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 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 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. Ó2012 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 liposar- coma/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 com- pared 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- Ó2012 American Association for Cancer Research. crimination of pleomorphic from DDLS based on morphology www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst February 7, 2013; DOI: 10.1158/0008-5472.CAN-12-2086 Zhang et al. 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