Amplification of FRS2 and Activation of FGFR/FRS2 Signaling Pathway in High-Grade Liposarcoma

Published OnlineFirst February 7, 2013; DOI: 10.1158/0008-5472.CAN-12-2086

Cancer Molecular and Cellular Pathobiology Research

Ampliﬁcation of FRS2 and Activation of FGFR/FRS2 Signaling Pathway 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

Abstract Fibroblast 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. The significance of FRS2 and FGFR signaling in high-grade liposarcomas is unknown. Herein, we first comparatively examined the amplification and expression of FRS2 with CDK4 and MDM2 in dedifferentiated liposarcoma (DDLS) and undifferentiated high-grade pleomorphic sarcoma (UHGPS). Amplification and expression of the three genes were identified in 90% to 100% (9–11 of 11) of DDLS, whereas that of FRS2, CDK4, and MDM2 were observed in 55% (41 of 75), 48% (36 of 75), and 44% (33/75) of clinically diagnosed UHGPS, suggesting that these "UHGPS" may represent DDLS despite lacking histologic evidence of lipoblasts. Immunohistochemical analysis of phosphorylated 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 and functional in three high-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2, and SW872. Importantly, the FGFR selective inhibitor NVP-BGJ-398 significantly inhibited the growth of FU-DDLS-1 and LiSa-2 cells with a concomitant suppression of FGFR signal transduction. Attenuation of FRS2 protein in FU-DDLS-1 and LiSa-2 cell lines decreased the phosphorylated extracellular signal–regulated kinase 1/2 and AKT and repressed cell proliferation. These findings indicate that analysis of FRS2 in combination with CDK4 and MDM2 will more accurately characterize pathologic features of high-grade liposarcomas. Activated FGFR/FRS2 signaling may play a functional role in the development of high-grade liposarcomas, therefore, serve as a potential therapeutic target. Cancer Res; 73(4); 1–10. 2012 AACR.

Introduction angiogenesis, and metastasis in multiple cancer types includ- Fibroblast growth factor (FGF) receptor (FGFR) substrate 2 ing breast cancer, bladder cancer, multiple myeloma, hepato- (FRS2) is a member of the adaptor/scaffold protein family that cellular, and renal cell carcinoma. Clinical studies have also binds to receptor tyrosine kinases (RTK) including FGFR, the revealed that aberrant FGFR signaling is associated with poor – neurotrophin receptor, RET, and ALK, and is required for signal outcome of different human cancers (4 7). These observations transduction from FGFRs (1). The FGFR family of 4 RTKs, make FGFRs signaling increasingly attractive as target for FGFR1/2/3/4, mediate numerous physiologic processes therapeutic intervention in various human cancers (2). including cell migration, proliferation, survival, and differen- Liposarcomas are a heterogeneous group of mesenchymal tiation (2). Deregulation of FGFR signaling is associated with tumors, and on the basis of morphologic features and cyto- ﬁ many developmental syndromes (3). Aberrant activation of genetic aberrations, liposarcoma is classi ed into 3 biologic FGFR signaling has been associated with tumor formation, types encompassing 5 subtypes: well-differentiated liposarcoma/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/ round cell liposarcoma, and undifferentiated high-grade pleo- Authors' Afﬁliations: Departments of 1Molecular Pharmacology and morphic sarcoma (UHGPS; refs. 8–10). Surgery serves as the 2 3 Pathology; Division of Information Sciences of Department of Molecular mainstay of therapy for localized liposarcoma. However, for Medicine; 4Solexa Core Lab; 5Translational Research Laboratory, Beck- man Research Institute of the City of Hope National Medical Center, Duarte, locally advanced and disseminated disease, there are few California; and 6Taipei Medical University, Taipei, Taiwan effective treatment options (11). Liposarcomas with similar Note: Supplementary data for this article are available at Cancer Research morphologic appearance can follow different clinical courses Online (http://cancerres.aacrjournals.org/). and show divergent responses to systemic therapy (12, 13). K. Zhang and K. Chu contributed equally to this work. DDLS and UHGPS share similar histologic features, but compared with DDLS, UHGPS displays more aggressive local Corresponding Author: Yun Yen, Department of Molecular Pharmacolo- gy, Beckman Research Institute, City of Hope Comprehensive Cancer behavior and strong resistance to systemic ifosfamide-based Center, 1500 E. Duarte Road, Duarte, CA 91010. Phone: 626-256-4673, ext chemotherapy, resulting in a 5-year survival rate of about 50% 65707; Fax: 626-301-8233; E-mail: [email protected] (10). DDLS tends to have a lower metastatic rate (15%–20%) doi: 10.1158/0008-5472.CAN-12-2086 than UHGPS (30%–50%; ref. 12). However, the accurate dis- 2012 American Association for Cancer Research. crimination of pleomorphic from DDLS based on morphology

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alone is often a challenge even for an experienced soft-tissue in vitro growth of FU-DDLS-1 and LiSa-2 cells with a concom- pathologist. Recent reports indicate that "UHGPS" has been itant suppression of FGFR signal transduction. Our findings for misused for several better classifiable, poorly differentiated the first time indicate that activated FGFR/FRS2 signaling may soft-tissue sarcomas (14). For example, a study reported that contribute to the progression of high-grade liposarcomas, 23% of 163 high-grade liposarcomas in the Netherlands Cancer therefore representing a potential therapeutic target that Institute were reclassified on the basis of molecular character- deserves further extensive study. Recent advances in molecular istics of liposarcoma (15). Similarly, another study re-evaluated characterization of liposarcomas have shown that aberrant 159 cases of previously diagnosed UHGPS and found that a amplification of MDM2 and CDK4 genes within chromosome total of 97 of these cases could be reclassified as other sarcomas 12q13-15 distinguishes WDLS/DDLS from UHGPS (16–18). and 20 were proven to be nonmesenchymal neoplasms (16). Accordingly, we also validated that a subset of clinically During the past decade, with the insight afforded by immu- diagnosed "UHGPS" of current study may represent DDLS. nohistochemistry as well as improved cytogenetic and molec- Importantly, we observed that 7% of these "UHGPS" were ular diagnostics, the term UHGPS has been restricted to a immunopositive for FRS2 only. Our study also suggests that group of soft-tissue malignancies without a specific line of analysis of FRS2 in combination with CDK4 and MDM2 will differentiation (17). Therefore, it is critical to better classify and even more accurately classify high-grade liposarcomas and target various high-grade liposarcomas based on their specific lead to better prognostication. pathologic features. Amplification of MDM2 and CDK4 genes on chromosome Materials and Methods 12q13-15 is noted to frequently occur in WDLS/DDLS and may Cellular proliferation and FRS2 siRNA knockdown assays also serve as genetic characteristics of DDLS from UHGPS. For FU-DDLS-1 DDLS cell line was a kind gift from Dr. Hiroshi example, Taylor and colleagues, using high-resolution genomic Iwasaki (Fukuoka University, School of Medicine, Fukuoka, analysis, proved that there was no CDK4 amplification in Japan; ref. 23) and was maintained in Dulbeccos' Modified UHGPS (18). The chromosome 12q13-15 amplicon is discon- Eagle's Medium (DMEM):F12 medium with 10% FBS. SW872 tinuous and spans several megabases of DNA and contains DDLS cell line was purchased from the American Type Culture multiple genes (19). The discontinuous amplification may lead Collection and was cultured in DMEM with 10% FBS. LiSa-2 to the patterns for overexpressed proteins that may provide pleomorphic liposarcoma cell line was a kind gift from Dr. Silke growth advantages to tumors quite different between patients. Bruderlein (University of Ulm, Ulm, Germany; ref. 24) and was Among genes on chromosome 12q13-15, CDK4 gene, a cyclin- maintained in Iscove's modified Dulbecco's medium/RPMI in a dependent kinase promoting G1-phase cell-cycle progression, 4:1 ratio supplemented with 10% FBS, 2 mmol/L L-glutamine, and MDM2 gene, a transcriptional target of p53, which med- and 0.1 mg/mL gentamicin. All cells were newly DNA finger- iates both ubiquitin-dependent proteasomal degradation of printed to confirm identity as previously described (25). p53 and ubiquitin-independent proteasomal degradation of Human adipose tissue total RNA survey Lot #07505959A and p21, causing unchecked cell-cycle progression and prolifera- #0903009 were purchased from Ambion Inc. Human FRS2 tion, have been well-studied because of their critical function in gene–specific siRNA and scrambled siRNA (Life Technologies oncogenesis of liposarcoma (20, 21). The FRS2 gene is located Corporation) were transfected into cells with Lipofectamine close to CDK4 and MDM2 on chromosome 12q13-15. Most siRNA Maximum (Life Science, Inc.) according to the user recently, a study showed that the FRS2 gene is also frequently manual. FRS2 siRNA–mediated inhibition of FU-DDLS-1 and amplified in WDLS, and the FRS2 protein was overexpressed in LiSa-2 cells proliferation was monitored by using a W200 real- WDLS, but not in normal fat or preadipocytes, which then time cell electronic sensing analyzer (RT-CES) 16X workstation raises the possibility that FRS2 could be a useful target in (Acea Biosciences) (26). A total of 2,500 cells were seeded into malignant liposarcomas (22). each well of RT-CES device in multiple duplicate of 8 and In the present study, we first comparatively analyzed the transfected with siRNAs. Twenty-four hours later, the index amplification and overexpression of FRS2 with that of CDK4 and curve of cell proliferation were monitored and plotted and MDM2 in clinically diagnosed DDLS/UHGPS. We discov- every half hour. At 72 hours after siRNA transfection, cells were ered a high frequency of amplification and overexpression of also collected for Western blot analysis. FRS2 in these high-grade liposarcomas. Considering that FRS2 acts as "a conning center" in FGFR signaling (1, 2), we further FGFR-specific inhibitor NVP-BGJ-398 treatment and investigated the activation of FGFR signaling in these malig- MTS assay nant liposarcomas. We found that FGFR signaling was acti- FGFR in FU-DDLS-1 and LiSa-2 cells was specifically inhib- vated in 75% of high-grade liposarcomas expressing FRS2 ited using NVP-BGJ-398 (BGJ-398) purchased from Novartis protein. The biologic significance of FRS2 and FGFR signaling Oncology Inc (27). BGJ-398 concentration ranged from 0.125 to in the development of these malignant liposarcomas has not 4 mmol/L, dimethyl sulfoxide (DMSO) was served as vehicle previously been evaluated, to the best of our knowledge. We controls. MTS assays were done according to manufacturer's examined expression of FRS2 and FGFRs genes in three human instructions (CellTiter 96 AQueous Assay reagent; Promega) to high-grade liposarcoma cell lines: FU-DDLS-1, LiSa-2 and quantify the number of viable cells. Briefly, a total of 2,500 cells SW872 and found that FRS2 and FGFRs are ubiquitously were seeded in a 96-well plate in multiples of 10 wells treated expressed and functional in these cell lines. Furthermore, we with NVP-BGJ-398. At 72 hours after treatment, cells were improved that targeting FGFR-FRS2 signaling may inhibit the subjected to MTS assays. A function of the logarithm of

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inhibitor concentration was used to produce logistic fitof CDK4 and MDM2 genes of the region to measure their copy percentage cell viability. The IC50 value was determined as the number by q-PCR analysis. For each gene, 2 primer pairs for concentration of compound needed to reduce cell viability to 2 exons were designed. The primer sequences of 2 exons of 50% of a DMSO control. CDK4, MDM2,andFRS2 genes are listed in Supplementary Table S3. q-PCR analysis was conducted in 32 randomly Case selection selected UHGPS cases (Supplementary Table S4) and 15 The diagnosis of DDLS and UHGPS was done by pathologist DDLS cases (Supplementary Table S5) using ABI Prism P. Chu. The criterions for UHGPS were as follows: showing 7900 HT Sequence Detection System (Applied Biosystems). marked cytologic and nuclear pleomorphism, admixed b-Globin was used as reference gene. Relative gene quanti- with spindle cells, without histologic (lipogenic) or immuno- fication method was used to calculate the fold change of histochemical (IHC; epithelial, neuromelanocytic, lymphohe- gene copy number in a liposarcoma tissue (based on average matopoietic, and muscular) evidence of specific lineage of 2 exons of each gene) to gDNA extracted from the paired differentiation (28). Seventy-five such cases arising in various adjacent fat tissue (26). anatomic locations were selected from the surgical pathology files at City of Hope National Medical Center from 1990 to 2010 Immunohistochemistry (Supplementary Table S1). Twenty-six of 75 UHGPS from Commercially available monoclonal antibodies against retroperitoneum and thigh were previously reported (28). The CDK4 (Clone DCS-31, 1:50, Invitrogen), MDM2 (Clone IF-2, mean age was 57 years with 1.2:1 male predominance and all 75 1:75, Zymed Laboratories), and FRS2 (Clone H-91, 1:100, Santa cases were resection specimens: 37 cases from lower extremity, Cruz Biotechnology Inc.) and a FRS2-rabbit polyclonal (1:50, from upper extremity, 11 from abdomen/retroperitoneum, 8 Proteintech) were used. Anti-total FGFR3 (D2G7E), phosphor- metastases in lung, 8 from chest/trunk, 2 from head/neck FGF receptor (Tyr653/654), and phosphor-FRS2 (Tyr436) anti- (Supplementary Table S1). In addition, 11 cases of previously body were purchased from Cell Signaling. Anti-FRS2 antibodies diagnosed DDLS were also enrolled in the study (Supplemen- for Western blotting were purchased from R&D Systems tary Table S2). All tumor specimens were resected. All tissues (Clone 462910) and GeneTex (N1N3/GTX103288). All UHGPS were routinely fixed in 10% neutral-buffered formalin and and DDLS cases were stained for CDK4, MDM2, and FRS2 by embedded in paraffin (FFPE). One paraffin tissue block with immunohistochemistry. Only FRS2-postive cases were sub- tumor was selected from each case. For these cases with jected to phospho-FRS2 staining. Sections were deparaffinized quantitative real-time PCR (q-PCR) analysis and genome-wide and rehydrated in graded alcohol. For heat-induced epitope exon capture sequencing, an adjacent normal fat tissue block retrieval (HIER), the sections were subjected to DIVA retrieval was also selected. All samples were selected under City of Hope buffer (pH 6.0) in a Pressure Cooker (Biocare Medical) at 98C IRB-06206. for 60 minutes. The sections were then brought to an auto- mated stainer (DAKO) following the vendor's protocol. EnVi- Illumina Genome Analyzer (Solexa) whole-exome sion Plus and peroxidase detection methods were used and sequencing, sequence alignment, various calling, counterstained in 50% Mayer's hematoxylin for 1 minute. and annotation Nuclear CDK4 immunoreactivity, nuclear and cytoplasmic Three micrograms of genomic DNA was sheared and end- MDM2 immunoreactivity, and cytoplasmic FRS2 immunore- repaired. Illumina adaptor oligonucleotides were ligated to the activity were assessed. Those cases with more than 1% of ends. Ligation products were purified and enriched with a 12- tumor cells showing positive staining were assessed as positive. cycle PCR. The enriched PCR products were subjected to the The staining was graded as þ (1%–5% tumor cells positive), exon capture procedure using the SureSelect Human All Exon þþ (5%–24% tumor cells positive), and þþþ (>25% tumor Kit. The captured products were then used for sequencing and cells positive; ref. 11). cluster generation by synthesis using the Illumina Genome Analyzer IIx. Image Analysis and base calling, which was FISH conducted by using Illumina default pipeline. The sequences To further validate FRS2 gene amplification, dual color were aligned to the human genome reference sequence FISH was conducted using the FISH-mapped confirmed BAC (NCBI36) using Bowtie 0.12.7 (29). All subsequent analysis was probe, RP11-1130G21 (12q15) for FRS2 labeled in Spectrum done using customized R scripts. The log2 coverage ratio of Orange (Abbott Laboratories, Inc.) and RP11-433j6 (12p13) each exon was calculated for tumor versus normal tissue and for chromosome 12 centromere labeled in Spectrum Green median-centered. Log2 ratios were smoothed by DNAcopy 25 (Abbott Laboratories, Inc.) were selected for FISH assays. using default values and copy number variation was detected After the unstained positron emission tomographic (PET) by DNAcopy's circular binary segmentation algorithm (30). slides were hybridized with FISH probes, cells were counter- stainedwith40,6-diamidino-2-phenylindole (DAPI; Sigma). gDNA preparation and q-PCR AnAppliedImagingSystemwasusedtorecordimagesof The gDNAs of liposarcoma and the paired adjacent nor- representative cells. Two hundred interphase cells were mal fat tissue were isolated from FFPE tissue sections using scored per sample. All sections were scorable. Images were the QIAamp FFPE DNA Mini Kit (Qiagen) according to the captured with the BioView Imaging System (BioView). Two user manual. To characterize the amplification of the chro- red and 2 green signals (2R2G) are considered normal in a mosome 12q13-15 region in UHGPS, we selected FRS2 and diploid genome. Because of truncation and overlapping cells,

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at least 20% of cells must show an abnormal pattern to be analyses indicated that these cells expressed relatively high considered abnormal. level of FGFR1 and various levels of FGFR2, FGFR3, and FGFR4 mRNA (Fig. 1B). The levels of FRS2, FGFR1, FGFR3, and FGFR4 Statistics mRNA were substantially higher in these cell lines than that of Data were collected using an MS Excel spreadsheet. Data normal adipose tissues; especially in FU-DDLS-1 and LiSa-2 were analyzed using the JMP Statistical Discovery Software cells. Western blot analysis showed the relatively high expres- version 6.0 (SAS Institute). Group comparisons for continuous sion of FGFR1 and FGFR3 proteins in these 3 liposarcoma cell data were done with the Student t test for independent means lines (Fig. 1C). Although the copy number and mRNA of FRS2 or 2-way ANOVA. Statistical significance was set at P < 0.05. gene were higher in FU-DDLS-1, the level of FRS2 protein was slightly higher in FU-DDLS-1 cells than that in other 2 cells, Results which may indicate the presence of posttranscriptional regu- FRS2 and FGFR expression and activation of the FGFR/ lation of FRS2 expression. Coincidently, a previous article FRS2 axis in DDLS/UHGPS cell lines reported that for the distinction of liposarcomas, genomic pro- We first measured the copy numbers of CDK4, MDM2, and filing appears to be more useful than RNA expression analysis FRS2 genes in 3 human high-grade liposarcoma cell lines: FU- (19). We found that phosphorylated FRS2 protein was generally DDLS-1 (DDLS), LiSa-2 (UHGPS), and SW872 (DDLS). Quan- present in these 3 liposarcoma cell lines, and the levels of titative PCR analysis showed that the ratios of copy numbers of phosphorylated FRS2 protein in FU-DDLS-1 and LiSa-2 cells in CDK4, MDM2, and FRS2 to b-globin were about 7 to 10 in FU- serum were relatively higher (Fig. 1C). Consistently, a most DDLS-1, whereas the ratios of these 3 genes were less than 2 in recent study showed that high level of phosphorylated FRS2 LiSa-2 and SW872, indicating that the amplification of these 3 protein was only present in liposarcoma but not in normal fat genes was only present in FU-DDLS-1 cells (Fig. 1A). FGFR/ or preadipocytes (22). To determine the cellular responses to FRS2 signaling has not been studied previously in high-grade bFGF2 stimulation, these 3 cell lines were serum starved for liposarcoma cell lines. We then examined the mRNA expres- about 72 hours and then stimulated with 10 ng/mL of basic sion of both FRS2 and 4 FGFR genes, including isoforms of FGF2 (bFGF2) for 30 minutes. As shown in Fig. 1D, bFGF2 FGFR1, FGFR2, and FGFR3 in these 3 cell lines in comparison stimulation promptly increased phosphorylated total FGFR with that of human normal adipose tissues by quantitative (p-FGFR), FRS2 (p-FRS2), extracellular signal–regulated kinase reverse transcription PCR (qRT-PCR) analysis. The qRT-PCR (ERK)1/2 (p-ERK1/2), and AKT (p-AKT) in all 3 high-grade

Figure 1. Expression and activation of FGFR/FRS2 in DDLS/UHGPS cell lines. A, q-PCR analysis showed relative copy numbers of CDK4, MDM2, and FRS2 in 3 high- grade liposarcoma cell lines: FU- DDLS-1, LiSa-2, and SW872. Data for gene copy number are presented as ratio to b-globin. B, q- PCR analysis of FRS2 and FGFR1– 4 mRNA expression in FU-DDLS-1, LiSa-2, and SW872 cells. Shown are expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal loading control. Adipose tissue cells (Adipose) were used as normal control. C, Western blot analysis showed the expression of total and phosphorylated FRS2, as well as FGFR1/3 proteins in FU- DDLS-1 (Fu), LiSa-2 (Li), and SW872 (Sw) cells. D, bFGF2 stimulation activated phosphorylation of total FGFR, FRS2, AKT, and ERK1/2 in the 3 cells. FU-DDLS-1 was serum-starved for 48 hours; LiSa-2 and SW872 cells were starved for 72 hours before being stimulated with 10 ng/mL bFGF2 for 30 minutes.

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liposarcoma cell lines, especially in FU-DDLS-1 and LiSa-2, Effect of knockdown of FRS2 on cell proliferation and showing the presence of a functional FGFR/FRS2 signaling response to BGJ-398 treatment after bFGF2 stimulation. FRS2 is an adaptor protein absolutely necessary for transfer of signal from activated FGFR. Knockdown of FRS2 blocks Effects of FGFR inhibitor BGJ-398 on FU-DDLS-1 and FGFR signaling and suppresses cancer cells proliferation (32, LiSa-2 cell proliferation 33). Therefore, we further asked whether knockdown of FRS2 The FGFR pathway is an important stimulus to cancer can also disrupt FGFR signaling and then attenuate cellular development and progression in many epithelial tumors, and proliferation of these high-grade liposarcoma cells. Western targeted inhibition of FGFR signaling has shown promise in blot analysis showed that knockdown of FRS2 substantially in vitro studies (2, 31). As FGFR signaling was highly activated decreased the p-ERK1/2 and p-AKT in both FU-DDLS-1 (Fig. in FU-DDLS-1 and LiSa-2 in serum, we tested the effects of an 3A) and LiSa-2 cells (Fig. 3B) in serum and dramatically FGFR inhibitor on the proliferation and activation of AKT and reduced bFGF2-induced p-AKT and p-ERK1/2 in both ERK signaling in these 2 cell lines. As shown in Fig. 2A and B, the serum-starved FU-DDLS-1 (Fig. 3A) and LiSa-2 cells (Fig. selective FGFR inhibitor BGJ-398 significantly decreased FU- 3B), which suggested that knockdown of FRS2 may suppress DDLS-1 and LiSa-2 cell proliferation in a dose-dependent the bFGF2-stimulated, FGFR-mediated activation of p-AKT manner. The IC50 of BGJ-398 was about 0.25 to 0.50 mmol/L and p-ERK1/2. We further examined the impact of knockdown for FU-DDLS-1 and LiSa-2 cells, respectively. After cells were of FRS2 on cellular proliferation of both FU-DDLS-1and LiSa-2 exposed to BGJ-398 for 1 to 8 hours, Western blot analysis cells. Knockdown of FRS2 significantly decreased the prolifer- showed that p-FRS2, p-AKT, and p-ERK1/2 proteins rapidly ation of FU-DDLS-1 (Fig. 3C) and LiSa-2 cells (Fig. 3D) mon- decreased in a dosage-dependent manner in FU-DDLS-1 (Fig. itored in a real-time growth assay. In all, these data strongly 2C) and LiSa-2 cells (Fig. 2D). However, p-ERK1/2 and p-AKT in indicate that knockdown of FRS2 may specifically block FGFR FU-DDLS-1 (Fig. 2C) and LiSa-2 cells (Fig. 2D) started to signaling in these high-grade liposarcoma cell lines. rebound after 24 to 48 hours of exposure to BGJ-398, which indicated that undefined feedback mechanisms might reacti- Amplification of FRS2, CDK4, and MDM2 genes in high- vate these pathways. In consistent with the low level of p-FRS2 grade liposarcoma in SW872 cells in serum, the same concentration of BGJ-398 We initiated a pilot study to explore genomic abnormalities barely inhibited the proliferation of SW872 cells, suggesting its of UHGPS by conducting whole-exome sequencing on 7 cases proliferation in serum may be less dependent on FGFR sig- of UHGPS. Sequencing results unveiled 2 large and discontin- naling (data not shown). Overall, above data indicated that uous amplified regions of chromosome 12q13-15 and 12q22- BGJ-398 may suppress cellular proliferation and survival 24 in one "UHGPS" (Fig. 4A). q-PCR analysis showed that through blocking FGFR-mediated signaling. 3 selected genes CDK4, MDM2, and FRS2 on chromosome

Figure 2. Effect of FGFR inhibitor BGJ-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 treated with 0.016, 0.063, 0.25, 1.0, or 4.0 mmol/L BGJ-398 for 72 hours. C and D, Western blot analyses of FRS2, p-FRS2, AKT, p-AKT, ERK1/2, and p-ERK1/2 in cell lysates of FU-DDLS-1 and LiSa-2 cells treated with 0.25 and 0.5 mmol/L BGJ-398 for 1, 4, 8, 24, and 48 hours.

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Figure 3. Effect of knockdown of FRS2 on the response of FU- DDLS-1 and LiSa-2 cells to bFGF2- activated FGFR signaling and cellular proliferation. A and B, knockdown of FRS2 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 FRS2 siRNA, FU-DDLS-1 and LiSa-2 were starved for 48 hours, stimulated with 10 ng/mL bFGF2 for 30 minutes, and then lysates were immunoblotted with indicated antibodies. Lysates from cells in serum were harvested at 48 hours posttransfection of FRS2 siRNA (Con-siR for control siRNA; FRS2-siR for FRS2 siRNA). C and D, plot of RT-CES showed that knockdown of FRS2 signiﬁcantly decreased the proliferation of FU- DDLS-1 and LiSa-2 cells at 72 hours posttransfection of FRS2 siRNA. Results are presented as mean SD of 8 duplicates. , P < 0.01 compared with control siRNA.

12q13-15 were amplified 10-fold compared with saliva DNA of and FRS2 genes in DDLS and clinically diagnosed UHGPS the same patient (Fig. 4B), suggesting the "UHGPS" may respectively, indicating the presence of co-amplification of represent a DDLS. The chromosome 12q13-15 amplicon con- FRS2 with CDK4 and MDM2 on chromosome 12q13-15. tains several important genes such as CDK4 and MDM2, which may play important roles in the development of liposarcoma, Immunohistochemical analysis of expression FRS2, and has been well studied in WDLS/AL/DDLS (34, 35). We CDK4, and MDM2 proteins in high-grade liposarcoma selected 8 genes (DDIT3, TSPAN31, CDK4, HMGA2, DYRK2, Furthermore, we measured the amplification and overex- MDM2, YEATS4, and FRS2) mapping to chromosome 12q13- pression of FRS2 in high-grade liposarcomas by FISH and 15 to compare their amplification patterns in 15 cases of DDLS immunohistochemistry. The IHC staining results for each case and 32 cases of UHGPS using q-PCR assays. The results of of clinically diagnosed high-grade liposarcomas are listed in q-PCR analysis indicated that the frequency for amplification Supplementary Tables S1 and S2. All 11 of 11 cases of DDLS of DDIT3 and DYRK2 was much lower than others (Supple- were positive for CDK4 (100%), 10 of 11 for MDM2 (91%), and 9 mentary Table S3), indicating that the amplicon was discon- of 11 for FRS2 (82%). Accordingly, these 44 of 75 of "UHGPS" tinuous. Details for gene amplification in individual sample of with positive IHC staining for one of CDK4, MDM2, and FRS2 UHGPS and DDLS are listed in Supplementary Tables S4 and proteins should be reclassified as DDLS (14, 16, 17). Of note, 5 of S5. The frequency of amplification of CDK4, MDM2, and FRS2 in 75 cases UHGPS (7%) were positive for FRS2 protein only. DDLS was 100% (15 of 15), 93% (14 of 15), and 93% (14 of 15), Therefore, we concluded that analysis of FRS2 in combination respectively, whereas the frequency of amplification of CDK4, with CDK4 and MDM2 will more accurately characterize MDM2, and FRS2 in UHGPS was 44% (14 of 32), 25% (8 of 32), pathologic features of high-grade liposarcomas. Table 1 sum- and 34% (11 of 32), respectively, indicating that a subgroup of marized semiquantitative analysis of immunostaining of these clinically diagnosed "UHGPS" may represent DDLS despite 3 proteins in these high-grade liposarcomas. Hematoxylin and lacking histologic evidence of lipoblasts (14, 16, 17). Figure eosin (H&E) and IHC staining for 4 typical example cases are 4C and D show the copy number distribution of CDK4, MDM2, presented in Fig. 5. Dual-color FISH analysis showed that FRS2

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FRS2 and FGFR Signaling Pathway in High-Grade Liposarcoma

Figure 4. Amplification of FRS2, CDK4, and MDM2 genes in a case of high-grade liposarcoma. A, whole- exome sequencing was conducted on DNA sample from an FFPE liposarcoma tissue and saliva to determine copy number variants. The copy number variant analysis showed a large and discontinuous amplification on chromosome 12, locations of FRS2, CDK4, and MDM2 genes are shown by arrows. B, q- PCR analysis was conducted to quantitatively analyze the copy number of CDK4, MDM2, and FRS2 genes within the 12q13-15 amplicon; the result showed that all 3 genes were about 10-fold of that of germ line control (saliva). C and D, distribution and amplification of CDK4, MDM2, and FRS2 genes determined by q-PCR analysis in clinically diagnosed DDLS (n ¼ 15) and UHGPS (n ¼ 32) tissues.

gene ampliﬁcation was present in case 1 (originally diagnosed these high-grade liposarcoma tissues, we investigated the as DDLS) or case 2/3 (originally diagnosed as "UHGPS"), phosphorylated FRS2 protein by immunohistochemistry. We whereas immunostaining analysis revealed that these cases found that 75% of 48 cases of high-grade liposarcoma with were FRS2-positive. FISH of case 4 (originally diagnosed as FRS2-positive IHC staining were immunopositive for phos- "UHGPS") displayed a normal pattern of 2 red signals of FRS2 phorylated FRS2 protein, which strongly suggests that the gene with 2 green signals of centromere in the diploid genome, FGFR signaling was activated and might function in these suggesting no FRS2 gene ampliﬁcation was present in the liposarcoma tissues. The morphology of a high-grade liposar- liposarcoma tissue, which corresponded to the negative FRS2 coma was revealed by H&E staining (Fig. 6A), whereas IHC IHC staining. analysis showed strong staining for FRS2 protein (Fig. 6B) and moderately positive staining for phosphorylated FRS2 protein Phosphorylated FRS2 protein in high-grade liposarcoma (Fig. 6C). Quantitative analysis showed that 37.5% of phos- Activation of FGFR results in phosphorylation of FRS2 phorylated FRS2-positive high-grade liposarcomas were grad- tyrosine residues and subsequent sustained levels of ERK ed as þ (1%–5% tumor cells positive), whereas 35.4% of them activation and also activates the phosphoinositide 3-kinase were graded as þþ (5%–24% tumor cells positive), and 2.1% of (PI3K)/AKT pathway (36). Phosphorylated FRS2 protein may them were graded as þþþ (>25% tumor cells positive). The therefore serve as a marker for activated FGFR signaling. To IHC staining grade of p-FRS2 for each high-grade liposarcoma further assess whether FGFR/FRS2 signaling was activated in is also listed in Supplementary Tables S1 and S2.

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

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". The immunostaining was graded as þ, (1%–5% tumor cells positive), þþ (5%–24% tumor cells positive), and þþþ (>25% tumor cells positive).

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

Case-1

Figure 5. IHC staining of CDK4, MDM2, and FRS2 proteins in high-grade liposarcoma tissues. H&E staining (200) shows the morphologic characters of 4 cases Case-2 of high-grade liposarcomas. IHC staining of CDK4, MDM2, and FRS2 protein showed variable expression patterns in these high- grade liposarcoma tissues. Dual- color FISH assay validated Case-3 ampliﬁcation of FRS2 gene in 3 cases, and IHC analysis showed that FRS2 stain was positive in these tissues.

Case-4

Discussion growth advantages to tumors quite different between patients. FISH analysis or immunohistochemistry analysis of ampli- Consistently, we also observed that the patterns for amplifi- fication and overexpression of MDM2 and CDK4 on chromo- cation and overexpression of genes on chromosome 12q13-15 some 12q13-15 have been proven useful adjuncts in diagnosing are quite heterogeneous among high-grade liposarcoma tis- WDLS/DDLS (11, 35, 37) and may also represent genetic sues. Of note, 7% of UHGPS were immunopositive for FRS2 characteristics of DDLS from UHGPS. For example, Taylor only, suggesting molecular and/or IHC analysis of FRS2 gene, in and colleagues proved that there was no CDK4 gene amplifi- combination with MDM2 and CDK4 genes, may more precisely cation in UHGPS (18). Accordingly, in the present study, we characterize and stratify high-grade liposarcomas. also found that 44 of 75 cases of "UHGPS" should be reclassified The FGFR signaling pathways play a pivotal role in normal as DDLS. The FRS2 gene is located close to CDK4 and MDM2 on embryonic development. FRS2 gene knockout in mice causes chromosome 12q13-15 but has not been well-studied in high- lethality due to the interruption of the FGFR signaling pathway grade liposarcoma. The amplification of chromosome 12q13- (36). FRS2 acts as "a conning center" in FGF signaling mainly 15 is typically large and discontinuous (20), which may lead to because it induces sustained levels of activation of ERK the profiles for overexpressed proteins that may provide (31, 38, 39). FGF ligands and their receptors are overexpressed

AB C

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

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FRS2 and FGFR Signaling Pathway in High-Grade Liposarcoma

in a variety of cancers, including breast, stomach, prostate, in SW872 cells, which do not. We also found that FGFR pancreas, bladder, and colon (4–7). Aberrant activation of inhibitors suppressed the activation of ERK and PI3K sig- FGFR signaling has been associated with tumor formation, naling pathways. Similarly, studies on breast cancer cells angiogenesis, and metastasis of these human cancers. A showed that PD173074 selectively inhibits FGFR tyrosine recent study has proven that amplification of FGFR1 was kinase activity, mitogen-activated protein kinase (MAPK), the strongest independent predictor of poor outcome in a and PI3K signaling pathways (41, 42). FRS2 itself is also a cohort of unselected breast carcinomas (4). Increasing stud- potentially attractive target for disruption of the mitogenic ies have uncovered solid evidences that activated FGFRs are and tumorigenic effects of multiple FGFs. For example, driving oncogenes in certain cancers and act in a cell- studies in prostate cancer showed that knockdown of FRS2 autonomous fashion to maintain the malignant properties might block global FGFR signaling in vitro and in vivo (32, of tumor cells. These observations make FGFRs increasingly 33). Our finding that knockdown of FRS2 in a UHGPS cell attractive as targets for therapeutic intervention in cancer line inhibited cell growth and activation of ERK and AKT are (27, 31, 36). Many small compounds that inhibit RTKs and consistent with this. FGFR signaling can be constitutively humanized antibodies against RTKs have been developed, activated by upregulation of FGFs or by amplification or and a number of potent inhibitors of the FGF receptors are mutation of FGFRs (4–7). Currently, the underlying mechan- in early-phase clinical trials (2, 27, 40). Most recently, Wang isms responsible for activation of the FGFR/FRS2 signaling and colleagues showed that the FRS2 gene was co-amplified, axis in these liposarcomas remain unknown, therefore, is overexpressed, and activated in WDLS, but not in benign deserving of further intensive study. However, our findings lipomas (22). To further investigate the biologic significance open a door that leads to dissecting and targeting FGFR/ of FGFR-FRS2 signaling in high-grade liposarcomas, we FRS2 signaling in high-grade liposarcoma. examined phosphorylation of FRS2 protein: the functional form of FRS2, in these tissues expressing FRS2 protein. Disclosure of Potential Conflicts of Interest Interestingly, we identified a very high frequency of phos- No potential conflicts of interest were disclosed. phorylation of FRS2 in these high-grade liposarcomas, indicating the FGFR signaling was generally activated. Authors' Contributions Conception and design: K. Zhang, Y.-C. Yuan, P. Chu, Y. Yen Furthermore, we hypothesized that activated FGFR-FRS2 Development of methodology: K. Zhang, K. Chu, H. Gao, J. Wang, Y.-C. Yuan, signaling might have a positive contribution to the devel- S. Lorea, P. Chu, Y. Yen opment of high-grade liposarcoma and serve as a potential Acquisition of data (provided animals, acquired and managed patients, fi provided facilities, etc.): K. Zhang, K. Chu, H. Gao, K. Ho, W. Chow, P. Chu, therapeutic target. To test the hypothesis indirectly, we rst Y. Yen measured FRS2 and FGFR expression in human high-grade Analysis and interpretation of data (e.g., statistical analysis, biostatistics, liposarcoma cell lines, whereas we found that FRS2 and computational analysis): K. Zhang, X. Wu, H. Gao, Y.-C. Yuan, K. Ho, P. Chu, Y. Yen FGFR1/3 transcripts and proteins are expressed in all 2 Writing, review, and/or revision of the manuscript: K. Zhang, K. Chu, X. Wu, liposarcoma cell lines. The phosphorylated FRS2 in these H. Gao, W. Chow, F. Un, Y. Yen fi Administrative, technical, or material support (i.e., reporting or orga- cells was signi cantly upregulated by bFGF2 stimulation. nizing data, constructing databases): Y. Wang, P. Chu, Y. Yen Activated FGFR signaling in FU-DDLS-1 and LiSa-2 cells was Study supervision: Y.-C. Yuan, P. Chu, Y. Yen indicated by the strong phosphorylated FRS2 signal in the presence of serum, whereas phosphorylated FRS2 was very Acknowledgments low in the SW872 cell line. We next investigated the effects of The authors thank Dr. Lynne Smith for critical reading of the article and editing and Victoria Bedell at the City of Hope Cytogenetic Core Laboratory for an FGFR inhibitor on the proliferation and activation of AKT assistance in FRS2 FISH assay. and ERK signaling of these 3 cell lines. Consistent with the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement differences observed for phosphorylated FRS2 between these in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cell lines, we found that an FGFR-selective inhibitor significantly suppressed the proliferation of FU-DDLS-1 and LiSa- Received June 1, 2012; revised November 12, 2012; accepted November 19, 2012; 2 cells, which have highly activated FGFR signaling, but not published OnlineFirst February 7, 2013.

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Amplification of FRS2 and Activation of FGFR/FRS2 Signaling Pathway in High-Grade Liposarcoma

Keqiang Zhang, Kevin Chu, Xiwei Wu, et al.

Cancer Res Published OnlineFirst February 7, 2013.

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