A Recurrent Activating PLCG1 Mutation in Cardiac … · Molecular and Cellular Pathobiology A Recurrent Activating PLCG1 Mutation in Cardiac Angiosarcomas Increases Apoptosis Resistance

Molecular and Cellular Pathobiology

A Recurrent Activating PLCG1 Mutation in CardiacAngiosarcomas Increases Apoptosis Resistance andInvasiveness of Endothelial Cells

Kristin Kunze1, Tilmann Spieker2, Ulrike Gamerdinger1, Kerstin Nau1, Johannes Berger1, Thomas Dreyer1,J€urgen R. Sindermann3, Andreas Hoffmeier3, Stefan Gattenl€ohner1, and Andreas Br€auninger1

AbstractPrimary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations

are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arraysand targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gainsof chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNParray analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymousmutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation(R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCg1 is usually activatedby Y783 phosphorylation and activates protein kinase C and Ca2þ-dependent second messengers, witheffects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCg1-R707Q mutantin endothelial cells revealed reduced PLCg1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca2þ-dependent calcineurin activationcompared with ectopic expressed PLCg1-wild-type. Furthermore, cofilin, whose activation is associated withactin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression ofPLCg1-R707Q compared with PLCg1-wild-type. At the cellular level, expression of PLCg1-R707Q inendothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migrationand invasiveness in in vitro assays. Together, these findings indicate that the PLCg1-R707Q mutation causesconstitutive activation of PLCg1 and may represent an alternative way of activation of KDR/PLCg1 signalingbesides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies. Cancer Res;74(21); 6173–83. �2014 AACR.

IntroductionPrimary malignant cardiac tumors are rare and encom-

pass several sarcoma entities (1). Besides undifferentiatedsarcomas angiosarcomas are most frequent. Angiosarcomasare derived from endothelial cells and encompass a hetero-geneous group of tumors with variable histologic character-istics and several clinical forms besides cardiac angiosarco-mas, such as the skin and scalp angiosarcomas, angiosarco-mas arising in irradiated fields, and hepatica angiosarcomasrelated to occupational causes (2–4). Cardiac angiosarcomas

are characterized by a high proliferation rate and a markedpropensity to metastasize, mostly to the lung (5, 6). Patientshave usually developed metastases at the time of diagnosisand prognosis is extremely adverse due to the inoperablelocalization and a distinct insensitivity toward chemo- andradiotherapy. Because of a lack of clinical studies, thecurrent therapy is oriented on morphologic similar sarco-mas of other localizations and mainly based on the inhibi-tion of angiogenesis (7, 8).

Knowledge regarding the pathogenesis of cardiac sarcomasis so far very limited and only studies of few or single cases ofcardiac sarcomas of various entities using cytogenetics andmutational analyses of KRAS andTP53 are available (9–11). Forcardiac angiosarcomas, these studies described several numer-ical abnormalities and KRAS mutations in 2 of 3 and p53mutations in 2 of 4 cases analyzed (12). For angiosarcomas ofother locations, recently recurrent mutations in PLCg1 andPTPRB were detected (13).

To gain insight into the pathogenesis of primary cardiacangiosarcomas, we analyzed a collection of formalin-fixedparaffin-embedded (FFPE) biopsies of 10 cases for genomicaberrations. SNP arrays were used to detect genomic gains andlosses and targeted next-generation sequencing (tNGS), which

1Department of Pathology, Justus-Liebig-University Giessen, Giessen,Germany. 2Institute for Pathology, St. Francis Hospital, M€unster, Germany.3Department of Cardiothoracic Surgery, Division of Cardiac Surgery, Uni-versity Hospital M€unster, M€unster, Germany.

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

Corresponding Author: Andreas Br€auninger, Department of Pathology,Justus-Liebig-University Giessen, Langhansstr. 10, 35392 Giessen, Ger-many. Phone: 49-641-98541130; Fax: 49-641-98541139; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-1162

�2014 American Association for Cancer Research.

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is suitable for the analysis ofDNA fromFFPEbiopsies, was usedto detect single-nucleotide variants (SNV) and small insertionsand deletions (indel) in 409 frequently mutated oncogenes andtumor-suppressor genes.

Materials and MethodsTumor samples and cell lines

Primary cardiac angiosarcomas were collected by theDepartments of Heart Surgery and Pathology, WestphalianWilhelms-University (M€unster, Germany), from 2002 to 2013.The study was conducted in accordance with the Declara-tion of Helsinki and approved by the local Ethics commis-sion (Ethics commission of the Medical Faculty of theWestphalian-Wilhelms University M€unster, 2009-283-f-S).HEK293 were obtained from the DSMZ (Braunschweig,Germany) and the identity was regularly (each 6 months)confirmed using short tandem repeat (STR) profiling(AmpFLSTR Profiler Plus; Life Technologies). Human umbil-ical vein endothelial cells (HUVEC) were obtained fromPromoCell, cultured in standard endothelial cell growthmedium [ECGM; containing 0.1 ng/mL EGF and 1 ng/mLbasic fibroblast growth factor (bFGF); PromoCell] and usedat passage 1 to 10. For stimulation of PLCg1 ECGM withhigher concentrations of growth factors (ECGM2; containing5 ng/mL EGF, 10 ng/mL bFGF, 20 ng/mL IGF, and 0.5 ng/mLVEGF 165; PromoCell) was used. Cell numbers were countedusing a hemocytometer.

DNA extractionTumor cell–rich regions (fraction of tumor cells >80%) and

tumor cell–free regions were microdissected from 5-mm tissuesections of the FFPE biopsies and genomic DNA was extractedusing the Maxwell 16 FFPE LEV DNA Purification Kit(Promega).

SNP array analysisSNP array analysis was based on Illumina's HumanOmniEx-

press-FFPE BeadChip (Illumina) adapted for DNA extractedfrom FFPE tissue. The procedure comprises a quantitative-PCR quality control, a FFPE DNA restoration step, and theInfinium HD FFPE assay, based on whole-genome amplifica-tion, DNA fragmentation, hybridization of fragmented DNA to50mer probes bound to beads on the array, and enzymaticsingle base extension with differently labeled nucleotides todiscriminate between AT and GC SNPs. Subsequently, colorand signal intensity was detected using Illumina's iScan imag-ing system.

Log R ratios (LRR) and B-allele frequencies (BAF) wereanalyzed with the Illumina Genome Studio software. LRRis the ratio between observed and expected probe intensity andinformative regarding copy number alterations, while BAF is theB-allele frequency and informative regarding heterozygosity.

ImmunohistochemistryImmunohistochemical analyses were performed with the

DakoREALDetection System (Dako). Details for the antibodiesused are given in Supplementary Table S1.

Targeted next-generation sequencingFor the detection of somatic mutations in 409 frequently

mutated tumor-suppressor and oncogenes, massive parallelsemiconductor sequencing with an Ion Torrent personalgenome machine (Ion Torrent; Life Technologies) wasapplied. DNA (40 ng) from six FFPE primary cardiac angio-sarcomas and from the corresponding normal tissue wastarget enriched with an initial multiplex PCR (IonTorrentAmpliSeq Comprehensive Cancer Panel) and quantifiedusing an Agilent Bioanalyzer (Agilent Technologies). Thetemplate preparation was performed with the Ion OneTouch200 Template Kit and the massive parallel semiconductorsequencing reaction was executed with the Ion PGM 200Sequencing Kit. Sequence reads were aligned to Human hg19and the analysis of the alignments was performed with theIon Torrent Variant Caller plugin. For identification of SNPs,somatic SNV, and indels, the alignments of tumor tissue andcorresponding normal tissue were compared and visualizedwith the Integrative Genomic Viewer (Broad Institute, Cam-bridge, MA).

PCR and Sanger sequencingPCRs were performed with a Fast PCR System (Life Tech-

nologies) at 95�C for 2 minutes and 40 cycles at 94�C for 10seconds and 63�C for 20 seconds. PCR products were purifiedwith ExoSAP-IT (Affymetrix) and sequenced with the BigDyeCycle Sequencing Kit and an ABI 3730 Genetic Analyzer (LifeTechnologies). Primer sequences are given in SupplementaryTable S2.

Site-directed mutagenesisThe PLCG1 expression vector (pCMV6-C-Myc-DDK) was

obtained from OriGene (RC216448). The GeneArt Site-Directed Mutagenesis Kit (Life Technologies) was used togenerate the mutant PLCg1 (PLCg1-R707Q). Primersequences for site-directed mutagenesis are given in Sup-plementary Table S2.

Transfection of HEK293 cells and HUVECsFor transfection of HEK293 cells, the FuGENE HD Trans-

fection Reagent (Promega), and for HUVECs, PromoFectin-HUVEC was used (PromoKine). HUVECs were seeded in 12-well plates (40,000–70,000 cells/well) and transfected with 3-mgplasmid using 6-mL PromoFectin reagent or seeded in 6-wellplates (100,000–150,000 cells/well) and transfected with 6-mgplasmid using 12-mL PromoFectin. Transfection efficiencieswere determined with a plasmid encoding EGFP (pcDNA3-EGFP, length 6 kb; Addgene) and flow cytometry after 24 hours,and were 72% transfected cells for HEK293 and 59% forHUVECs (Supplementary Fig. S1).

Inhibition of protein tyrosine phosphatasesTransfected cells were treated with 100 mmol/L pervanadate

for 1 hour. A 30-mmol/L pervanadate stock solution wasfreshly prepared by dilution of 200-mmol/L sodium orthova-nadate in PBS and addition of hydrogene peroxide to a finalconcentration of 0.18%. The mixture was incubated for 15

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minutes at room temperature and diluted in the cell culturemedium to the final concentration.

Western blot analysisWhole-cell lysates were prepared by lysis of 100,000 cells in

50-mL SDS sample buffer (Life Technologies) and analyzed aspreviously described (14). Details for the antibodies used aregiven in Supplementary Table S1.

IP-1 ELISAIP1 amounts inHEK293 cells weremeasuredwith the IP-One

ELISA Kit (Cisbio) at 450 nm.

Flow cytometryFor apoptosis, detection HUVECs were stained with

Annexin V–Alexa Fluor-488 and propidium iodide (PI; Life

Technologies) and analyzed by flow cytometry. For cell-cycleanalysis, HUVECs were fixed in 70% ethanol for 2 hours at�20�C, washed in PBS/1% BSA, followed by staining with PI/RNase Staining Buffer (Calbiochem, Merck Millipore), andanalyzed by flow cytometry.

Migration and invasion assayHUVECs were transfected with PLCg1-R707Q, PLCg1-WT,

or GFP. After 24 hours, 2,500, 3,000, or 5,000 cells wereseeded in a two-chamber invasion assay system in whichthe chambers are separated by a membrane with 8-mm poresalone or coated with a Matrigel layer (BD Biosciences). After24 hours, the migrated or invaded cells were fixed, stainedwith the Differential Quik Stain Kit (Polysciences Inc.) andcounted.

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Figure 1. Detection of a recurrent gain encompassing KDR and KIT by SNP array analysis. A, the recurrent gain of chromosome 4 was identified by SNP arrayanalysis using the Illumina HumanOmniExpress-FFPE BeadChips with an overlapping region of about 2Mb (highlighted as red box). LRR data and BAFwereexamined by visual inspection using the Illumina Genome Studio software. Genes within the region of gain were identified by the Illumina KaryostudioSoftware and the twoRTKs are highlighted in red. B andC, the expression of KDRwas analyzed by immunohistochemistry. Both caseswith the recurrent gainof chromosome 4 and strong expression are shown in B and one case with weak expression is shown in C.

Recurrent Activating PLCg1 Mutation in Cardiac Angiosarcoma

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 6175




ResultsDetection of a recurrent gain encompassing KDR andKIT by SNP array analysis

We performed SNP array analysis for genome-wide detec-tion of genomic gains and losses with DNA from eight cardiacangiosarcomas using the Illumina HumanOmniExpress-FFPEBeadChip examining �700,000 SNPs with a median markerspacing of 2.1 kb. Gains and losses were detected in all caseswith a range of two to 15 and an average of nine aberrations percase (Supplementary Table S3). Besides repeated 1q gains, arecurrent gain of a small region of chromosome 4 in two cases(overlapping region 2Mb) encompassing two receptor tyrosinekinase (RTK) genes, KIT and KDR (Fig. 1A), was identified. KDRand KIT are known oncogenes and we therefore performedimmunohistochemistry to determine whether the genomicgains were accompanied by an increased expression (Fig.1B). KDR was expressed in all 8 cases analyzed ranging fromweak (1 case) over intermediate (4 cases) to strong (3 cases)staining, and both cases with the gain showed strong expres-sion (Fig. 1B and C). KIT expression was investigated in 5 cases,including the two cases with the gain. However, only one of thetwo cases showed weak expression and KIT was not expressedin any of the other cases.

Identification of recurrent mutations in PLCG1, KDR,and MLL2 by tNGS

Because of the rarity of cardiac sarcomas, access to nativetumor samples and corresponding normal tissue is limited.Usually only small amounts of FFPE biopsies are available andtNGS with an initial multiplex PCR is then a suitable approachfor the detection of somatic SNVs and small indels (15). Wetherefore used the IonTorrent AmpliSeq Comprehensive Can-cer Panel for the detection of somatic mutations in 409frequently mutated oncogenes and tumor-suppressor genes(16,000 amplicons covering about 1.6 Mbp). Six cardiac angio-sarcomas for which corresponding normal tissue was availablewere analyzed with an average coverage of 298 reads per base(range of average reads per base, 210–359) and a >20� cov-erage of 92% of the bases analyzed. Altogether 148 somaticvariants (128 SNVs, 20 indels) were detected with at least 11mutations and an average of 25 mutations per case (range, 11–33 mutations; Supplementary Table S4). A total of 133 of themutations were silent, in untranslated regions or in introns.

All 15 replacement mutations were confirmed by Sangersequencing of DNA from tumor and corresponding normaltissue and affected 11 genes, PLCG1,ERBB4, FGFR1,NRAS,KDR,RAF1, MLL2, ASXL1, DCC, LRP1B, and POT1 (SupplementaryTable S5). Upon analysis of four additional cases, for which nocorresponding normal tissuewas available, by Sanger sequenc-ing for all nonsynonymous mutations identified by tNGS, onefurther PLCG1 mutation was detected.

Three genes were recurrently mutated, KDR, MLL2, andPLCG1. KDR, which is also frequently mutated in angiosar-comas of other locations (16), was mutated in two samples,in one case in an extracellular immunoglobulin domain(R720W), and in the other case in the transmembranedomain (T771K), where several mutations were alsodetected in other tumors [Catalogue of Somatic Mutationsin Cancer (COSMIC), release 68; Wellcome Trust SangerInstitute, Hinxton, United Kingdom]. MLL2 was mutated intwo samples and in one case two mutations were detected.Each mutation created a nonsense codon (Q1613�, R5454� ,and R4904� in the 5537-amino acid–long protein), and thusmost likely inactivated MLL2. In line with our results, recentstudies identified loss-of-function mutations in MLL2 dis-seminated over the whole coding region in several cancertypes, indicating that MLL2 is most likely a tumor suppres-sor (COSMIC; ref. 17).

A recurrent missense mutation in PLCG1 (c.2120G>A; p.R707Q) was detected in three angiosarcomas. PLCg1 possesstwo SH2 domains, the N-terminal (nSH2) for binding to otherproteins and the C-terminal (cSH2) involved in autoinhibition(18), and the mutated R707 is located within the evolutionaryhighly conserved cSH2 domain (Fig. 2). The samemutation hasrecently been detected in 9% of angiosarcomas of other loca-tions (13).

The R707Q mutation activates PLCg1To examine the consequences of the R707Q mutation in the

PLCg1-cSH2 domain, we used a vector in which PLCg1 expres-sion is driven by the cytomegalovirus (CMV) promotor (PLCg1-WT) and performed site-directed mutagenesis to generate themutated PLCg1 variant (PLCg1-R707Q). PLCg1-WT andPLCg1-R707Q were then transiently transfected into HUVECas angiosarcomas are derived from endothelial cells. Twenty-four hours after transfection, strong expression of both PLCg1forms was observed in Western blot analysis (Fig. 3A), with

Figure 2. Localization of the PLCg1 mutation and evolutionary conservation of C-terminal SH2 domain. Schematic description of the functional domains ofPLCg1, with an expanded view of the cSH2 domain. Sequence alignment of the cSH2 domain among various species shows that the entire cSH2 domain(R707 highlighted in red) is highly conserved.

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weaker expression of the PLCg1-R707Qmutant, whichwas alsoobserved in all further experiments although identical plasmidamounts from various preparations were used.PLCg1 is usually in an inactive state, in which the catalytic

center is blocked by an intramolecular interaction withthe autoinhibitory cSH2 domain. After ligand-inducedRTK activation and autophosphorylation, PLCg1 can bindvia its nSH2 domain to phosphorylated RTKs, whichthen phosphorylate PLCg1 at Y783. Because of subsequentintramolecular binding of phosphorylated (p)-Y783 to thePLCg1-cSH2 domain, autoinhibition is relieved and PLCg1activated (19). When PLCg1-WT- and PLCg1-R707Q–trans-fected HUVECs were cultured in standard ECGM with low

amounts of growth factors, a significantly decreased phos-phorylation of PLCg1-R707Q at Y783 compared with PLCg1-WT was observed (Fig. 3A). Incubation for 5 minutes inmedium with higher growth factor concentrations inducedstronger increases of p-Y783 in PLCg1-WT (60%) than inPLCg1-R707Q (17%)–transfected cells.

The strongly reduced Y783-phosphorylation in PLCg1-R707Q could be due to mutation-induced conformationalchanges preventing either Y783 phosphorylation by RTKs orintramolecular binding of p-Y783 to the cSH2 domain, whichwould protect p-Y783 from dephosphorylation by phospha-tases. To distinguish between both possibilities, we added theprotein tyrosine phosphatase inhibitor pervanadate toHUVECs 24 hours after transfection. Incubation with 100-mmol/L pervanadate for 1 hour caused significant increasesof p-Y783 in PLCg1-WT and PLCg1-R707Q (Fig. 3B). Together,these findings suggest that the R707Q mutation causes aconformational change that does not prevent phosphorylationof Y783 by RTKs but prohibits the interaction of p-Y783 withthe cSH2 domain.

As the intramolecular interaction of p-Y783 with theautoinhibitory cSH2 domain is necessary for PLCg1-WTactivation, we next analyzed whether PLCg1-R707Q is stillactive. Activated PLCg1 cleaves PIP2 to generate DAG andIP3 and DAG can activate the RAF–MEK–ERK pathway viaprotein kinase C and IP3 via Ca2þ release Ca2þ-dependentenzymes, such as the serine/threonine phosphatase calci-neurin, which dephosphorylates nuclear factor of activated Tcells (NFAT) in endothelial cells (20–22). Expression of thePLCg1-R707Q mutant in HUVECs and HEK293 cells causedstronger phosphorylation of c-RAF, MEK, and ERK1/2 thanPLCg1-WT (Fig. 4A). Furthermore, expression of PLCg1-R707Q resulted in increased amounts of IP1, which wasquantified as a surrogate for IP3 (Fig. 4B; ref. 23), anddecreased NFAT phosphorylation compared with wild-typePLCg1 (Fig. 4C).

Taken together, these observations indicate that thePLCg1-R707Q mutation causes conformational changes thatprevent intramolecular binding of p-Y783 to the autoinhi-bitory cSH2 domain with concomitant increased activationof DAG and IP3-dependent signaling compared with PCLg1-WT, indicating that the R707Q mutation causes constitutiveactivation of PLCg1 independent of RTKs. These experimen-tal findings are in line with results of structure modeling forthe PLCg1-R707Q mutant, which predict a loss of autoinhi-bition (13).

The PLCg1 R707Q mutation increases cell survivalActivation of the KDR–PLCg1 pathway has been reported to

increase proliferation of endothelial cells (20, 24). To analyzewhether the PLCg1-R707Q mutation influenced cellular pro-liferation, we expressed PLCg1-R707Q and PLCg1-WT inHUVECs and counted cell numbers after 24 and 48 hours.Small increases of the cell numbers in PLCg1-R707Q–trans-fected cells compared with PLCg1-WT were observed after 24(16%) and 48 hours (28%; Fig. 5A). As potential influences of theR707Q mutation on cell numbers could become more obviousinmediawith reduced supplements, transfected cells were also

Figure 3. The PLCg1-R707Q mutation causes decreased phoshorylationof PLCg1 at Y783. A, PLCg1-R707Q and PLCg1-WT were transientlyexpressed in HUVECs cultured for 24 hours in media supplemented withlow (ECGM) concentrations of growth factors and either left in ECGM orincubated for 5minutes with ECGM2 containing higher concentrations ofgrowth factors. As a control, untransfectedHUVECswere used. Amountsof p-Y783 were determined by Western blot analyses. Bottom, the foldchanges of p-PLCg1 amounts between PLCg1-R707Q and PLCg1-WTnormalized to PLCg1 expression. Mean values were calculated from twoexperiments. B, to investigate whether the reduced phosphorylation ofPLCg1 results from dephosphorylation of p-Y783 by phosphatases, weused the protein tyrosine phosphatase inhibitor pervanadate. HUVECswere transfected, and after 24 hours in ECGM, 100-mmol/L pervanadatewas added for 1 hour. Western blot analyses were used to determine theamounts of p-PLCg1.






cultured in media with less supplement (1:9 diluted). However,although the number of control cells were halved after 48 hourscompared with full media, only small increases in cell numbersof PLCg1-R707Q–transfected compared with PLCg1-WT–transfected cells were observed (9%; Fig. 5B).

To determine whether the increases in cell numberwere dueto increased proliferation rates, we analyzed the cell-cycle

distribution by quantification of the cellular DNA contentsusing PI. However, despite the increases in cell numbers, nodifferences in the fractions of cells in the G1, S, and G2 phaseswere observed between HUVECs transfected with PLCg1-R707Q and PLCg1-WT (Supplementary Fig. S2).

Previous studies also reported an antiapoptotic role ofPLCg1 (25–27), and the increased cell numbers could

Figure 4. The PLCg1 R707Qmutation activates PLCg1 signaling. We transiently expressed PLCg1-R707Q and PLCg1-WT in HEK293 andHUVECs to analyzethe effects of the R707Q mutation on intracellular signaling. A, Western blot analyses with HEK293 and HUVEC lysates were performed 24 hours aftertransfection to verify ectopic PLCg1 expression and to investigate the phosphorylation of PLCg1-R707Q at Y783, which causes activation ofWT PLCg1. Activated PLCg1 can activate the RAF–MEK–ERK pathway via DAG and PKC, and the R707Q mutation increased phosphorylation of cRAF,MEK, and ERK1/2 compared with PLCg1-WT. Right, the fold changes of p-PLCg1, p-cRAF, p-MEK, and p-ERK1/2 amount between PLCg1-R707Q-and PLCg1-WT–transfected HEK293 normalized to PLCg1 expression. For HEK293, experiments were repeated at least three times; for HUVECs two times.B, activated PLCg1 generates IP3 via PIP2 hydrolysis. To determine IP3 amounts in transiently transfected HEK293, the amounts of IP1, which is adownstream metabolite of IP3 and accumulates in cells upon PLCg1 activation, were measured by an IP1-ELISA. Mean values were calculated from threeexperiments. C, IP3 causes release of Ca2þ from intracellular stores and activation of Ca2þ-dependent enzymes such as calcineurin. We used Westernblot analysis to determine the amount of phosphorylation of the calcineurin substrateNFAT.Right, the fold changeof p-NFATnormalized toPLCg1 expressioncalculated from three experiments.

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therefore be due to an antiapoptotic effect of PLCg1-R707Q.To investigate whether the R707Q mutation affects cellsurvival, we quantified the amount of apoptotic cells inHUVECs transfected with PLCg1-R707Q and PLCg1-WT, andalso in transfected cells that were treated with 45-mmol/Lcisplatin for 24 hours to induce apoptosis. Annexin V/PIstaining revealed an increased cell survival of the PLCg1-R707Q–expressing cells without and with cisplatin com-pared with PLCg1-WT (increases of 6% and 11% in thefractions of living cells, respectively; Fig. 5C and D).When total cell numbers were determined after 24 hoursof cisplatin treatment, a 50% increase was observed incells expressing PLCg1-R707Q compared with PLCg1-WT(Fig. 5E).Together, these findings indicate that the R707Q mutation

increases apoptosis resistance but not the proliferation rate ofendothelial cells.

The PLCg1 R707Q mutation increases migration andinvasiveness of HUVECs

PLCg1 is involved in the regulation of the reorganizationof the cytoskeleton and migration and invasiveness of cells(28–30). For reorganization of the cytoskeleton, cofilin playsan important role (31, 32). In its dephosphorylated form,cofilin can bind to actin filaments causing severing withsubsequent reorganization. Active PLCg1 can influence theavailable amounts of cofilin by hydrolysis of PIP2, withwhich cofilin forms complexes, and the dephosphorylationof cofilin by calcineurin or PKC-dependent activation of thecofilin phosphatase SSH1 (32). We therefore examined thephosphorylation of cofilin in HUVECs transfected withPLCg1-R707Q and PLCg1-WT after 24 hours. Western blotanalyses revealed a decreased phosphorylation of cofilin incells that expressed PLCg1-R707Q compared with PLCg1-WT (Fig. 6A).

Figure 5. The PLCg1 R707Qmutation increases cell survival. Toanalyze the effect of the R707mutation on proliferation and cellsurvival, HUVECswere transfectedwith plasmids encoding PLCg1-R707Q or PLCg1-WT and asreference for cells without anyectopic PLCg1 expression alsowith a plasmid encoding GFP. A,thecell numbersweremeasured24and 48 hours after transfection andmean values calculated from threeexperiments. B, cells were alsoincubated in reduced ECGM (1:9dilution) after transfection and cellnumbers were measured after 48hours. The mean values werecalculated from three experiments.C and D, to analyze cell survival,cells were transfected and culturedfor 48 hours with or withoutcisplatin (45 mmol/L) after 24 hours,stained with Annexin V and PI, andanalyzed by flow cytometry.Numbers of live (Annexin V�/PI�),apoptotic (Annexin Vþ/PI�), andnecrotic (Annexin Vþ/PIþ) cellswithout cisplatin treatment areshown in C and with cisplatintreatment in D (mean values fromthree experiments). E, total cellnumbers were determined 24hours after cisplatin treatment(mean values from fourexperiments).






To analyze the effects of the R707Q mutation on migrationand invasiveness, we used transiently transfected HUVECs anda two-chamber invasion assay in which the chambers areseparated by a membrane with 8-mm pores alone or coatedwith a Matrigel layer. Compared with PLCg1-WT, PLCg1-R707Q transfected cells showed increased migration and inva-sion (Fig. 6B and C). However, the extent of the effects variedlargely between different experiments (Supplementary TableS6), and although increased migration was observed in allseven experiments and increased invasion in six of sevenexperiments comparing PLCg1-R707Q with PLCg1-WT, no

statistical significance was reached. This is likely due to thevery different behavior of HUVECs, which are primary cellsisolated from different donors, regarding migration and inva-sion in individual experiments, as already the numbers ofmigrating and invading cells transfected with the controlGFP-plasmid varied more than 4- and 15-fold, respectively,even when the same numbers of cells were used for the assay.

Taking the decreased cofilin phosphorylation, the increasedmigration in all seven and the increased invasiveness in six ofseven experiments comparing PLCg1-R707Q and PLCg1-WTtogether, we conclude that PLCg1-R707Q increases cytoskel-eton reorganization and migration and invasiveness ofHUVECs.

Increased expression of PLCg1 in primary cardiacangiosarcomas

As our functional analyses revealed that not only thePLCg1-R707Q had effects on cellular behavior but alsostrong expression of PLCg1-WT, we investigated PLCg1expression in the available cardiac angiosarcomas by immu-nohistochemistry (Fig. 7). Although the expression of PLCg1in endothelial cells of normal blood vessels was below thedetection limit (Fig. 7A), PLCg1 expression was observed inseven of eight angiosarcomas ranging from weak (3 cases)over intermediate (2 cases) to strong (2 cases). The threeangiosarcomas with the PLCg1-R707Q mutation showed noor weak staining (Fig. 7B) compared with PLCg1-WT cases(Fig. 7C).

DiscussionThe pathogenesis of primary cardiac angiosarcomas is

largely unexplored and we therefore analyzed a collection ofFFPE biopsies from10 patients for genomic aberrations by SNParray analysis and tNGS. The SNP array analysis revealed formost cases complex karyotypes and recurrent gains of theentire long arm of chromosome 1 in 3 cases and a small regionof chromosome 4 encompassing KDR andKIT in 2 cases. So far,only few cytogenetic studies of angiosarcomas of variouslocations have been published and gain of 1q was describedfor one further cardiac angiosarcoma (9, 10). KDR plays animportant role in the regulation of endothelial cell proliferationand migration and is activated by mutations in about 10% ofangiosarcomas of other locations (16, 33). As the cases withgenomic gains encompassing KDR also showed strong KDRexpression, genomic gains could besides nonsynonomousmutations be an additional way of aberrant KDR activationin angiosarcomas.

Using tNGS, nonsynonymous mutations were identified inall cases and affected 11 genes (Supplementary Table S3).Among these were the chromatin modifiers MLL2 andASXL1, and all mutations detected in both genes in threecases were nonsense mutations, and in one case even twononsense MLL2 mutations were detected. Loss-of-functionmutations in MLL2 and ASXL1 disseminated over the wholecoding region were also identified in recent studies of severalcancer types (17, 34), and the detection of inactivatingmutations in chromatin modifiers in 3 of 6 cases analyzed

Figure 6. The PLCg1 R707Q mutation increases migration andinvasiveness of HUVECs. PLCg1 is involved in the regulation of thereorganization of the cytoskeleton and migration and invasiveness ofcells. To analyze the effects of the R707Q mutation on actin remodeling,migration, and invasion, PLCg1-R707Q and PLCg1-WT were transientlyexpressed in HUVECs. In addition, as reference for cells without anyectopic PLCg1 expression,HUVECswere also transfectedwith a plasmidencoding GFP. A, cofilin plays an important role in actin filamentremodeling and is activated by dephosphorylation. Phosphorylation ofcofilin was analyzed 24 hours after transfection. Right, the fold change ofp-cofilin normalized to PLCg1 expression calculated from threeexperiments. B and C, to analyze the effects on migration andinvasiveness, we used a two-chamber invasion assay in which thechambers were separated by a membrane with 8-mm pores alone orcoated with a Matrigel layer. The HUVECs were transfected with PLCg1-R707Q,PLCg1-WT, andascontrolwithGFP.After 24hours, 2,500, 3,000,or 5,000 cells were seeded in the migration and invasion chambers. Thechambers were incubated for additional 24 hours. After incubation, themigrated or invaded cells were fixed, stained, and counted. Cell numbersfor each single experiment are given in Supplementary Table S6. Notethat the numbers of migrating and invading cells for PLCg1-R707Q arelikely underestimated, as we observed in all experiments where PLCg1expressionwascontrolled byWesternblot analysis a stronger expressionof PLCg1-WT, and the cell numbers were not corrected for amounts ofPLCg1-WT and PLCg1-R707Q expressed.

Kunze et al.





likely indicates that, as recently shown for several othermalignancies, epigenetic dysregulation plays an importantrole in angiosarcoma.Several replacement mutations were found in genes con-

tributing to tyrosine kinase signaling. Besides mutations inthe RTKs ERBB4 and FGFR1 and in RAF1, whose conse-quences are currently unknown, and an activating NRASmutation, KDR and PLCG1 were repeatedly affected bymutations. KDR is one of the endothelial-specific RTK, playsan important role in angiogenesis, and is frequently acti-vated by mutations in angiosarcomas of other locations(16, 35). Taking our SNP array and tNGS results together,KDR is also in cardiac angiosarcomas in a significant frac-tion of cases affected by genomic aberrations, although theconsequences of the KDR mutations and genomic gainsremain to be investigated.PLCg1 is an important second messenger of KDR signal-

ing in endothelial cells. It binds to autophosphorylated KDRvia its nSH2 domain, is phosphorylated at Y783 and p-Y783,then binds to the PLCg1 cSH2 domain, causing a confor-mational change, which terminates autoinhibition (18, 19).The recurrent PLCg1 mutation affects R707 in the evolu-tionary highly conserved autoinhibitory cSH2 domain. WhenPLCg1-WT and the PLCg1-R707Q mutant were ectopicallyexpressed in cell lines, phosphorylation at Y783, most likelydue to constitutive tyrosine kinase activity in the culturedcells, was significantly reduced in the PLCg1-R707Q mutantcompared with PLCg1-WT. However, an increase of p-Y783was observed in both, the PLCg1-WT and the PLCg1-R707Qmutant, when a tyrosine phosphatase inhibitor was applied,suggesting that conformational changes in PLCg1 caused byR707Q do not prevent phosphorylation by RTKs but ratherprevents intramolecular binding of p-Y783 to the cSH2domain. Interestingly, in a study in which the regulation ofPLCg1 was analyzed by generation of various PLCg1mutants, replacements of other residues in the cSH2 domainalso resulted in decreased phosphorylation of Y783 in theabsence of phosphatase inhibitors (19).Despite the significant decreases in the PLCg1-WT–acti-

vating Y783-phosphorylation in PLCg1-R707Q, the effects onactivation of intracellular signaling pathways were always

significantly stronger upon ectopic expression of PLCg1-R707Q compared with ectopically expressed PLCg1-WT. Asthe intramolecular interaction of the cSH2 with p-Y783 isessential for a conformational change and full activation ofPLCg1 (19), the R707Q mutation seems to cause a confor-mational change in PLCg1, which causes constitutive acti-vation independent of Y783-phosphorylation by RTKs, in linewith structure modeling for PLCg1-R707Q (13).

At the cellular level, PLCg1-R707Q increased apoptosisresistance of HUVECs compared with PLCg1-WT under nor-mal culture conditions and more pronounced when an apo-ptosis-inducing agent was applied. In line with previous stud-ies, ectopic expression of PLCg1-WT also increased migrationand invasiveness of HUVECs without additional RTK stimu-lation, and this effect, along with the cytoskeleton reorgani-zation inducing cofilin dephosphorylation, was significantlystronger upon ectopic expression of PLCg1-R707Q (28–30).However, in contrast to previous studies in which KDR-medi-ated PLCg1 stimulation increased proliferation (20, 24), weobserved no influence of ectopically expressed PLCg1-R707Qor PLCg1-WT without additional RTK stimulation on prolif-eration rates of the HUVEC preparations that we used. Takentogether, these findings show that the R707Q mutation causesRTK-independent, constitutive PLCg1 activation, and, togeth-er with the stronger PLCg1 expression compared with endo-thelial cells in nearly all angiosarcomas, indicate that PLCg1plays an important role in the pathogenesis of primary cardiacangiosarcomas.

Activating mutations in most second messengers of RTKssuch as K-, N- and HRAS, BRAF and PIK3CA have been knownfor years and are frequent in several tumor entities. Interest-ingly, despite the actual efforts for mutation identification intumors, a recurrent potentially PLCg1-activating mutationaffecting the catalytic domain (S345F) has only been detectedin cutaneous T-cell lymphoma (36), and the same mutationthat we describe has recently also been identified in 9% ofangiosarcomas of other locations (13). As the S345F mutationwas neither detected in our study of cardiac angiosarcomas norin the study of angiosarcomas of other locations, it seems thatdifferent PLCg1 activation mechanisms are selected in differ-ent tumor entities.

Figure 7. PLCg1 expression in primary cardiac angiosarcomas. The expression of PLCg1 was analyzed by immunohistochemistry. A, the endothelial cells ofnormal vessels (black arrows) showed no staining of PLCg1. B, weak expression of PLCg1 in a casewith the R707Qmutation and strong expression of PLCg1in a case without the R707Q mutation (C).






In summary, our analysis indicates that epigenetic dysre-gulation and activated KDR/PLCg1 signaling contributeto the pathogenesis of a significant fraction of cardiacangiosarcomas. Furthermore, we show that the R707Q muta-tion confers KDR-independent PLCg1 activation and maythus cause primary resistance against VEGF/KDR-directedtherapies.

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

Authors' ContributionsConception and design: T. Spieker, J.R. Sindermann, A. Br€auningerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Kunze, T. Spieker, K. Nau, J. Berger, A. Hoffmeier,S. Gattenl€ohnerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Kunze, T. Spieker, U. Gamerdinger, T. Dreyer,A. Br€auningerWriting, review, and/or revision of themanuscript:K. Kunze, T. Spieker, J.R.Sindermann, A. Br€auninger

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): T. Spieker, U. Gamerdinger,S. Gattenl€ohnerStudy supervision: T. SpiekerOther (Interpretation of immunochistochemistry): T. Dreyer

AcknowledgmentsThe authors thank Kirsten Bommersheim, Sebastian Sch€afer, Ann-Katrin

Jest€adt, and Cindy Arnold for excellent technical assistance.

Grant SupportThis work was supported by the Deutsche Forschungsgemeinschaft (SP 1249/

2-1 to T. Spieker and A. Br€auninger and HO 4295/2-1 to J.R. Sindermann andA. Hoffmeier) and the Westphalian Heart Foundation (T. Spieker).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 15, 2014; revised August 14, 2014; accepted August 21, 2014;published OnlineFirst September 24, 2014.

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2014;74:6173-6183. Published OnlineFirst September 24, 2014.Cancer Res Kristin Kunze, Tilmann Spieker, Ulrike Gamerdinger, et al. CellsIncreases Apoptosis Resistance and Invasiveness of Endothelial

Mutation in Cardiac AngiosarcomasPLCG1A Recurrent Activating

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