AmpliﬁcationofFRS2andActivationofFGFR/FRS2Signaling ......Pathway in High-Grade Liposarcoma Keqiang Zhang 1 , Kevin Chu 1 , Xiwei Wu 3 , Hanlin Gao 4 , Jinhui Wang 4 , Yate-Ching

Molecular and Cellular Pathobiology

Amplification of FRS2 andActivation of FGFR/FRS2 SignalingPathway in High-Grade Liposarcoma

Keqiang Zhang1, Kevin Chu1, Xiwei Wu3, Hanlin Gao4, Jinhui Wang4, Yate-Ching Yuan3, Sofia Loera2,Kimberley Ho1, Yafan Wang5, Warren Chow1, Frank Un1, Peiguo Chu2, and Yun Yen1,6

AbstractFibroblast growth factor (FGF) receptor (FGFR) substrate 2 (FRS2) is an adaptor protein that plays a critical role

in FGFR signaling. FRS2 is located on chromosome 12q13-15 that is frequently amplified in liposarcomas. Thesignificance of FRS2 and FGFR signaling in high-grade liposarcomas is unknown. Herein, we first comparativelyexamined the amplification and expression ofFRS2withCDK4 andMDM2 in dedifferentiated liposarcoma (DDLS)and undifferentiated high-grade pleomorphic sarcoma (UHGPS). Amplification and expression of the three geneswere identified in 90% to 100% (9–11 of 11) of DDLS, whereas that of FRS2,CDK4, andMDM2were observed in 55%(41 of 75), 48% (36 of 75), and 44% (33/75) of clinically diagnosed UHGPS, suggesting that these "UHGPS" mayrepresent DDLS despite lacking histologic evidence of lipoblasts. Immunohistochemical analysis of phosphor-ylated FRS2 protein indicated that the FGFR/FRS2 signaling axis was generally activated in about 75% of FRS2-positive high-grade liposarcomas. Moreover, we found that FRS2 and FGFRs proteins are highly expressed andfunctional in three high-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2, and SW872. Importantly, the FGFRselective inhibitor NVP-BGJ-398 significantly inhibited the growth of FU-DDLS-1 and LiSa-2 cells with aconcomitant suppression of FGFR signal transduction. Attenuation of FRS2 protein in FU-DDLS-1 and LiSa-2cell lines decreased the phosphorylated extracellular signal–regulated kinase 1/2 and AKT and repressed cellproliferation. These findings indicate that analysis of FRS2 in combination with CDK4 and MDM2 will moreaccurately characterize pathologic features of high-grade liposarcomas. Activated FGFR/FRS2 signalingmay playa functional role in the development of high-grade liposarcomas, therefore, serve as a potential therapeutic target.Cancer Res; 73(4); 1298–307. �2012 AACR.

IntroductionFibroblast growth factor (FGF) receptor (FGFR) substrate 2

(FRS2) is a member of the adaptor/scaffold protein family thatbinds to receptor tyrosine kinases (RTK) including FGFR, theneurotrophin receptor, RET, andALK, and is required for signaltransduction from FGFRs (1). The FGFR family of 4 RTKs,FGFR1/2/3/4, mediate numerous physiologic processesincluding cell migration, proliferation, survival, and differen-tiation (2). Deregulation of FGFR signaling is associated withmany developmental syndromes (3). Aberrant activation ofFGFR signaling has been associated with tumor formation,

angiogenesis, and metastasis in multiple cancer types includ-ing breast cancer, bladder cancer, multiple myeloma, hepato-cellular, and renal cell carcinoma. Clinical studies have alsorevealed that aberrant FGFR signaling is associated with pooroutcome of different human cancers (4–7). These observationsmake FGFRs signaling increasingly attractive as target fortherapeutic intervention in various human cancers (2).

Liposarcomas are a heterogeneous group of mesenchymaltumors, and on the basis of morphologic features and cyto-genetic aberrations, liposarcoma is classified into 3 biologictypes encompassing 5 subtypes: well-differentiated liposar-coma/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma, and undifferentiated high-grade pleo-morphic sarcoma (UHGPS; refs. 8–10). Surgery serves as themainstay of therapy for localized liposarcoma. However, forlocally advanced and disseminated disease, there are feweffective treatment options (11). Liposarcomas with similarmorphologic appearance can follow different clinical coursesand show divergent responses to systemic therapy (12, 13).DDLS and UHGPS share similar histologic features, but com-pared with DDLS, UHGPS displays more aggressive localbehavior and strong resistance to systemic ifosfamide-basedchemotherapy, resulting in a 5-year survival rate of about 50%(10). DDLS tends to have a lower metastatic rate (15%–20%)than UHGPS (30%–50%; ref. 12). However, the accurate dis-crimination of pleomorphic from DDLS based on morphology

Authors' Affiliations: Departments of 1Molecular Pharmacology and2Pathology; 3Division of Information Sciences of Department of MolecularMedicine; 4Solexa Core Lab; 5Translational Research Laboratory, Beck-manResearch Institute of theCity ofHopeNationalMedical Center, Duarte,California; and 6Taipei Medical University, Taipei, Taiwan

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

K. Zhang and K. Chu contributed equally to this work.

Corresponding Author: Yun Yen, Department of Molecular Pharmacolo-gy, Beckman Research Institute, City of Hope Comprehensive CancerCenter, 1500 E. Duarte Road, Duarte, CA 91010. Phone: 626-256-4673, ext65707; Fax: 626-301-8233; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-2086

�2012 American Association for Cancer Research.

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alone is often a challenge even for an experienced soft-tissuepathologist. Recent reports indicate that "UHGPS" has beenmisused for several better classifiable, poorly differentiatedsoft-tissue sarcomas (14). For example, a study reported that23% of 163 high-grade liposarcomas in the Netherlands CancerInstitute were reclassified on the basis of molecular character-istics of liposarcoma (15). Similarly, another study re-evaluated159 cases of previously diagnosed UHGPS and found that atotal of 97 of these cases could be reclassified as other sarcomasand 20 were proven to be nonmesenchymal neoplasms (16).During the past decade, with the insight afforded by immu-nohistochemistry as well as improved cytogenetic and molec-ular diagnostics, the term UHGPS has been restricted to agroup of soft-tissue malignancies without a specific line ofdifferentiation (17). Therefore, it is critical to better classify andtarget various high-grade liposarcomas based on their specificpathologic features.Amplification of MDM2 and CDK4 genes on chromosome

12q13-15 is noted to frequently occur inWDLS/DDLS andmayalso serve as genetic characteristics of DDLS from UHGPS. Forexample, Taylor and colleagues, using high-resolution genomicanalysis, proved that there was no CDK4 amplification inUHGPS (18). The chromosome 12q13-15 amplicon is discon-tinuous and spans several megabases of DNA and containsmultiple genes (19). The discontinuous amplification may leadto the patterns for overexpressed proteins that may providegrowth advantages to tumors quite different between patients.Among genes on chromosome 12q13-15, CDK4 gene, a cyclin-dependent kinase promoting G1-phase cell-cycle progression,and MDM2 gene, a transcriptional target of p53, which med-iates both ubiquitin-dependent proteasomal degradation ofp53 and ubiquitin-independent proteasomal degradation ofp21, causing unchecked cell-cycle progression and prolifera-tion, have beenwell-studied because of their critical function inoncogenesis of liposarcoma (20, 21). The FRS2 gene is locatedclose to CDK4 and MDM2 on chromosome 12q13-15. Mostrecently, a study showed that the FRS2 gene is also frequentlyamplified inWDLS, and the FRS2 protein was overexpressed inWDLS, but not in normal fat or preadipocytes, which thenraises the possibility that FRS2 could be a useful target inmalignant liposarcomas (22).In the present study, we first comparatively analyzed the

amplification and overexpression of FRS2 with that of CDK4and MDM2 in clinically diagnosed DDLS/UHGPS. We discov-ered a high frequency of amplification and overexpression ofFRS2 in these high-grade liposarcomas. Considering that FRS2acts as "a conning center" in FGFR signaling (1, 2), we furtherinvestigated the activation of FGFR signaling in these malig-nant liposarcomas. We found that FGFR signaling was acti-vated in 75% of high-grade liposarcomas expressing FRS2protein. The biologic significance of FRS2 and FGFR signalingin the development of these malignant liposarcomas has notpreviously been evaluated, to the best of our knowledge. Weexamined expression of FRS2 and FGFRs genes in three humanhigh-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2 andSW872 and found that FRS2 and FGFRs are ubiquitouslyexpressed and functional in these cell lines. Furthermore, weimproved that targeting FGFR-FRS2 signaling may inhibit the

in vitro growth of FU-DDLS-1 and LiSa-2 cells with a concom-itant suppression of FGFR signal transduction. Our findings forthe first time indicate that activated FGFR/FRS2 signalingmaycontribute to the progression of high-grade liposarcomas,therefore representing a potential therapeutic target thatdeserves further extensive study. Recent advances inmolecularcharacterization of liposarcomas have shown that aberrantamplification of MDM2 and CDK4 genes within chromosome12q13-15 distinguishes WDLS/DDLS from UHGPS (16–18).Accordingly, we also validated that a subset of clinicallydiagnosed "UHGPS" of current study may represent DDLS.Importantly, we observed that 7% of these "UHGPS" wereimmunopositive for FRS2 only. Our study also suggests thatanalysis of FRS2 in combination with CDK4 and MDM2 willeven more accurately classify high-grade liposarcomas andlead to better prognostication.

Materials and MethodsCellular proliferationandFRS2 siRNAknockdownassays

FU-DDLS-1 DDLS cell line was a kind gift from Dr. HiroshiIwasaki (Fukuoka University, School of Medicine, Fukuoka,Japan; ref. 23) and was maintained in Dulbeccos' ModifiedEagle's Medium (DMEM):F12 medium with 10% FBS. SW872DDLS cell line was purchased from the American Type CultureCollection and was cultured in DMEM with 10% FBS. LiSa-2pleomorphic liposarcoma cell linewas a kind gift fromDr. SilkeBruderlein (University of Ulm, Ulm, Germany; ref. 24) and wasmaintained in Iscove'smodifiedDulbecco'smedium/RPMI in a4:1 ratio supplemented with 10% FBS, 2 mmol/L L-glutamine,and 0.1 mg/mL gentamicin. All cells were newly DNA finger-printed to confirm identity as previously described (25).Human adipose tissue total RNA survey Lot #07505959A and#0903009 were purchased from Ambion Inc. Human FRS2gene–specific siRNA and scrambled siRNA (Life TechnologiesCorporation) were transfected into cells with LipofectaminesiRNA Maximum (Life Science, Inc.) according to the usermanual. FRS2 siRNA–mediated inhibition of FU-DDLS-1 andLiSa-2 cells proliferation was monitored by using a W200 real-time cell electronic sensing analyzer (RT-CES) 16Xworkstation(Acea Biosciences) (26). A total of 2,500 cells were seeded intoeach well of RT-CES device in multiple duplicate of 8 andtransfected with siRNAs. Twenty-four hours later, the indexand curve of cell proliferation were monitored and plottedevery half hour. At 72 hours after siRNA transfection, cells werealso collected for Western blot analysis.

FGFR-specific inhibitor NVP-BGJ-398 treatment andMTS assay

FGFR in FU-DDLS-1 and LiSa-2 cells was specifically inhib-ited using NVP-BGJ-398 (BGJ-398) purchased from NovartisOncology Inc (27). BGJ-398 concentration ranged from 0.125 to4 mmol/L, dimethyl sulfoxide (DMSO) was served as vehiclecontrols. MTS assays were done according to manufacturer'sinstructions (CellTiter 96 AQueous Assay reagent; Promega) toquantify the number of viable cells. Briefly, a total of 2,500 cellswere seeded in a 96-well plate in multiples of 10 wells treatedwith NVP-BGJ-398. At 72 hours after treatment, cells weresubjected to MTS assays. A function of the logarithm of

FRS2 and FGFR Signaling Pathway in High-Grade Liposarcoma

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inhibitor concentration was used to produce logistic fit ofpercentage cell viability. The IC50 value was determined as theconcentration of compound needed to reduce cell viability to50% of a DMSO control.

Case selectionThe diagnosis of DDLS and UHGPS was done by pathologist

P. Chu. The criterions for UHGPS were as follows: showingmarked cytologic and nuclear pleomorphism, admixedwith spindle cells, without histologic (lipogenic) or immuno-histochemical (IHC; epithelial, neuromelanocytic, lymphohe-matopoietic, and muscular) evidence of specific lineagedifferentiation (28). Seventy-five such cases arising in variousanatomic locations were selected from the surgical pathologyfiles at City of Hope National Medical Center from 1990 to 2010(Supplementary Table S1). Twenty-six of 75 UHGPS fromretroperitoneum and thigh were previously reported (28). Themean agewas 57 years with 1.2:1male predominance and all 75cases were resection specimens: 37 cases from lower extremity,from upper extremity, 11 from abdomen/retroperitoneum, 8metastases in lung, 8 from chest/trunk, 2 from head/neck(Supplementary Table S1). In addition, 11 cases of previouslydiagnosed DDLS were also enrolled in the study (Supplemen-tary Table S2). All tumor specimens were resected. All tissueswere routinely fixed in 10% neutral-buffered formalin andembedded in paraffin (FFPE). One paraffin tissue block withtumor was selected from each case. For these cases withquantitative real-time PCR (q-PCR) analysis and genome-wideexon capture sequencing, an adjacent normal fat tissue blockwas also selected. All samples were selected under City of HopeIRB-06206.

Illumina Genome Analyzer (Solexa) whole-exomesequencing, sequence alignment, various calling,and annotation

Three micrograms of genomic DNA was sheared and end-repaired. Illumina adaptor oligonucleotides were ligated to theends. Ligation products were purified and enriched with a 12-cycle PCR. The enriched PCR products were subjected to theexon capture procedure using the SureSelect Human All ExonKit. The captured products were then used for sequencing andcluster generation by synthesis using the Illumina GenomeAnalyzer IIx. Image Analysis and base calling, which wasconducted by using Illumina default pipeline. The sequenceswere aligned to the human genome reference sequence(NCBI36) using Bowtie 0.12.7 (29). All subsequent analysis wasdone using customized R scripts. The log2 coverage ratio ofeach exon was calculated for tumor versus normal tissue andmedian-centered. Log2 ratios were smoothed by DNAcopy 25using default values and copy number variation was detectedby DNAcopy's circular binary segmentation algorithm (30).

gDNA preparation and q-PCRThe gDNAs of liposarcoma and the paired adjacent nor-

mal fat tissue were isolated from FFPE tissue sections usingthe QIAamp FFPE DNA Mini Kit (Qiagen) according to theuser manual. To characterize the amplification of the chro-mosome 12q13-15 region in UHGPS, we selected FRS2 and

CDK4 and MDM2 genes of the region to measure their copynumber by q-PCR analysis. For each gene, 2 primer pairs for2 exons were designed. The primer sequences of 2 exons ofCDK4, MDM2, and FRS2 genes are listed in SupplementaryTable S3. q-PCR analysis was conducted in 32 randomlyselected UHGPS cases (Supplementary Table S4) and 15DDLS cases (Supplementary Table S5) using ABI Prism7900 HT Sequence Detection System (Applied Biosystems).b-Globin was used as reference gene. Relative gene quanti-fication method was used to calculate the fold change ofgene copy number in a liposarcoma tissue (based on averageof 2 exons of each gene) to gDNA extracted from the pairedadjacent fat tissue (26).

ImmunohistochemistryCommercially available monoclonal antibodies against

CDK4 (Clone DCS-31, 1:50, Invitrogen), MDM2 (Clone IF-2,1:75, Zymed Laboratories), and FRS2 (Clone H-91, 1:100, SantaCruz Biotechnology Inc.) and a FRS2-rabbit polyclonal (1:50,Proteintech) were used. Anti-total FGFR3 (D2G7E), phosphor-FGF receptor (Tyr653/654), and phosphor-FRS2 (Tyr436) anti-bodywere purchased fromCell Signaling. Anti-FRS2 antibodiesfor Western blotting were purchased from R&D Systems(Clone 462910) and GeneTex (N1N3/GTX103288). All UHGPSand DDLS cases were stained for CDK4, MDM2, and FRS2 byimmunohistochemistry. Only FRS2-postive cases were sub-jected to phospho-FRS2 staining. Sections were deparaffinizedand rehydrated in graded alcohol. For heat-induced epitoperetrieval (HIER), the sections were subjected to DIVA retrievalbuffer (pH 6.0) in a Pressure Cooker (Biocare Medical) at 98�Cfor 60 minutes. The sections were then brought to an auto-mated stainer (DAKO) following the vendor's protocol. EnVi-sion Plus and peroxidase detection methods were used andcounterstained in 50% Mayer's hematoxylin for 1 minute.Nuclear CDK4 immunoreactivity, nuclear and cytoplasmicMDM2 immunoreactivity, and cytoplasmic FRS2 immunore-activity were assessed. Those cases with more than 1% oftumor cells showing positive stainingwere assessed as positive.The staining was graded as þ (1%–5% tumor cells positive),þþ (5%–24% tumor cells positive), and þþþ (>25% tumorcells positive; ref. 11).

FISHTo further validate FRS2 gene amplification, dual color

FISH was conducted using the FISH-mapped confirmed BACprobe, RP11-1130G21 (12q15) for FRS2 labeled in SpectrumOrange (Abbott Laboratories, Inc.) and RP11-433j6 (12p13)for chromosome 12 centromere labeled in Spectrum Green(Abbott Laboratories, Inc.) were selected for FISH assays.After the unstained positron emission tomographic (PET)slides were hybridized with FISH probes, cells were counter-stained with 40,6-diamidino-2-phenylindole (DAPI; Sigma).An Applied Imaging System was used to record images ofrepresentative cells. Two hundred interphase cells werescored per sample. All sections were scorable. Images werecaptured with the BioView Imaging System (BioView). Twored and 2 green signals (2R2G) are considered normal in adiploid genome. Because of truncation and overlapping cells,

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at least 20% of cells must show an abnormal pattern to beconsidered abnormal.

StatisticsData were collected using an MS Excel spreadsheet. Data

were analyzed using the JMP Statistical Discovery Softwareversion 6.0 (SAS Institute). Group comparisons for continuousdata were done with the Student t test for independent meansor 2-way ANOVA. Statistical significance was set at P < 0.05.

ResultsFRS2 and FGFR expression and activation of the FGFR/FRS2 axis in DDLS/UHGPS cell linesWe first measured the copy numbers of CDK4, MDM2, and

FRS2 genes in 3 human high-grade liposarcoma cell lines: FU-DDLS-1 (DDLS), LiSa-2 (UHGPS), and SW872 (DDLS). Quan-titative PCR analysis showed that the ratios of copy numbers ofCDK4, MDM2, and FRS2 to b-globin were about 7 to 10 in FU-DDLS-1, whereas the ratios of these 3 genes were less than 2 inLiSa-2 and SW872, indicating that the amplification of these 3genes was only present in FU-DDLS-1 cells (Fig. 1A). FGFR/FRS2 signaling has not been studied previously in high-gradeliposarcoma cell lines. We then examined the mRNA expres-sion of both FRS2 and 4 FGFR genes, including isoforms ofFGFR1, FGFR2, and FGFR3 in these 3 cell lines in comparisonwith that of human normal adipose tissues by quantitativereverse transcription PCR (qRT-PCR) analysis. The qRT-PCR

analyses indicated that these cells expressed relatively highlevel of FGFR1 and various levels of FGFR2, FGFR3, and FGFR4mRNA (Fig. 1B). The levels of FRS2, FGFR1, FGFR3, and FGFR4mRNA were substantially higher in these cell lines than that ofnormal adipose tissues; especially in FU-DDLS-1 and LiSa-2cells. Western blot analysis showed the relatively high expres-sion of FGFR1 and FGFR3 proteins in these 3 liposarcoma celllines (Fig. 1C). Although the copy number and mRNA of FRS2gene were higher in FU-DDLS-1, the level of FRS2 protein wasslightly higher in FU-DDLS-1 cells than that in other 2 cells,which may indicate the presence of posttranscriptional regu-lation of FRS2 expression. Coincidently, a previous articlereported that for the distinction of liposarcomas, genomic pro-filing appears to be more useful than RNA expression analysis(19).We found that phosphorylated FRS2 proteinwas generallypresent in these 3 liposarcoma cell lines, and the levels ofphosphorylated FRS2 protein in FU-DDLS-1 and LiSa-2 cells inserum were relatively higher (Fig. 1C). Consistently, a mostrecent study showed that high level of phosphorylated FRS2protein was only present in liposarcoma but not in normal fator preadipocytes (22). To determine the cellular responses tobFGF2 stimulation, these 3 cell lines were serum starved forabout 72 hours and then stimulated with 10 ng/mL of basicFGF2 (bFGF2) for 30 minutes. As shown in Fig. 1D, bFGF2stimulation promptly increased phosphorylated total FGFR(p-FGFR), FRS2 (p-FRS2), extracellular signal–regulated kinase(ERK)1/2 (p-ERK1/2), and AKT (p-AKT) in all 3 high-grade

Figure 1. Expression and activationof FGFR/FRS2 in DDLS/UHGPS celllines. A, q-PCR analysis showedrelative copy numbers of CDK4,MDM2, and FRS2 in 3 high-gradeliposarcoma cell lines: FU-DDLS-1,LiSa-2, and SW872. Data for genecopy number are presented as ratioto b-globin. B, q-PCR analysis ofFRS2 and FGFR1–4 mRNAexpression in FU-DDLS-1, LiSa-2,and SW872 cells. Shown areexpression levels relative toglyceraldehyde-3-phosphatedehydrogenase (GAPDH) as theinternal loading control. Adiposetissue cells (Adipose) were used asnormal control. C, Western blotanalysis showed the expression oftotal and phosphorylated FRS2, aswell as FGFR1/3 proteins in FU-DDLS-1 (Fu), LiSa-2 (Li), and SW872(Sw) cells. D, bFGF2 stimulationactivated phosphorylation of totalFGFR, FRS2, AKT, and ERK1/2in the 3 cells. FU-DDLS-1 wasserum-starved for 48 hours; LiSa-2and SW872 cells were starved for 72hours before being stimulated with10 ng/mL bFGF2 for 30 minutes.






liposarcoma cell lines, especially in FU-DDLS-1 and LiSa-2,showing the presence of a functional FGFR/FRS2 signalingafter bFGF2 stimulation.

Effects of FGFR inhibitor BGJ-398 on FU-DDLS-1 andLiSa-2 cell proliferation

The FGFR pathway is an important stimulus to cancerdevelopment and progression in many epithelial tumors, andtargeted inhibition of FGFR signaling has shown promise inin vitro studies (2, 31). As FGFR signaling was highly activatedin FU-DDLS-1 and LiSa-2 in serum, we tested the effects of anFGFR inhibitor on the proliferation and activation of AKT andERK signaling in these 2 cell lines. As shown in Fig. 2A andB, theselective FGFR inhibitor BGJ-398 significantly decreased FU-DDLS-1 and LiSa-2 cell proliferation in a dose-dependentmanner. The IC50 of BGJ-398 was about 0.25 to 0.50 mmol/Lfor FU-DDLS-1 and LiSa-2 cells, respectively. After cells wereexposed to BGJ-398 for 1 to 8 hours, Western blot analysisshowed that p-FRS2, p-AKT, and p-ERK1/2 proteins rapidlydecreased in a dosage-dependent manner in FU-DDLS-1 (Fig.2C) and LiSa-2 cells (Fig. 2D). However, p-ERK1/2 and p-AKT inFU-DDLS-1 (Fig. 2C) and LiSa-2 cells (Fig. 2D) started torebound after 24 to 48 hours of exposure to BGJ-398, whichindicated that undefined feedback mechanisms might reacti-vate these pathways. In consistent with the low level of p-FRS2in SW872 cells in serum, the same concentration of BGJ-398barely inhibited the proliferation of SW872 cells, suggesting itsproliferation in serum may be less dependent on FGFR sig-naling (data not shown). Overall, above data indicated thatBGJ-398 may suppress cellular proliferation and survivalthrough blocking FGFR-mediated signaling.

Effect of knockdown of FRS2 on cell proliferation andresponse to BGJ-398 treatment

FRS2 is an adaptor protein absolutely necessary for transferof signal from activated FGFR. Knockdown of FRS2 blocksFGFR signaling and suppresses cancer cells proliferation (32,33). Therefore, we further asked whether knockdown of FRS2can also disrupt FGFR signaling and then attenuate cellularproliferation of these high-grade liposarcoma cells. Westernblot analysis showed that knockdown of FRS2 substantiallydecreased the p-ERK1/2 and p-AKT in both FU-DDLS-1 (Fig.3A) and LiSa-2 cells (Fig. 3B) in serum and dramaticallyreduced bFGF2-induced p-AKT and p-ERK1/2 in bothserum-starved FU-DDLS-1 (Fig. 3A) and LiSa-2 cells (Fig.3B), which suggested that knockdown of FRS2 may suppressthe bFGF2-stimulated, FGFR-mediated activation of p-AKTand p-ERK1/2. We further examined the impact of knockdownof FRS2 on cellular proliferation of both FU-DDLS-1and LiSa-2cells. Knockdown of FRS2 significantly decreased the prolifer-ation of FU-DDLS-1 (Fig. 3C) and LiSa-2 cells (Fig. 3D) mon-itored in a real-time growth assay. In all, these data stronglyindicate that knockdown of FRS2 may specifically block FGFRsignaling in these high-grade liposarcoma cell lines.

Amplification of FRS2, CDK4, and MDM2 genes in high-grade liposarcoma

We initiated a pilot study to explore genomic abnormalitiesof UHGPS by conducting whole-exome sequencing on 7 casesof UHGPS. Sequencing results unveiled 2 large and discontin-uous amplified regions of chromosome 12q13-15 and 12q22-24 in one "UHGPS" (Fig. 4A). q-PCR analysis showed that3 selected genes CDK4, MDM2, and FRS2 on chromosome

Figure 2. Effect of FGFR inhibitorBGJ-398 on the proliferation of FU-DDLS-1 and LiSa-2 cells. A and B,MTS cell viability assays of FU-DDLS-1 and LiSa-2 cells treatedwith 0.016, 0.063, 0.25, 1.0, or4.0 mmol/L BGJ-398 for 72 hours.C and D, Western blot analysesof FRS2, p-FRS2, AKT, p-AKT,ERK1/2, and p-ERK1/2 in celllysates of FU-DDLS-1 and LiSa-2cells treated with 0.25 and 0.5mmol/L BGJ-398 for 1, 4, 8, 24, and48 hours.

Zhang et al.





12q13-15 were amplified 10-fold compared with saliva DNA ofthe same patient (Fig. 4B), suggesting the "UHGPS" mayrepresent a DDLS. The chromosome 12q13-15 amplicon con-tains several important genes such as CDK4 andMDM2, whichmay play important roles in the development of liposarcoma,and has been well studied in WDLS/AL/DDLS (34, 35). Weselected 8 genes (DDIT3, TSPAN31, CDK4, HMGA2, DYRK2,MDM2, YEATS4, and FRS2) mapping to chromosome 12q13-15 to compare their amplification patterns in 15 cases of DDLSand 32 cases of UHGPS using q-PCR assays. The results ofq-PCR analysis indicated that the frequency for amplificationof DDIT3 and DYRK2 was much lower than others (Supple-mentary Table S3), indicating that the amplicon was discon-tinuous. Details for gene amplification in individual sample ofUHGPS and DDLS are listed in Supplementary Tables S4 andS5. The frequency of amplification ofCDK4,MDM2, and FRS2 inDDLS was 100% (15 of 15), 93% (14 of 15), and 93% (14 of 15),respectively, whereas the frequency of amplification of CDK4,MDM2, and FRS2 in UHGPS was 44% (14 of 32), 25% (8 of 32),and 34% (11 of 32), respectively, indicating that a subgroup ofclinically diagnosed "UHGPS" may represent DDLS despitelacking histologic evidence of lipoblasts (14, 16, 17). Figure4C and D show the copy number distribution of CDK4,MDM2,

and FRS2 genes in DDLS and clinically diagnosed UHGPSrespectively, indicating the presence of co-amplification ofFRS2 with CDK4 and MDM2 on chromosome 12q13-15.

Immunohistochemical analysis of expression FRS2,CDK4, and MDM2 proteins in high-grade liposarcoma

Furthermore, we measured the amplification and overex-pression of FRS2 in high-grade liposarcomas by FISH andimmunohistochemistry. The IHC staining results for each caseof clinically diagnosed high-grade liposarcomas are listed inSupplementary Tables S1 and S2. All 11 of 11 cases of DDLSwere positive for CDK4 (100%), 10 of 11 forMDM2 (91%), and 9of 11 for FRS2 (82%). Accordingly, these 44 of 75 of "UHGPS"with positive IHC staining for one of CDK4, MDM2, and FRS2proteins should be reclassified asDDLS (14, 16, 17). Of note, 5 of75 cases UHGPS (7%) were positive for FRS2 protein only.Therefore, we concluded that analysis of FRS2 in combinationwith CDK4 and MDM2 will more accurately characterizepathologic features of high-grade liposarcomas. Table 1 sum-marized semiquantitative analysis of immunostaining of these3 proteins in these high-grade liposarcomas. Hematoxylin andeosin (H&E) and IHC staining for 4 typical example cases arepresented in Fig. 5. Dual-color FISH analysis showed that FRS2

Figure 3. Effect of knockdown ofFRS2on the response of FU-DDLS-1and LiSa-2 cells to bFGF2-activatedFGFR signaling and cellularproliferation. A and B, knockdown ofFRS2 decreased p-FRS2, p-AKT,and p-ERK1/2 in serum or serum-starved and bFGF2-stimulated FU-DDLS-1 and LiSa-2 cells. Twenty-four hours after transfection of FRS2siRNA, FU-DDLS-1 and LiSa-2 werestarved for 48 hours, stimulated with10 ng/mL bFGF2 for 30 minutes, andthen lysates were immunoblottedwith indicated antibodies. Lysatesfrom cells in serumwere harvested at48 hours posttransfection of FRS2siRNA (Con-siR for control siRNA;FRS2-siR for FRS2 siRNA). C andD, plot of RT-CES showed thatknockdown of FRS2 significantlydecreased the proliferation of FU-DDLS-1 and LiSa-2 cells at 72 hoursposttransfection of FRS2 siRNA.Results are presented as mean� SDof 8 duplicates. ��, P < 0.01compared with control siRNA.






gene amplification was present in case 1 (originally diagnosedas DDLS) or case 2/3 (originally diagnosed as "UHGPS"),whereas immunostaining analysis revealed that these caseswere FRS2-positive. FISH of case 4 (originally diagnosed as"UHGPS") displayed a normal pattern of 2 red signals of FRS2gene with 2 green signals of centromere in the diploid genome,suggesting no FRS2 gene amplification was present in theliposarcoma tissue, which corresponded to the negative FRS2IHC staining.

Phosphorylated FRS2 protein in high-grade liposarcomaActivation of FGFR results in phosphorylation of FRS2

tyrosine residues and subsequent sustained levels of ERKactivation and also activates the phosphoinositide 3-kinase(PI3K)/AKT pathway (36). Phosphorylated FRS2 protein maytherefore serve as a marker for activated FGFR signaling. Tofurther assess whether FGFR/FRS2 signaling was activated in

these high-grade liposarcoma tissues, we investigated thephosphorylated FRS2 protein by immunohistochemistry. Wefound that 75% of 48 cases of high-grade liposarcoma withFRS2-positive IHC staining were immunopositive for phos-phorylated FRS2 protein, which strongly suggests that theFGFR signaling was activated and might function in theseliposarcoma tissues. The morphology of a high-grade liposar-coma was revealed by H&E staining (Fig. 6A), whereas IHCanalysis showed strong staining for FRS2 protein (Fig. 6B) andmoderately positive staining for phosphorylated FRS2 protein(Fig. 6C). Quantitative analysis showed that 37.5% of phos-phorylated FRS2-positive high-grade liposarcomas were grad-ed as þ (1%–5% tumor cells positive), whereas 35.4% of themwere graded asþþ (5%–24% tumor cells positive), and 2.1% ofthem were graded as þþþ (>25% tumor cells positive). TheIHC staining grade of p-FRS2 for each high-grade liposarcomais also listed in Supplementary Tables S1 and S2.

Figure 4. Amplification of FRS2,CDK4, and MDM2 genes in a caseof high-grade liposarcoma. A,whole-exome sequencing wasconducted onDNA sample from anFFPE liposarcoma tissue andsaliva to determine copy numbervariants. The copy number variantanalysis showed a large anddiscontinuous amplification onchromosome 12, locations ofFRS2,CDK4, andMDM2 genes areshown by arrows. B, q-PCRanalysis was conducted toquantitatively analyze the copynumber of CDK4, MDM2, andFRS2 genes within the 12q13-15amplicon; the result showed that all3 genes were about 10-fold of thatof germ line control (saliva). C andD, distribution and amplification ofCDK4, MDM2, and FRS2 genesdetermined by q-PCR analysis inclinically diagnosed DDLS (n ¼ 15)and UHGPS (n ¼ 32) tissues.

Table 1. Quantitative analysis of IHC staining of CDK4, MDM2, and FRS2 proteins in high-gradeliposarcomas

Percentagea CDK4 MDM2 FRS2

� 16.4% (9/55) 23.6% (13/55) 12.7% (7/55)þ 36.3% (20/55) 43.6% (24/55) 34.5% (19/55)þþ 34.5% (19/55) 30.9% (17/55) 47.3% (26/55)þþþ 12.7% (7/55) 1.8% (1/55) 5.5% (3/55)Sum of positive 83.6% (46/55) 76.4% (42/55) 87.3% (48/55)

aTotal of 55 cases of high-grade liposarcoma included 11 cases of DDLS and 44 cases of clinically diagnosed "UHGPS". Theimmunostaining was graded as þ, (1%–5% tumor cells positive), þþ (5%–24% tumor cells positive), and þþþ (>25% tumor cellspositive).

Zhang et al.





DiscussionFISH analysis or immunohistochemistry analysis of ampli-

fication and overexpression of MDM2 and CDK4 on chromo-some 12q13-15 have been proven useful adjuncts in diagnosingWDLS/DDLS (11, 35, 37) and may also represent geneticcharacteristics of DDLS from UHGPS. For example, Taylorand colleagues proved that there was no CDK4 gene amplifi-cation in UHGPS (18). Accordingly, in the present study, wealso found that 44 of 75 cases of "UHGPS" should be reclassifiedas DDLS. The FRS2 gene is located close to CDK4 andMDM2 onchromosome 12q13-15 but has not been well-studied in high-grade liposarcoma. The amplification of chromosome 12q13-15 is typically large and discontinuous (20), which may lead tothe profiles for overexpressed proteins that may provide

growth advantages to tumors quite different between patients.Consistently, we also observed that the patterns for amplifi-cation and overexpression of genes on chromosome 12q13-15are quite heterogeneous among high-grade liposarcoma tis-sues. Of note, 7% of UHGPS were immunopositive for FRS2only, suggestingmolecular and/or IHC analysis of FRS2 gene, incombination withMDM2 and CDK4 genes, may more preciselycharacterize and stratify high-grade liposarcomas.

The FGFR signaling pathways play a pivotal role in normalembryonic development. FRS2 gene knockout in mice causeslethality due to the interruption of the FGFR signaling pathway(36). FRS2 acts as "a conning center" in FGF signaling mainlybecause it induces sustained levels of activation of ERK(31, 38, 39). FGF ligands and their receptors are overexpressed

Figure 5. IHC staining of CDK4,MDM2, and FRS2 proteins inhigh-grade liposarcoma tissues.H&E staining (�200) shows themorphologic characters of 4 casesof high-grade liposarcomas. IHCstaining of CDK4, MDM2, and FRS2protein showed variable expressionpatterns in these high-gradeliposarcoma tissues. Dual-colorFISH assay validated amplificationof FRS2 gene in 3 cases, and IHCanalysis showed that FRS2stainwaspositive in these tissues.

Cases

Case-1

H&E staining CDK4 IHC MDM2 IHC FRS2 IHC FRS2 FISH

Case-2

Case-3

Case-4

A B C

Figure 6. IHC staining of phosphorylated FRS2 protein in high-grade liposarcoma. A, H&E staining shows the morphologic features of a high-gradeliposarcoma. B, IHC staining shows strongly positive FRS2 protein in the high-grade liposarcoma tissue. C, IHC staining shows moderately positivephosphorylated FRS2 protein in the high-grade liposarcoma tissue.






in a variety of cancers, including breast, stomach, prostate,pancreas, bladder, and colon (4–7). Aberrant activation ofFGFR signaling has been associated with tumor formation,angiogenesis, and metastasis of these human cancers. Arecent study has proven that amplification of FGFR1 wasthe strongest independent predictor of poor outcome in acohort of unselected breast carcinomas (4). Increasing stud-ies have uncovered solid evidences that activated FGFRs aredriving oncogenes in certain cancers and act in a cell-autonomous fashion to maintain the malignant propertiesof tumor cells. These observations make FGFRs increasinglyattractive as targets for therapeutic intervention in cancer(27, 31, 36). Many small compounds that inhibit RTKs andhumanized antibodies against RTKs have been developed,and a number of potent inhibitors of the FGF receptors arein early-phase clinical trials (2, 27, 40). Most recently, Wangand colleagues showed that the FRS2 gene was co-amplified,overexpressed, and activated in WDLS, but not in benignlipomas (22). To further investigate the biologic significanceof FGFR-FRS2 signaling in high-grade liposarcomas, weexamined phosphorylation of FRS2 protein: the functionalform of FRS2, in these tissues expressing FRS2 protein.Interestingly, we identified a very high frequency of phos-phorylation of FRS2 in these high-grade liposarcomas, indi-cating the FGFR signaling was generally activated.

Furthermore, we hypothesized that activated FGFR-FRS2signaling might have a positive contribution to the devel-opment of high-grade liposarcoma and serve as a potentialtherapeutic target. To test the hypothesis indirectly, we firstmeasured FRS2 and FGFR expression in human high-gradeliposarcoma cell lines, whereas we found that FRS2 andFGFR1/3 transcripts and proteins are expressed in all 2liposarcoma cell lines. The phosphorylated FRS2 in thesecells was significantly upregulated by bFGF2 stimulation.Activated FGFR signaling in FU-DDLS-1 and LiSa-2 cells wasindicated by the strong phosphorylated FRS2 signal in thepresence of serum, whereas phosphorylated FRS2 was verylow in the SW872 cell line. We next investigated the effects ofan FGFR inhibitor on the proliferation and activation of AKTand ERK signaling of these 3 cell lines. Consistent with thedifferences observed for phosphorylated FRS2 between thesecell lines, we found that an FGFR-selective inhibitor signif-icantly suppressed the proliferation of FU-DDLS-1 and LiSa-2 cells, which have highly activated FGFR signaling, but not

in SW872 cells, which do not. We also found that FGFRinhibitors suppressed the activation of ERK and PI3K sig-naling pathways. Similarly, studies on breast cancer cellsshowed that PD173074 selectively inhibits FGFR tyrosinekinase activity, mitogen-activated protein kinase (MAPK),and PI3K signaling pathways (41, 42). FRS2 itself is also apotentially attractive target for disruption of the mitogenicand tumorigenic effects of multiple FGFs. For example,studies in prostate cancer showed that knockdown of FRS2might block global FGFR signaling in vitro and in vivo (32,33). Our finding that knockdown of FRS2 in a UHGPS cellline inhibited cell growth and activation of ERK and AKT areconsistent with this. FGFR signaling can be constitutivelyactivated by upregulation of FGFs or by amplification ormutation of FGFRs (4–7). Currently, the underlying mechan-isms responsible for activation of the FGFR/FRS2 signalingaxis in these liposarcomas remain unknown, therefore, isdeserving of further intensive study. However, our findingsopen a door that leads to dissecting and targeting FGFR/FRS2 signaling in high-grade liposarcoma.

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

Authors' ContributionsConception and design: K. Zhang, Y.-C. Yuan, P. Chu, Y. YenDevelopment of methodology: K. Zhang, K. Chu, H. Gao, J. Wang, Y.-C. Yuan,S. Lorea, P. Chu, Y. YenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Zhang, K. Chu, H. Gao, K. Ho, W. Chow, P. Chu,Y. YenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Zhang, X. Wu, H. Gao, Y.-C. Yuan, K. Ho, P. Chu,Y. YenWriting, review, and/or revision of themanuscript:K. Zhang, K. Chu, X.Wu,H. Gao, W. Chow, F. Un, Y. YenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Wang, P. Chu, Y. YenStudy supervision: Y.-C. Yuan, P. Chu, Y. Yen

AcknowledgmentsThe authors thank Dr. Lynne Smith for critical reading of the article and

editing and Victoria Bedell at the City of Hope Cytogenetic Core Laboratory forassistance in FRS2 FISH assay.

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

Received June 1, 2012; revisedNovember 12, 2012; acceptedNovember 19, 2012;published OnlineFirst February 7, 2013.

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2013;73:1298-1307. Published OnlineFirst February 7, 2013.Cancer Res Keqiang Zhang, Kevin Chu, Xiwei Wu, et al. Pathway in High-Grade LiposarcomaAmplification of FRS2 and Activation of FGFR/FRS2 Signaling

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AmpliﬁcationofFRS2andActivationofFGFR/FRS2Signaling ......Pathway in High-Grade Liposarcoma Keqiang Zhang 1 , Kevin Chu 1 , Xiwei Wu 3 , Hanlin Gao 4 , Jinhui Wang 4 , Yate-Ching