The Dermatofibrosarcoma Protuberans-associated Collagen ...cancerres.aacrjournals.org/content/canres/59/15/3719.full.pdf · The Dermatofibrosarcoma Protuberans-associated Collagen

[CANCER RESEARCH 59, 3719–3723, August 1, 1999]

The Dermatofibrosarcoma Protuberans-associated Collagen Type Ia1/Platelet-derived Growth Factor (PDGF) B-Chain Fusion Gene Generates aTransforming Protein That Is Processed to Functional PDGF-BB1

Akira Shimizu, 2 Kevin P. O’Brien,2 Tobias Sjoblom, Kristian Pietras, Elisabeth Buchdunger, V. Peter Collins,Carl-Henrik Heldin, Jan P. Dumanski, and Arne Ostman3

Ludwig Institute for Cancer Research, S-751 24 Uppsala, Sweden [A. S., T. S., K. P., C-H. H., A. O¨ .]; Department of Molecular Medicine, Clinical Genetics Unit, KarolinskaHospital, S-171 76 Stockholm, Sweden [K. P. O., J. P. D.]; Novartis Pharma AG, Oncology Research, CH-4002 Basel, Switzerland [E. B.]; and Department of Histopathology,University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom [V. P. C.]

ABSTRACT

Dermatofibrosarcoma protuberans (DFSP) displays chromosomal re-arrangements involving chromosome 17 and 22, which fuse the collagentype Ia1 (COLIA1) gene to the platelet-derived growth factor (PDGF)B-chain (PDGFB) gene. To characterize the functional and structuralproperties of the COLIA1/PDGFB fusion protein, we generated a stableNIH3T3 cell line that contained a tumor-derived chimeric gene resultingfrom a COLIA1 intron 7-PDGFB intron 1 fusion. Expression of the fusionprotein led to morphological transformation and increased growth rate ofthese cells. The PDGF receptor kinase inhibitor CGP57148B reversed thetransformed phenotype and reduced the growth rate ofCOLIA1/PDGFB-expressing cells but had no effects on control cells. The presence of dimericCOLIA1/PDGFB precursors was demonstrated through PDGFB immu-noprecipitations of metabolically labeled cells and also by PDGFB immu-noprecipitations followed by immunoblotting with COLIA1 antibodies.Pulse-chase studies demonstrated that the COLIA1/PDGFB precursorwas processed to an end product that was indistinguishable from wild-type PDGF-BB. Finally, COLIA1/PDGFB-expressing cells generated tu-mors after s.c. injection into nude mice, and tumor growth was reduced bytreatment with CGP57148B. We conclude that theCOLIA1/PDGFB fusionassociated with DFSP contributes to tumor development through ectopicproduction of PDGF-BB and the formation of an autocrine loop. Ourfindings, thus, suggest that PDGF receptors could be a target for phar-macological treatment of DFSP and giant cell fibroblastoma,e.g., throughthe use of PDGF receptor kinase inhibitors such as CGP57148B.

INTRODUCTION

DFSP4 and its juvenile form, GCF, are highly recurrent, infiltrativeskin tumors of intermediate malignancy (1). They are occasionallymisdiagnosed as benign lesions such as dermatofibroma and neurofi-broma, leading to improper primary management. The current treat-ment of DFSP/GCF is entirely surgical, and recurrence rates of up to50% following removal have been reported (2). The histogenesis ofDFSP/GCF is still controversial; however, the strongest evidencesupports a fibroblastic origin (2).

All DFSP and GCF cases studied to date have been found to containa fusion ofCOLIA1 to PDGFB, as a result of rearrangements involv-ing chromosomes 17 and 22 (3, 4). TheCOLIA1 gene, located on17q22, encodes the major component of type I collagen, the mostabundant protein in the body, which is produced primarily by fibro-

blasts.PDGFB (or c-sis proto-oncogene), located on 22q13, is thecellular equivalent of the v-sis oncogene, which causes simian sar-coma (5, 6). PDGF-BB, the homodimer formed by disulfide linking,is a potent growth factor that acts as a mitogen and chemoattractantfor a variety of connective tissue cells (7). It exerts its action throughtwo structurally similar tyrosine kinase receptors, the PDGFa- andb-receptors, on the surface of target cells (8). The coexpression ofPDGFB and its receptors has been described in several tumors,including DFSP, and involvement of a PDGFB autocrine loop intumorigenesis has been suggested (9). The PDGFb-receptor pathwayhas also been implicated in chronic myelomonocytic leukemia, inwhich a translocation gives rise to a ligand-independent constitutivelyactive form of the PDGFb-receptor (10, 11).

The transforming potential of theCOLIA1/PDGFBfusion gene wasrecently demonstrated (12). In this study, we have generated aNIH3T3 cell line that expresses a tumor-derived chimeric gene. Thecell line has been used to characterize the fusion protein functionallyand structurally.

MATERIALS AND METHODS

COLIA1/PDGFB Cosmid Isolation and Establishment of NIH3T3 CellLines. The COLIA1/PDGFB fusion transcript and genomic breakpoint ofTNM2, a DFSP recurrence, has been characterized previously, and a Super-Cos1 cosmid library has been constructed (4). This library was screened usingpreviously described probes: aCOLIA1 probe, 59COL, and aPDGFB probe,pex7 (13). The following primers were used in PCR amplification and se-quencing: fd.f2, 59-CCT CGG AGG AGT AAG TGG; SuperCos1, 59-GCAATT AAC CCT CAC TAA AG; SuperCos2, 59-CCG CAT AAT ACG ACTCAC TAT; and c1a1.int1, pdgfb.int, c1a1.ext1, C1A1.TS1, C1A1.TS3,C1A1.TS4, C1A1.TS5, PF.T1r, and PDGF.r6 (4).

NIH3T3 cells (ATCC CRL-1658) were maintained in DMEM supple-mented with 10% calf serum using standard conditions. Twomg of cosmidDNA were transfected into 33 105 NIH3T3 cells plated in 353 10 mm platesin serum-free DMEM using Lipofectamine, according to the recommendationsof the supplier (Life Technologies, Inc., Gaithersburg, MD). Transfectantswere selected in 600mg/ml geneticin (Life Technologies, Inc.) before passag-ing. Genomic DNA was extracted from cells using standard protease Kdigestion followed by phenol/chloroform extraction. The presence of thefusion gene was confirmed by PCR of genomic DNA using the primers fd.f2and pdgfb.int (see above). Total RNA was extracted from cells using acidicguanidinium thiocyanate-phenol-chloroform extraction (14). Reverse tran-scriptase-PCR and sequencing was performed as described (4). The cell linesB5/5 (COLIA1/PDGFBtransfectant) and CNEG-1 (negative control) wereused in this study.

Drugs and Immunological Reagents,in Vitro Growth Characterization,Metabolic Labeling, Immunoprecipitation, and Immunoblotting. Brefel-din A (Sigma Chemical Co., St. Louis, MO) was prepared as a stock solutionof 6 mM in ethanol. The PDGF receptor tyrosine kinase inhibitor CGP57148B(15) was prepared as a 100mM stock solution in PBS. Goat anti-PDGFb-receptor antibody P-20 (Santa Cruz Biotechnology, Santa Cruz, CA), mono-clonal antiphosphotyrosine antibody PY20 (Transduction Laboratories, Lex-ington, KY), and rat antihuman pro-collagen I NH2-terminal monoclonal

Received 12/29/98; accepted 5/21/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Cancer Foundation, the Swedish MedicalResearch Council, the Berth von Kantzow Fond, the Cancer Society in Stockholm, and theKarolinska Institute.

2 The first two authors contributed equally to this work.3 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer

Research, Box 595, S-751 24 Uppsala, Sweden. Phone: 46-18-160414; Fax: 46-18-160420; E-mail: [email protected].

4 The abbreviations used are: DFSP, dermatofibrosarcoma protuberans; GCF, giantcell fibroblastoma; COLIA1, collagen type Ia1; PDGF, platelet-derived growth factor,PDGFB, PDGF B-chain.

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antibody (Chemicon, Temicula, CA) were used as recommended by thesuppliers.

For studies of thein vitro growth rates, cells were plated in six-well plates(5 3 104 cells/well) in DMEM supplemented with 10% FCS in the presenceor absence of 1.0mM CGP57148B. Medium exchange was performed threetimes per week. After trypsinization, cells were counted using a Coulterparticle counter.

Metabolic labeling was performed as follows: cells were labeled for 2 h with0.1 mCi/ml [35S]cysteine in cysteine-free MCDB 104 medium supplementedwith 1 mg/ml BSA. In pulse-chase experiments, chase was performed inmedium containing 250mg/ml unlabeled cysteine. Immunoprecipitations wereperformed using a PDGF-BB antiserum (16), as described previously (17).

To detect the COLIA1/PDGFB fusion protein through COLIA1 immunoblot-ting, we incubated subconfluent CNEG-1 and B5/5 cells with 10mM brefeldin Afor 4 h at 37°C. Cell lysates were prepared and immunoprecipitated withPDGF-BB antiserum, as described above. After 7% SDS-PAGE and semidrytransfer to nitrocellulose membranes, membranes were incubated with rat antihu-man pro-collagen I antibody. After incubation with horseradish peroxidase-con-jugated antirat antibody (Amersham, Uppsala, Sweden), COLIA1/PDGFB fusionproteins were detected by enhanced chemiluminescence.

For immunoprecipitation and immunoblotting of PDGFb-receptors, cellswere incubated overnight in the absence or presence of CGP57148B inserum-free DMEM containing 1 mg/ml BSA, washed with PBS, and lysed by15 min of incubation in 1% NP40, 0.15M NaCl, 20 mM Tris (pH 7.5), 5 mM

EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol, and1 mM orthovanadate at 4°C. After centrifugation, lysates were incubated withPDGFb-receptor antiserum PDGFR-3 (18) for 2 h at4°C. After collection ofimmune complexes with protein A-Sepharose, complexes were washed threetimes with lysis buffer and once with 20 mM Tris-HCl (pH 7.5); immunecomplexes were then eluted and subjected to 7% SDS-PAGE. Proteins weretransferred to nitrocellulose membranes using semidry electrophoresis. Mem-branes were blocked and probed with phosphotyrosine antibody PY20, andafter incubation with horseradish peroxidase-conjugated antimouse antibodies,tyrosine-phosphorylated proteins were detected by enhanced chemilumines-cence (ECL; Amersham). For detection of PDGFb-receptor expression, mem-branes were stripped and incubated with PDGF receptor antiserum P-20.PDGF receptors were visualized by incubation with horseradish peroxidase-conjugated antigoat antibodies (Amersham) followed by ECL.

Tumorigenicity Assays and Histological Characterization of s.c. Tu-mors. B5/5 and CNEG-1 cells (23 106 in 50 ml of PBS) were injected s.c.into the left flank of 6–10-week-old male BALB/cnu/numice; 10 mice wereinjected with each cell type. Half of the mice in each group were given 50mg 3 kg21 3 day21 CGP57148B in 200ml of PBS, and the other half weregiven 200 ml of PBS only. Treatment was started on the day followinginjection of tumor cells. Drug was administered p.o. by tube feeding oncedaily. Animals were individually caged and fed animal chowad libitum.Tumor diameter was measured every 3 days, and tumor volume was subse-quently calculated using the formulaV 5 p 3 6-21 3 a2 3 b, wherea andbrepresent the shorter and the longer diameters of the tumor, respectively. Forhistological analysis, tumors were fixated in 4% paraformaldehyde and em-bedded in paraffin, and 5-mm sections were subjected to H&E and Picro-Ponceau stainings.

RESULTS

Isolation of a COLIA1/PDGFB Cosmid and Establishment ofCOLIA1/PDGFB-expressing NIH3T3 Cell Lines. TNM2, a DFSPcase displaying a ring chromosome, contains aCOLIA1/PDGFBchimeric gene with aCOLIA1 intron 7-PDGFBintron 1 fusion. Froma SuperCos1 cosmid library of tumor TNM2, 15 positive clones wereisolated and characterized by end-sequencing. One cosmid, CC12,was found to contain the full chimeric gene. The position of the 59 and39 ends of the fusion gene was extrapolated from end-sequencing,primer walking, and insert size estimation (35 kb). The 59 end of thechimeric gene was located in the middle of the cosmid, and the 39 endwas located 1 kb from the end of the cosmid. A negative controlcosmid, CNEG, was randomly picked from the TNM2 cosmid libraryand confirmed by PCR to contain neitherCOLIA1 nor PDGFB

segments. Cosmids CC12 and CNEG were transfected into NIH3T3cells using Lipofectamine and, after G418 selection, clones of resist-ant cells were obtained. CC12-transfected clones were confirmed byPCR to contain theCOLIA1/PDGFB fusion gene. Reverse tran-scriptase-PCR analysis revealed that these cells produced the expectedchimeric transcript with aCOLIA1 exon 7-PDGFBexon 2 fusion.This was confirmed by Northern blot analysis, which detected achimeric 3.4-kb transcript (data not shown). OneCOLIA1/PDGFB-positive clone, B5/5, was chosen for further analysis together withCNEG-1, a clone derived from the transfection with the CNEGcosmid.

Expression of COLIA1/PDGFB Protein Leads to Transforma-tion of NIH3T3 Cells through Activation of PDGF b-Receptors.To investigate the potential transforming ability of the COLIA1/PDGFB protein, we compared the morphology and growth pattern ofB5/5 and CNEG-1 cells. As shown in Fig. 1A, B5/5 cells grew in adisorganized manner and displayed a spindle-shaped morphology thatis typical of transformed cells, with loss of contact inhibition. Addi-tion of the PDGF receptor kinase inhibitor CGP57148B dramaticallyaltered the morphology of B5/5 cells to a phenotype that is indistin-guishable from that of the control cells, indicating that the alteredmorphology occurred as a consequence of PDGF receptor activation

Fig. 1. COLIA1/PDGFB expression leads toin vitro transformation through activationof PDGF receptors.A, CNEG-1 (control) and B5/5 (COLIA1/PDGFB) cells were culturedin the absence or presence of 1.0mM CGP57148B for 6 days in DMEM containing 10%FCS. Microphotographs were taken with a phase contrast microscope.B, CNEG-1 cells(M andf) and B5/5 cells (E andF) were plated in six-well plates at 2.53 104 cells/wellin DMEM containing 10% FCS and grown in the absence (M andE) or presence (f andF) of 1.0mM CGP57148B. At indicated times, cells were trypsinized, and cell number wasdetermined with a Coulter counter.C, CNEG-1 (control) and B5/5 (COLIA1/PDGFB)cells were grown in the absence or presence of CGP57148B, lysed, and subjected toPDGF b-receptor immunoprecipitation and SDS-gel electrophoresis. After transfer tomembranes immunoblotting with phosphotyrosine antibodies (top) and PDGFb-receptorantiserum (bottom) was performed.

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(Fig. 1A). The effects of CGP57148B on the growth rate of B5/5 andCNEG-1 cells were also analyzed (Fig. 1B). Whereas the growth rateof CNEG-1 cells was unaffected by CGP57148B, the growth of B5/5cells was dramatically reduced in the presence of the PDGF receptorkinase inhibitor. The difference in growth rate between CGP57148B-treated CNEG-1 and B5/5 cells most likely reflect clonal variationsbetween the two NIH3T3 cell line derivatives. However, the cleardifference between the two cell lines with regard to CGP57148Bsensitivity strongly indicate that autocrine PDGF receptor signalingcontributes to the growth of B5/5 cells but not to the growth ofCNEG-1 cells.

To directly demonstrate the presence of activated PDGFb-recep-tors in B5/5 cells, receptors were isolated, from cells grown in theabsence or presence of CGP57148B, by immunoprecipitations andwere subjected to phosphotyrosine immunoblotting (Fig. 1C). Uponimmunoblotting, B5/5 extracts display a strong signal, indicatingautocrine stimulation of PDGFb-receptors by theCOLIA1/PDGFB-encoded protein. Furthermore, the steady-state level of PDGFb-re-ceptors in B5/5 cells was reduced compared to CNEG-1 cells, con-sistent with a ligand-induced receptor down-regulation. Finally, theinhibitory effect of CGP57148B on PDGF receptor activation wasclearly demonstrated by the finding of dramatically reduced tyrosinephosphorylation of PDGF receptors after treatment (Fig. 1C, Lanes 2and4). Studies using mutant forms of PDGFb-receptors have dem-onstrated that kinase-inactive receptors still display down-regulationupon ligand binding (19). Similarly, it is likely that that the reducedreceptor levels in CGP57148B treated B5/5 cells also occur as aconsequence of ligand-induced internalization, despite the absence oftyrosine kinase activation.

The COLIA1/PDGFB Fusion Protein Forms a Disulfide-linkedPrecursor Dimer That Is Processed to Mature PDGF-BB.Tocharacterize the protein(s) encoded by theCOLIA1/PDGFB fusiongene, we metabolically labeled the B5/5 cells with [35S]cysteine, andcell lysates were subjected to immunoprecipitations using aPDGF-BB antiserum (Fig. 2A). To enrich for precursor forms, weperformed the labeling in the presence of brefeldin A, a drug thatblocks ER to Golgi transport and that enriches for PDGF precursorforms (20). For comparison,PDGFB-expressing NIH3T3 cells (21)were included in the analysis. An unreducedMr 110,000 component,which was converted to aMr 60,000 component after reduction, wasidentified in the lysates of B5/5 cells (Fig. 2A,Lanes 4and8). Thesefindings indicate that the COLIA1/PDGFB fusion protein, likePDGFB, forms a dimeric precursor. To strengthen the notion that theMr 110,000 protein observed in B5/5 cells represents a COLIA1/PDGFB fusion protein, we subjected lysates of B5/5 cells to PDGFimmunoprecipitations followed by immunoblotting with a collagenantibody. A Mr 110,000 component was identified in lysates ofbrefeldin A-treated B5/5 cells but not in CNEG-1 cells (Fig. 2B).

To study the processing of the COLIA1/PDGF fusion protein inmore detail, we performed pulse-chase experiments usingCOLIA1/PDGFB- andPDGFB-expressing cells. Analysis ofPDGFB-express-ing cells revealed a gradual decrease in the cell-associated unproc-essedMr 56,000 precursor, with an accompanying increase in cell-associatedMr 40,000 andMr 24,000 forms over time (Fig. 3,Lanes6–8), which is consistent with previous reports (20, 22). Similarly, inthe case of B5/5 cells, processed forms ofMr 40,000 and 24,000 werealso found to accumulate with time. In both cell types, very smallfractions of the processed forms were secreted into the medium (Fig.3, Lanes 4,5, 9, and10). The distribution between cell-associated andsecreted forms of theMr 40,000 species did not vary in a consistentway betweenCOLIA1/PDGFB- andPDGFB-expressing cells (datanot shown).

The COLIA1/PDGFB precursor, thus, forms a COLIA1/PDGFB

dimeric precursor that is processed to mature forms indistinguishablefrom wild-type PDGF-BB, both with regard to structure and compart-mentalization.

In Vivo Tumorigenesis by COLIA1/PDGFB-expressingNIH3T3 Cells. To investigate the tumorigenic potential of the CO-LIA1/PDGFB protein, we injected B5/5 cells and CNEG-1 cells intonude mice and monitored them for the appearance of tumor growth.B5/5 cells, in contrast to CNEG-1 cells, formed tumors of measurablesize within 2 weeks (Fig. 4). The PDGF receptor kinase inhibitorCGP57148B significantly reduced the growth of B5/5 derived tumors,indicating that an autocrine PDGF receptor stimulation contributed to

Fig. 2. Detection of COLIA1/PDGFB fusion protein inCOLIA1/PDGFB-expressingcells. A, NIH3T3 cells expressingPDGFB (Lanes 1,2, 5, and6) or COLIA1/PDGFB(Lanes 3,4, 7, and8) were metabolically labeled with [35S]cysteine for 2 h in thepresenceof brefeldin A. Lysates were immunoprecipitated with preimmune serum (pi) orPDGF-BB antiserum (BB) and analyzed under nonreducing (Lanes 1–4) or reducing(Lanes 5–8) conditions by 5–15% SDS-PAGE.Arrows, positions of nonreduced andreduced precursor forms. Positions of molecular weight markers are indicated in themiddle. B, CNEG-1 (control) and B5/5 (COLIA1/PDGFB) cells were cultured in theabsence or presence of 10mM brefeldin A for 4 h and immunoprecipitated with anti-PDGF-BB serum. After SDS-gel electrophoresis under nonreducing conditions and trans-fer to membranes, immunoblotting with pro-COLIA1 antibody was performed. Positionsof molecular weight markers are indicated to theleft.

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tumor development (Fig. 4). The CGP57148B effect on the growth ofB5/5 derived tumors is similar to the previously published effects onv-sis-transformed BALB/c 3T3 cells (15). Finally, sections of tumorsderived fromCOLIA1/PDGFB-expressing cells were analyzed histo-logically. Tumors displayed a malignant phenotype similar to humansarcomas, with invasion of surrounding tissues, high vascularization,and necrotic areas. When the histological appearance of tumors de-rived from PDGFB- and COLIA1/PDGFB-expressing NIH3T3 cellswas compared, no major differences in growth pattern between thetwo was observed (data not shown), suggesting that the COLIA1 partof the COLIA1/PDGFB fusion protein does not contribute to thetumor phenotype.

DISCUSSION

DFSP and GCF display chromosomal rearrangements involvingchromosome 17q22 and 22q13 (23). These rearrangements fuse theCOLIA1 gene (17q22) to thePDGFB gene (22q13; 3, 4). There iswide variation in the position of the fusion position inCOLIA1, but italways occurs within the region encoding thea-helical coding do-main. The exons ofCOLIA1 in this region consistently end at the lastbase of a codon. The breakpoint inPDGFB is always in intron 1. Theresulting COLIA1/PDGFB fusion is, therefore, in-frame, becauseexon 2 ofPDGFBstarts at the first base of codon 22. This fusion genethus encodes a protein consisting of a COLIA1 NH2-terminal peptidefused to residues 22–241 of the PDGFB propeptide. The DFSP caseTNM2 used in this study displays a ring chromosome, which containsa chimeric gene, the result of aCOLIA1 intron 7-PDGFBintron 1fusion (4). This fusion gene was sufficiently compact to allow cloningin a cosmid vector. The cosmid CC12 was subsequently found tocontain the entire chimeric gene, including promoter and regulatoryregions. Although the DFSP/GCF histogenesis is still controversial,evidence from both electron microscopy and the involvement ofCOLIA1, suggest that DFSP/GCF is of fibroblastic origin (2). Be-cause DFSP cells, fibroblasts, and NIH3T3 have all been shownpreviously to produce collagen and express PDGFb-receptors, wechose NIH3T3 cells as a model for this study (24–27).

To study the functional and structural properties of the COLIA1/PDGFB protein, we stably transfected NIH3T3 cells with CC12 andestablished a number of cell lines, from which one, B5/5, was se-lected. The fusion gene transformed NIH3T3 cells into spindle-shapedcells, with a more disorganized growth pattern and the ability to formtumors in nude mice (Figs. 1A and 4). Expression of the fusion genealso made the cells sensitive to a PDGF receptor kinase inhibitor withregard to growth rate. These changes are all consistent with anautocrine PDGFB stimulation. We demonstrated the expression of anin-frameCOLIA1/PDGFBfusion transcript in B5/5 cells, identical tothe fusion transcript produced by TNM2 tumor cells. Furthermore,this transcript is translated into the endoplasmic reticulum to form aCOLIA1/PDGFB fusion peptide, with COLIA1 providing the signalpeptide. This fusion peptide forms a disulfide-linkedMr 110,000homodimeric precursor protein that is further processed toMr 40,000andMr 24,000 forms. The two latter forms appear identical to theMr

40,000 andMr 24,000 forms previously identified in studies onPDGF-BB biosynthesis (28). TheMr 40,000 form of PDGF-BB isgenerated by cleavage between amino acid residues 81 and 82 of thePDGFB precursor and theMr 24,000 form represent a dimer ofsubunits that have undergone additional NH2- and COOH-terminalprocessing (28). We, thus, conclude that the COLIA1/PDGFB dimeris processed in a way that removes the entire COLIA1 portion of thefusion protein and that the final protein product(s) of the COLIA1/PDGFB fusion gene is a mature PDGF-BB dimer.

We also demonstrated the activation of PDGFb-receptors in B5/5cells but not in control cells (Fig. 1C), a finding that confirms that theCOLIA1 fusion protein is processed to functional PDGF-BB. Little orno PDGF-BB is secreted from B5/5cells (Fig. 3), which is consistentwith previous observations onPDGFB or v-sis-expressing cells (22,28, 29). It is, thus, possible that, as in the case of v-sis-transformedcells, PDGF receptors are activated intracellularly and subsequentlytransported to the cell membrane, where they couple with the signaltransduction machinery (30).

The tumors formed by B5/5 cells in nude mice were identical ingrowth, appearance, and collagen distribution as compared to tumorsformed by PDGFB-expressing cells. This suggests that the COLIA1part of the fusion protein does not contribute to the phenotype of thetumor. Most likely the COLIA1 part of the fusion gene thus serves toprovide an active promoter and signal peptide for PDGFB. In addi-tion, the translocation removes negative regulatory elements in the 59

Fig. 4. COLIA1/PDGFB-expressing cells are tumorigenicin vivo and show sensitivityto CGP57148B. B5/5 cells (E andF) and CNEG-1 cells (M andf) were injected s.c. intonude mice; 10 mice were injected with each cell type. Tumor growth was monitored inmice receiving CGP57148B in PBS (f andF) or PBS only (M andE). Treatment wasstarted the day after tumor cell injection. Drug was administered p.o., by tube-feeding,once daily at a dose of 100 mg3 kg21 3 day21. Bars, SE. At the end of the study, tumorsof CGP57148B-treated animals were significantly smaller than tumors of control-treatedanimals (two-tailed Mann-WhitneyU test;P , 0.01).

Fig. 3. Pulse-chase analysis of the processing of PDGF-like protein inCOLIA1/PDGFB-expressing cells. Cells were labeled for 2 h with [35S]cysteine and cultured forthe indicated time periods in medium containing an excess of unlabeled cysteine. Celllysates and condition media were subjected to immunoprecipitation using PDGF-BBantiserum and analyzed under nonreducing conditions by 5–15% SDS-PAGE. Positions ofmolecular weight markers are indicated in themiddle.

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end of the PDGFB gene and, thereby, potentiates protein production(31).

Experiments involving the PDGF receptor tyrosine kinase inhibitorCGP57148B suggested that activation of PDGF receptors was respon-sible for the transformation induced byCOLIA1/PDGFBexpressionin NIH3T3 cells. This inhibitor reversed the transformed phenotype,giving B5/5 a morphology that was indistinguishable from that of thenegative control CNEG-1 (Fig. 1). This inhibitor also reduced thegrowth of B5/5 derived tumors in nude mice (Fig. 4).

In conclusion, we show that the DFSP/GCF-associatedCOLIA1/PDGFB fusion gene results in the production of a mature PDGF-BBin a collagen-producing cell and thereby leads to autocrine growthstimulation. Expression of PDGFb-receptors has been demonstratedin DFSP and GCF, both on tumor sections and on cultured tumor-derived cells (26, 32). A PDGF autocrine loop, therefore, most likelyoccurs in DFSP/GCF tumor cells. On the basis of our findings, thereseems to be a strong motivation to explore the possibility of targetingPDGF receptors in pharmacological treatment of DFSP/GCF, forexample, by investigating the effects of kinase inhibitors, likeCGP57148B, on primary cultures of DFSP and GCF tumor cells.

ACKNOWLEDGMENTS

We thank Dr. Nils Mandahl for supplying the tumor TNM2, Mari-AnneCarlsson for embedding the tumors, and Ingegard Schiller for secretarialassistance.

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PROCESSING OF COLIA1/PDGFB PROTEIN TO PDGF-BB

Research. on June 30, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

1999;59:3719-3723. Cancer Res   Akira Shimizu, Kevin P. O'Brien, Tobias Sjöblom, et al.   Processed to Functional PDGF-BBFusion Gene Generates a Transforming Protein That Is

1/Platelet-derived Growth Factor (PDGF) B-ChainαType IThe Dermatofibrosarcoma Protuberans-associated Collagen

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