinicalrelevance of T5 critically emerged from analysis of renalcell carcinoma biopsies, where T5 and heparanase expres-sion appeared to be induced in 75% of the cases (Fig. 6).

Compelling evidence indicates that heparanase expres-sion is elevated in the vast majority of primary hematolog-ical and solid malignancies (7, 12, 17), yet the role thatheparanase plays in primary tumor progression is incom-pletely understood. In several instances, heparanase in-duction was associated with tumors larger in size, attrib-uted, in part, to the proangiogenic function of heparanase(8, 9, 12). The discovery of the T5 splice variant providesanother explanation for the protumorigenic andproangiogenic properties of heparanase. This probablyis because T5 is expressed along with heparanase intumor-derived cells (Fig. 2A) and in clinical specimens(Fig. 6) to comparable levels and appears to promotecell proliferation (Fig. 4A), colony formation (Fig. 4B),xenograft progression (Fig. 5A), and tumor angiogen-esis (Fig. 5C). Thus, while inhibitors directed againstthe enzymatic activity of heparanase are currently eval-uated in clinical trials (44–47), T5 as well as the hepara-nase C domain responsible for enzymatic activity-indepen-dent functions of heparanase (26, 34) are not expectedto be affected by these inhibitors. It appears, therefore,that a well-defined enzymatic activity thought to berelatively easy to target, turned, at least in certain tumorsystems, into a complex matter as more knowledgeaccumulates and the biology of the protein is revealed.

Heparanase and T5 share several biochemical charac-teristics. The signal peptide of heparanase is retained in

Figure 6. Clinical relevance of T5. Total RNA was extracted from biopsies of renal cell carcinoma (C) and adjacent normallooking tissue (N) and subjected to RT-PCR analysis applying T5 (top panel), heparanase (middle panel), and GAPDH primers(bottom panel).


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T5, enabling its delivery to the ER and Golgi apparatus(Fig. 2C), glycosylation (evident by sensitivity to tunicamy-cin and binding to Con A; Fig. 2B and Supplemental Fig.1A), and secretion (Fig. 2B). Protein N-glycosylation likelyoccurs on a single asparagine residue (Asn162) preservedin T5 and shown to be required for protein secretion(48). Not surprisingly, T5 is devoid of heparanase enzy-matic activity, nor did it compete with the endogenousheparanase activity (Fig. 2D). This is largely because T5 isincapable of heparin binding (Supplemental Fig. 1).Although the basic heparin binding domain of hepara-nase (Lys158-Asn162) is retained in T5 and is followed byadditional positively charged amino acids (KK) thought tocreate an even more potent heparin binding domain, T5failed to interact with heparin (Supplemental Fig. 1A).Similarly, a peptide corresponding to this sequence in-cluding the unique amino acids introduced in T5 (CKKK;Supplemental Fig. 1B) failed to interact with heparin andto inhibit heparanase enzymatic activity, as opposed to theKKDC peptide, which efficiently accomplished both tasks(Supplemental Fig. 1B, C) (38). Thus, the mere presenceof Cardin-Weintraub consensus sequence (49) appearsinsufficient to ensure high-affinity binding to heparin andneighboring amino acids dictate the functional outcome(38). Therefore, Src activation by T5 cannot be explainedby interaction with HS and/or clustering of syndecans.The rational behind this function is likely structural.According to this notion, the alternative splicing gener-ated a truncated protein that acquired a novel conforma-tion not normally present in the heparanase molecule(50). This may also be the case with Spalax heparanasesplice variant 7, noted to enhance U87 xenograft devel-opment (51), while Spalax splice variant 36, which did notadvance tumor progression, assume an inactive conforma-tion. It should be noted that Spalax splice variants (splice7 and splice 36) appear unique to this mammal, andequivalent splice variants could not be identified in hu-mans, which leaves T5 the only functional human hepara-nase splice variant reported so far. Both heparanase andT5 still await detailed structural analysis.

Interestingly, heparanase and T5 appear to be localizedin endocytic vesicles (Supplemental Fig. 3B). Whileheparanase is subjected to cellular uptake together withsyndecans and accumulates in the lysosomal compart-ment (52, 53), where it undergoes proteolytic processingby cathepsin L (54–56), T5 does not interact with HS. Itsdelivery to endocytic vesicles is, therefore, likely mediatedby interaction with other cell surface molecules responsi-ble for its signaling and protumorigenic properties.

Taken together, we describe here, for the first time, afunctional splice variant of heparanase, discoveredthrough Compugen’s LEADS platform. The T5 splicevariant possesses protumorigenic properties, facilitatingcell proliferation, colony formation, and tumor xenograftdevelopment. Furthermore, T5 is expressed in the major-ity of renal cell carcinoma biopsies examined, stronglysupporting the clinical relevance of this protein variant.T5 appears, therefore, as a valid target for the develop-ment of anticancer drugs, introducing another level ofcomplexity to the heparanase field, and the attempts toneutralize its growing repertoire of biological activities.Prospective analysis of a large cohort of tumor biopsies isrequired to appreciate adequately the outcome of pa-

tients expressing high vs. low levels of T5. In addition,staining of tumor biopsies with monoclonal antibodyspecific for T5, but not heparanase will enable, possibly, todistinguish between T5-positive and -negative cases andreveal its association with patient outcome.

This work was supported by grants from the Israel ScienceFoundation (549/06); the National Cancer Institute, U.S. Na-tional Institutes of Health (RO1-CA106456); the DKFZ-MOSTcooperation program in cancer research; the Technion FinePostdoctoral Fellowship; and the Israel Cancer Research Fund(ICRF) Postdoctoral Fellowship. I.V. is a Research Professor ofthe ICRF.

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Received for publication September 28, 2009.Accepted for publication November 5, 2009.


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A novel human heparanase splice variant, T5, endowed with protumorigenic characteristics

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