The Tyrosine Kinase Adaptor Protein FRS2 Is Oncogenic and Amplified in High-Grade Serous Ovarian Cancer

Published OnlineFirst November 3, 2014; DOI: 10.1158/1541-7786.MCR-14-0407

Genomics Molecular Cancer Research The Tyrosine Kinase Adaptor Protein FRS2 Is Oncogenic and Ampliﬁed in High-Grade Serous Ovarian Cancer Leo Y. Luo1,2,3, Eejung Kim2,3, Hiu Wing Cheung2,4, Barbara A. Weir2, Gavin P. Dunn2,5, Rhine R. Shen3,6, and William C. Hahn2,3,7,8

Abstract

High-grade serous ovarian cancers (HGSOC) are character- andastumorsinimmunodeficient mice. FRS2, an adaptor ized by widespread recurrent regions of copy-number gain and protein in the FGFR pathway, induces downstream activation loss. Here, we interrogated 50 genes that are recurrently ampli- of the Ras–MAPK pathway. These observations identify FRS2 fied in HGSOC and essential for cancer proliferation and as an oncogene in a subset of HGSOC that harbor FRS2 survival in ovarian cancer cell lines. FRS2 is one of the 50 amplifications. genes located on chromosomal region 12q15 that is focally FRS2 fi amplified in 12.5% of HGSOC. We found that FRS2-amplified Implications: These studies identify as an ampli ed onco- FRS2 cancer cell lines are dependent on FRS2 expression, and that gene in a subset of HGSOC. expression is essential to ovarian fi FRS2 overexpression in immortalized human cell lines con- cancer cells that harbor 12q15 ampli cation. Mol Cancer Res; 13(3); ferred the ability to grow in an anchorage-independent manner 502–9. 2014 AACR.

Introduction To catalog the molecular aberrations present in HGSOC, The Cancer Genome Atlas (TCGA) network performed a large-scale, Ovarian cancer is the second most common gynecologic malig- multiplatform genomic profiling study of HGSOC (2). Analysis of nancy and the most common cause of gynecologic cancer–related 489 HGSOC primary tumors identified large number of recurrent death in the United States (1). Histologically, ovarian epithelial somatic copy-number alterations that include 31 focal amplifica- carcinomas can be divided into high-grade serous, low-grade tions. These amplified regions encode 1,825 genes, including serous, endometroid, mucinous, and clear cell types. Clinically, known oncogenes such as CCNE1 and MYC. However, the driver high-grade serous ovarian cancer (HGSOC) accounts for 70% to genes in the majority of the recurrently amplified regions remain 80% of all ovarian carcinomas and is characterized by its de novo unidentified. invasive nature and initial sensitivity to platinum treatment. The In parallel to these genome characterization efforts, we molecular features of HGSOC include BRCA1/2 and TP53 muta- initiated Project Achilles, a systematic effort to identify cancer tions and widespread DNA copy-number alterations (2). The lack dependencies at genome scale (3, 4). Here, by combining of readily targetable mutations found in HGSOC has contributed the output of ovarian cancer genome analysis with Project to slow progress in developing molecularly targeted therapies for Achilles, we systematically interrogated 1,825 recurrently this subset of ovarian cancers. amplified genes in ovarian cancer to identify genes that are essential in ovarian cancer cell lines that harbor such amplifica- fi FRS2 fi 1Health Sciences and Technology Program, Harvard Medical School, tions and identi ed as an ampli ed and essential gene in Boston, Massachusetts. 2Broad Institute of Harvard and Massachu- HGSOC. setts Institute of Technology, Cambridge, Massachusetts. 3Depart- ment of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. 4Department of Pathology and Laboratory Medicine, Materials and Methods Medical University of South Carolina, Charleston, South Carolina. 5Department of Neurological Surgery, Washington University School Analysis of TCGA primary tumor data of Medicine, St. Louis, Missouri. 6Astellas Pharma U.S. Inc., Santa Regions of copy-number amplification identified by Genomic 7 Monica, California. Center for Cancer Genome Discovery, Dana-Far- Identification of Significant Targets in Cancer (GISTIC) analyses ber Cancer Institute, Boston, Massachusetts. 8Department of Medi- cine, Brigham and Women's Hospital, Boston, Massachusetts. were used from the TCGA study on HGSOC (2). All RefSeq genes within these regions of amplification (n ¼ 1,825) were identified Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). and cross-referenced with genes interrogated in the Achilles screening library (n ¼ 582). All primary HGSOC data were L.Y. Luo, E. Kim, and H.W. Cheung contributed equally to this article. downloaded from the TCGA portal (http://tcga-data.nci.nih. Corresponding Author: William C. Hahn, Brigham and Women's Hospital, gov/tcga). Genomic characterization data were visualized using Medical Oncology, Dana 1538, 450 Brookline Avenue, Boston, MA 02215. Phone: the Integrative Genome Browser (http://www.broadinstitute.org/ 617-632-2641; Fax: 617-632-4005; E-mail: [email protected] igv). Mutual exclusivity analysis was performed using the cBio doi: 10.1158/1541-7786.MCR-14-0407 Portal for Cancer Genomics (5, 6), which uses different thresholds 2014 American Association for Cancer Research. for scoring regions of copy-number alteration.

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FRS2 Is an Ovarian Cancer Oncogene

Analysis of shRNA screening data after infection by using CellTiter-Glo luminescent cell viability Data from genome-scale loss-of-function screening were assay (Promega). processed as described previously (3). Briefly, 54,000 shRNAs were lentivirally delivered to 102 cancer cell lines, and the Anchorage-independent growth assay fi degree of representation of each shRNAs in the nal cell Growth in soft agar was determined by plating 5 104 cells population was measured by custom Affymetrix array. Normal- in triplicate in 4 mL of medium containing 0.35% Noble agar ization, variance stabilization, and expression score calculation (BD Biosciences), which was placed on top of 4 mL of solidified fi fi were conducted as speci ed in the modi ed dCHIP method 0.6% agar. Unstained colonies greater than 100 mmindiameter (4). Scores were median-adjusted per cell lines. Ovarian-spe- were counted 4 weeks after plating using Cell Profiler software fi ci c gene dependencies were determined with three comple- (10). mentary methods: (i) 150 best single shRNA or (ii) 300 second best shRNA or (iii) composite of all shRNAs for the gene using Immunoblotting KS statistics. Genes (582; 5.2%) were selected from the union of Cell lysates were prepared by scraping cells in lysis buffer [50 three methods above. mmol/L Tris HCl (pH 8), 150 mmol/L NaCl, 1% Nonidet P40, To identify genes that were both amplified in ovarian tumors 0.5% sodium deoxycholate, and 0.1% SDS] containing complete and essential in amplified cancer cell lines, each gene identified as protease inhibitors (Roche) and phosphatase inhibitors (10 amplified in primary ovarian tumors (1,825 genes) was tested mmol/L sodium floride and 5 mmol/L sodium orthovanadate). across the entire panel of 102 cell lines screened. Only genes Protein concentration was measured by using the BCA Protein with more than five amplified cell lines were included in the Assay Kit (Pierce). An equal amount of protein (20 mg) was study. Amplified genes that had mapped shRNAs with a value of separated by NuPAGE Novex Bis-Tris 4% to 12% gradient gels P < 0.05 were identified as candidate genes. (Invitrogen), and then transferred onto a polyvinylidene difluor- Cell culture and generation of stable cell lines ide membrane (Amersham). Antibodies against FRS2 (sc-8318) All human cancer cell lines were cultured in previously were purchased from Santa Cruz Biotechnology. Antibodies for described media supplemented with 10% FBS (Sigma; ref. 3). PARP (#9532), phospho-ERK1/2 (#9101), ERK1/2(#9102) were fi Immortalized human ovarian surface epithelial cells (IOSE; purchased from Cell Signaling Technology and antibody speci c b ref. 7) were maintained in 1:1 medium 199: DMEM supple- for -actin was obtained from Santa Cruz Biotechnology (sc- mented with 10% FBS. CAL120, COV644, COV362, and 8432-HRP). – CAOV3 cells were cultured in DMEM (Invitrogen) with 10% After incubation with the appropriate horseradish peroxidase FBS. HCC1143 and EFO21 cells were cultured in RPMI-1640 linked secondary antibodies (Bio-Rad), signals were visualized by medium (Invitrogen) with 10% FBS. NIH/3T3 cells were cul- enhanced chemiluminescence plus Western blotting detection tured in DMEM with 10% bovine calf serum. Lentiviruses were reagents (Amersham). Alternatively, membrane was incubated fl produced by transfection of 293T packaging cells with a three- with IRDye uorescent secondary antibodies (LI-COR) and visu- fl plasmid system. To generate stable cell lines, cells were seeded alized by Odyssey quantitative uorescence imaging system (LI- into 6-well dishes for 24 hours before infection with 0.3 mL of COR). lentiviruses for 12 hours in the presence of 8 mg/mL polybrene. After the incubation, medium was replaced with fresh medium Real-time quantitative RT-PCR for another 24 hours before selection in media containing 2 mg/ Total RNA was extracted with the RNeasy Mini Kit (Qiagen). mL of puromycin or 10 mg/mL of blasticidin until the control Reverse transcription was performed using SuperScript III First- cells were no longer viable. Strand Synthesis System (Invitrogen). Quantitative RT-PCR reactions were performed using SYBR green PCR Master Mix (Applied Plasmids Biosystems). The primer sequences used were obtained from 0 Human FRS2 from the CCSB human ORFeome collection (8) MGH PrimerBank: FRS2 (forward: 5 -CTGTCCAGATAAAGA- 0 0 was cloned into pLenti6.3-blast (BamHI and BsrGI sites). The CACTGTCC-3 , reverse: 5 -CACGTTTGCGGGTGTATAAAATC- 0 0 0 pLX304–LacZ was used as a control vector. The human MEKD218, 3 ); GAPDH (forward: 5 -CCTGTTCGACAGTCAGCCG-3 , 0 0 D222 (or MEKDD) fragment was removed from pBabe-puro- reverse: 5 - CGACCAAATCCGTTGACTCC-3 ). Triplicate reactions MEKDD plasmid (9) with BamHI and SalI and inserted for the gene of interest and the endogenous control (GAPDH) into pLX304–blasticidin. Lentiviral pLKO.1-puro-shRNA con- were performed separately on the same cDNA samples by using structs were obtained from The RNAi Consortium or designed the ABI 7900HT real-time PCR instrument (Applied Biosystems). C DDC by custom oligo synthesis (IDT). The shRNA constructs used are as The mean cycle threshold ( t) was used for the t analysis follows: control shRNA targeting LacZ (TRCN0000231710), method. FRS2-specific shRNAs (shFRS2#1: TRCN0000370440, shFRS2#2: 0 0 5 -CTCTAAATGGCTACCATAATA-3 ). Flow cytometry Cells were collected, washed, and fixed with 70% ethanol at Cell proliferation assay 20C for 4 hours. Fixed cells were washed, rehydrated, and CAL120, COV644, HCC1143, EFO21, CAOV3, and COV362 resuspended in propidium iodide staining solution (25 mg/mL cells (3 103) were seeded into each well of 96-well plates propidium iodide, Sigma P4862, 50 mg/mL RNase A, Invitrogen 24 hours before infection. Six replicate infections were performed 12091-021, in PBS) at room temperature for 30 minutes. for control shRNAs and each gene-specific shRNA in the presence Flow cytometry was done on BD LSR II flow cytometer (BD of 8 mg/mL polybrene for 24 hours followed by selection with Biosciences). Debris and aggregates were gated out, and the 2 mg/mL of puromycin. The ATP content was measured at 6 days sub-G1 population was analyzed using FlowJo software.

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Tumorigenicity assay We used Genomic Identification of Significant Targets in Cancer Female NCR/nude mice (Charles River Laboratories) were Version 2.0 (GISTIC 2.0) algorithm to identify the peak of obtained at 6 weeks of age. All animal experiments were approved amplification, which corresponds to the highest level of copy- by the Dana-Farber Institutional Animal Care and Use Commit- number gain. In ovarian cancer samples, we observed the overlap tee. Tumor xenograft experiments were performed as described between the peak of amplification and the location of the FRS2 previously (9). NIH/3T3 cells expressing indicated constructs gene. Furthermore, the focal amplification of 12q15 region in were trypsinized and collected in fresh media. Cells were washed HGSOC is correlated with increased mRNA expression of FRS2, and resuspended in PBS at 106 cells per 100 mL. Cells were injected suggesting the functional relevance of the copy-number gain s.c. on left and right flanks, and upper back. Two mice were used (Fig. 1C). In addition, we also observed frequent amplification for each experimental condition. A total of 2 106 cells were of FGFR family of tyrosine kinase receptor genes in HGSOC. injected per site, three sites per mouse. Tumor injection sites were Strikingly, using the cBio portal, we found that HGSOC samples monitored for 3 months for tumor formation. Mice were eutha- that harbor 12q15 amplifications were often mutually exclusive nized when the largest tumor on mouse reached 2 cm in largest with HGSOC that harbor FGFR1, FGFR2, FGFR3, and FGFR4 dimension. We attempted tumor experiments with HA1E-A cells, amplifications (Fisher exact test P ¼ 0.028; Fig. 1D). This pattern but encountered a high background in control cells. of mutations is observed in commonly mutated genes in the same pathway, such as KRAS and EGFR mutations or TP53 and MDM2 mutations. These observations implicate FGF signaling Statistical analysis through amplifications of FGFRs and FRS2 as a common event in Unless otherwise indicated, one-way ANOVA was used (Graph- HGSOCs. Pad). A P value of <0.05 was considered statistically significant. The Fisher exact test was used for tumor formation assays and mutual exclusivity analysis. A two-tailed Student t test was used FRS2 is essential in cancer cell lines that harbor 12q15 for pairwise comparisons. A log-rank test was performed for amplification animal survival studies. To confirm that FRS2 was essential in FRS2-amplified cancer cell lines, we used two independent shRNAs to suppress FRS2 Results expression in three cell lines with 12q15 amplification (CAL120_- BREAST, COV644_OVARY, HCC1143_BREAST) and three cancer Identification of FRS2 as an amplified and essential gene in cell lines that contain normal copies of 12q15 (CAOV3_OVARY, ovarian cancer EFO21_OVARY, COV362_OVARY). We used both breast and HGSOCs are characterized by high frequency, recurrent ovarian cancer cell lines because we found focal amplification regions of copy-number gain and loss. Recent genome-scale of 12q15 in a large subset of the primary breast cancers (Fig. 1B). effort to characterize structural alterations in HGSOC has Copy-number data for these cell lines were obtained from the identified 31 recurrently amplified chromosomal regions con- Broad Institute/Novartis Cancer Cell Line Encyclopedia (Fig. 2A; taining total of 1,825 genes (2). To systematically study pre- ref. 14). We found that FRS2 suppression by two independent viously unknown lineage-specific dependencies, we initiated a shRNAs significantly decreased the proliferation of cancer cell genome-scale effort (Project Achilles) to identify genes essential lines that harbor the 12q15 amplification, when compared with for proliferation/survival of a large number of well-character- cells that exhibit diploid copy number at 12q15 or cells infected ized cancer cell lines using loss-of-function genetics with with control shRNA (Fig. 2B). The degree of FRS2 suppression in shRNAs (ref. 4). Although recent studies suggest that estab- 12q15-amplified cell lines was validated by quantitative real-time lished ovarian cancer cell lines do not fully recapitulate PCR (Fig. 2C). To demonstrate that FRS2 suppression induced the genetic alterations found in high-grade ovarian cancers apoptotic cell death in 12q15-amplified cell lines, we interrogated (11, 12), here we have focused on those alterations found by PARP cleavage after suppression of FRS2. We found increased the TCGA in human cancers and shared by these ovarian cancer level of cleaved PARP in 12q15-amplified cell lines compared cell lines. Using data from 102 cell lines of which 25 were from with cell lines without 12q15 amplification (Fig. 2D). Together, the ovarian lineage, we identified 582 ovarian lineage–specific these findings demonstrate that cancer cells that harbor 12q15 gene dependencies (3). By looking at the intersection of genes amplification require FRS2 expression for proliferation and involved in regions of recurrent copy number and essential in survival. ovarian cancer cell lines, we identified 50 genes (Fig. 1A; Supplementary Table S1). Two of the 50 genes were previously identified as ovarian specific oncogenes (PAX8 and CCNE1) FRS2 induces oncogenic transformation with similar analytic approach (3, 13). To determine whether FRS2 can contribute to tumorigenesis by Among the remaining genes, we focused on fibroblast growth inducing transformation, we performed anchorage-independent receptor substrate 2 (FRS2) because FRS2 is (i) an adaptor protein growth assays and tumor xenograft experiments. In our prior in the FGFR pathway, (ii) is located on chromosomal region studies, we have shown that human kidney epithelial cells are 12q15, which is focally amplified in 12.5% of 559 primary immortalized by coexpression of the human catalytic subunit of HGSOCs characterized by TCGA (Fig. 1B), and (iii) was among telomerase (hTERT) and the SV40 Early Region (HA1E cell), and the top 100 genes that scored by our analysis of Project Achilles the expression of oncogenic alleles of RAS confers the ability to and copy-number data in HGSOC. We also found a structurally grow in anchorage-independent manner (15). We had previously similar chromosomal region amplification in other cancer types demonstrated that the RAS oncogene can be replaced by combi- such as breast invasive carcinoma, lung adenocarcinoma, lung nation of downstream effectors of the RAS signaling pathway, squamous cell carcinoma, head and neck squamous cell carcino- such as constitutively activated MEK1 (MEK-DD) and AKT1 ma, gastric adenocarcinoma, and bladder urothelial carcinoma. (myristoylated AKT; ref. 9). In addition, we used the same genetic

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FRS2 Is an Ovarian Cancer Oncogene

A C

15 Amplified genes in ovarian cancer Ovarian-specific (TCGA) essential genes 10 (Project Achilles)

1,825 50 582 0 FRS2 mRNA Z-score

−5

Loss Normal Gain Amp FRS2 copy number

B D

q21.31 Ovarian serous cystadenocarcinoma

q21.2 FRS2 q21.1 FGFR1 q15 FGFR2 q14.2 FGFR3 Chromosome 12 q13.3 FGFR4

q13.13

Ovarian Breast Lung Lung squamous AC H&N

Bladder Deletion Neutral Amplification Stomach

Figure 1. Amplification and overexpression of FRS2 in primary HGSOCs and ovarian cancer cell lines. A, FRS2 is one of the 50 genes that are recurrently amplified in primary ovarian tumors and essential for ovarian cancer cell proliferation and survival. B, copy-number profile along chromosome 12q of human tumor samples. FRS2 was amplified in multiple cancer types, including ovarian, breast, lung squamous, lung adenocarcinoma, stomach, head and neck (H&N), and bladder. Each vertical line represents one tumor sample. Red, copy-number gain; blue, copy-number loss. C, level of FRS2 mRNA expression in primary tumors correlates with the copy number. Copy number is divided into four categories based on log2 of copy numbers. "Amplification" is defined as log2 (copy number) more than 1; "gain" is between 0.2 and 1; "normal" is between 0.2 and 0.2; "loss" is less than 0.2. D, FRS2 amplification and FGFR1, FGFR2, FGFR3,andFGFR4 amplifications are mutually exclusive in HGSOCs. Data were analyzed using the cBio portal rather than GISTIC. elements to IOSE cells and fallopian tube epithelial cells and LacZ (Fig. 3A). The number of colonies formed with FRS2 used this cell line to identify ovarian cancer oncogenes such as overexpression is significantly higher (P < 0.001) compared ID4 (16). We note that recent reports suggest that both fallo- with constitutively activated MEK, suggesting possible activa- pian tube and ovarian surface epithelial cells can serve as tion of additional pathways that contribute to the transforma- the origin for HGSOC and have not noted differences in the tion process. We also conducted the same experiment in IOSE transformation potential of cells from either lineage (16–20). cells to show that FRS2 also induced transformation in ovarian As previous studies have shown that FRS2 preferentially acti- epithelial cells (Fig. 3B). vates the MAPK pathway, we overexpressed FRS2 in HA1E cell Next, we determined whether expression of FRS2 also induced lines expressing constitutively active myristoylated AKT (HA1E- tumor formation in vivo by expressing FRS2 in NIH3T3 mouse A) to determine whether FRS2-mediated MAPK pathway acti- fibroblast cells and implanting these cells subcutaneously in vation complemented AKT pathway activation to induce trans- immunodeficient mice. At 11 weeks, we observed that tumors formation. We measured anchorage-independent growth with formed in 33% (2 of 6) of the injection sites harboring cells FRS2 overexpression and found that FRS2 overexpression was expressing FRS2, but failed to observe any tumors in sites har- sufficient to induce anchorage-independent colony formation boring control cells (Fig. 3C). We note that because we implanted of HA1E-A cells compared with cells expressing the control tumors in several sites in each mouse, and we terminated the

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A B ** 0.8 CAL120

Genomic position on chromosome 12 HCC1143 FRS2 0.6 COV644 66 Mb 86 bM 70 Mb 72 Mb 47 bM CAL120 CAOV3 COV644 HCC1143 0.4 COV362 EFO21 COV362 EFO21 CAOV3 0.2 Relative proliferation ShFRS2 #1 Deletion Neutral Amplification ShFRS2 #2

0.0 Amplified Nonamplified

C CAL120 COV644 HCC1143 COV362 EFO21 CAOV3

1 Relative FRS2 mRNA level 0 shLacZ shLacZ shLacZ shLacZ shLacZ shLacZ shFRS2#2 shFRS2#2 shFRS2#2 shFRS2#1 shFRS2#1 shFRS2#2 shFRS2#1 shFRS2#2 shFRS2#1 shFRS2#2 shFRS2#1 shFRS2#1

D CAL120 COV644 HCC1143 COV362 EFO21 CAOV3 shLacZ shLacZ shFRS2 #1 shFRS2 #2 shLacZ shLacZ shFRS2 #1 shFRS2 #2 shFRS2 #1 shFRS2 #2 shFRS2 #1 shFRS2 #2 shLacZ shFRS2 #1 shFRS2 #2 shFRS2 #1 shFRS2 #2 shLacZ

PARP

Cleaved PARP

FRS2

Actin

Figure 2. Suppression of FRS2 decreases the proliferation of ovarian and breast cancer cells harboring 12q15 amplification. A, SNP array colorgram showing genomic amplification of chromosome 12q15 in ovarian and breast cancer cell lines. Red, copy-number amplification; blue, copy-number deletion. B, consequences of FRS2 suppression on the proliferation of cancer cell line that either harbor 12q15 amplification (CAL120, HCC1143, and COV644) or normal copy number of 12q15 (CAOV3, COV362, and EFO21) normalized to cells treated with shLacZ. Red, cell lines treated shFRS2 #1; black, cell lines treated with shFRS2#2; , P < 0.01 compared with control shLacZ, the Student t test was used. C, quantitative RT-PCR of FRS2 expression in FRS2-amplified (red) and -nonamplified (black) cell lines. D, increased apoptosis in FRS2-amplified cell lines (red) upon FRS2 suppression, shown by increased PARP cleavage.

experiment before observing tumor growth in all sites, these oncogenic transformation in human kidney fibroblasts or mouse experiments may underestimate the tumorigenicity of these cells. fibroblasts by promoting anchorage-independent growth or in These observations confirm that FRS2 overexpression can induce vivo xenograft tumor formation.

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300 A *** phospho-ERK levels (Fig. 4C). In contrast, we failed to observe a *** change in phospho-AKT when we overexpressed FRS2 (Fig. 4B). 250 These observations suggest that FRS2 overexpression preferentially activates the MAPK pathway in this context. This ﬁnding 200 LacZ corroborates anchorage-independent growth assays in which we observed that FRS2 was able to induce increased colony growth 150 when expressed with Myr–AKT as compared with coexpression with MEK-DD in HEK cells. 100 Colony number FRS2

50 Discussion Here, we identified FRS2 as one of the 50 genes that are 0 recurrently amplified in HGSOCs and essential to survival in LacZ FRS2 MEK-DD ovarian cancer cell lines. FRS2 belongs to the 12q15 genomic HA1E + AKT region that is focally amplified in 12.5% of HGSOC. Using independent shRNAs targeting against FRS2, we showed the FRS2 B 60 expression of was essential for survival in cancer cells with 12q15 amplification. We also discovered that overexpression of 50 FRS2 in immortalized kidney fibroblast or ovarian epithelial cells 40 ** promoted anchorage-independent growth and tumorigenesis in 30 mice. Together, these observations nominate FRS2 as an amplified 20 oncogene in a subset of HGSOCs. In addition to HGSOC, 12q15 amplification containing Colony number 10 FRS2 is found in other cancer types. 12q15 amplicon contain- 0 ing FRS2 is focally amplified in 9.2% of breast invasive carci- LacZ FRS2 GAB2 KRAS nomas. Indeed, we found that breast cancer cell lines that IOSE harbor 12q15 amplification are also sensitive to suppression of FRS2. Furthermore, new evidence has suggested the onco- C genic role of FRS2 and 12q15 amplification in high-grade liposarcomas through whole-exome sequencing and demon- ) 3 5,000 strated sensitivity of FRS2-amplified high-grade liposarcoma cell lines to FRS2 suppression through shRNAs (23, 24). These studies support FRS2 as a bona fide oncogene in a variety of cancers and a potential therapeutic target for a subset of cancers 3,000 that harbor such amplification. The discovery of FRS2 as an amplified oncogene adds to the family of FGFR signaling components that are critical to tumor- Tumor volume (mm 1,000 igenesis in many cancer types. For example, mutations or ampli- 0 fications of multiple FGFR have been reported in bladder cancer Control FRS2 KRAS G13D (25), gastric cancer (26), endometrial cancer (27), and non–small cell lung cancer (NSCLC; refs. 28, 29). Large-scale genome-wide Figure 3. FRS2 overexpression potentiates tumorigenicity. A, FRS2 promotes association studies have also linked breast cancer risk loci to anchorage-independent growth in HA1E-A cells compared with LacZ control. FGFR2 (30). Moreover, FGF ligands may also contribute to cancer MEK-DD, a constitutively active MEK, is positive control. Right, images of soft or therapy resistance as evidenced by FGF19 amplifications in agar colonies formed by HA1E-A with either FRS2 or control vector liver cancer (31) and the observed therapeutic effect of neutral- overexpression. B, FRS2 promotes anchorage-independent growth of IOSE izing anti-FGF antibodies (32). cells. GAB2 is a similar adaptor protein known to transform ovarian epithelial The 12q15 genomic region contains 15 genes and FRS2 resides cells; , P < 0.01; , P < 0.001 compared with respective control vectors, the Student t test. C, FRS2 overexpression promotes tumorigenicity in vivo. at the peak of this amplicon (Supplementary Table S2). Prior work 3T3 cells with FRS2 overexpression were able to form tumor in mouse in high-grade liposarcoma, which exhibits a broader region of xenograft models compared with LacZ control. Constitutively active KRAS amplification (12q13-12q15) than HGSOC, suggested that in G13D was used as positive control. Within each condition, tumors from the addition to FRS2, other genes such as CDK4 and MDM2 may same mouse were annotated with same color. contribute to cell transformation (23). Although neither CDK4 nor MDM2 is located within the 12q15-amplified region in FRS2 amplification activates the MAPK pathway HGSOC, this finding does not preclude the possibility that Previous studies have shown that FRS2 is a critical mediator of other genes in the genomic region may cooperate to drive various FGFR signaling and plays an important role in activating MAPK stages of tumorigenesis. Indeed, we recently demonstrated that and PI3K pathways (Fig. 4A; refs. 21, 22). We have confirmed that multiple genes resident in a recurrently amplified region (3q26) FRS2 overexpression induces activation of the MAPK pathway in contribute to cell transformation by inducing different cancer- 293 HEK cells and IOSE cells by assessing phospho-Thr202/ associated phenotypes, suggesting that further studies involving Tyr204 ERK1/2 levels (Fig. 4B). Conversely, suppression of FRS2 other assays will be necessary to investigate the function of these in the FRS2-amplified cancer cell line resulted in a decrease in other genes (33).

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FGFR1 A adaptor proteins include GRB2 in receptor tyrosine kinase (RTK) FGFR2 k FGFR3 signaling (34) and MYD88 in NF- B signaling (35). FRS2 was FGFR4 originally discovered as a docking site for coordinated assembly of a multiprotein complex that includes GRB2, GAB1, and SOS1 and FGF FGF serves a critical role in the FGFR signaling pathway (Fig. 4A; refs. 21, 36). Amplification or overexpression of the adaptor proteins may amplify signaling downstream of receptors and may mediate resistance to RTK-targeted therapies or confer de FRS2 GRB2 RafRasSos novo sensitivity to signaling pathway inhibitors. We previously P P identified CRKL, an adaptor protein involved in RAS and RAP P P signaling, as an amplified oncogene in NSCLC (37) that also MEK EGFR P P mediates resistance to an EGFR inhibitor in -mutant lung MAPK in vivo P P Pathway cancer cells. More recently, through a multiplexed transformation screen, we found another adaptor protein, GAB2, as an MAPK amplified ovarian cancer oncogene that activates PI3K signaling (38). Ovarian cancer cells with GAB2 alteration are sensitive to PI3K pathway inhibition. An independent analysis of TCGA B datasets across 16 cancer types identified 75 amplified and poten- FRS2 LacZ FRS2 tially druggable genes, including and EGFR family adaptors FRS2 LacZ GRB2 and GRB7 (39). Together, these findings suggest that FRS2 FRS2 adaptor proteins such as FRS2 play key roles in cell transformation and resistance to therapy. Identifying alterations in these pERK1/2 pERK1/2 adapter proteins may allow for the identification of resistant tumors and represent novel targets. ERK1/2 ERK1/2

pAKT Disclosure of Potential Conflicts of Interest pAKT W.C. Hahn is a consultant to Novartis and RRS is an employee of Astellas AKT Pharma U.S. No potential conflicts of interest were disclosed by the other AKT authors. 293T Actin Authors' Contributions Conception and design: L.Y. Luo, H.W. Cheung, G.P. Dunn, W.C. Hahn IOSE Development of methodology: B.A. Weir, G.P. Dunn, R.R. Shen Acquisition of data (provided animals, acquired and managed patients, C provided facilities, etc.): L.Y. Luo, E. Kim, G.P. Dunn Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): L.Y. Luo, E. Kim, H.W. Cheung, B.A. Weir, W.C. Hahn shLacZ shFRS2 #1 shFRS2 #2 Writing, review, and/or revision of the manuscript: L.Y. Luo, E. Kim, W.C. Hahn FRS2 Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B.A. Weir, W.C. Hahn Study supervision: W.C. Hahn pERK1/2 Acknowledgments ERK1/2 The authors thank the Broad Genetic Perturbation Platform for cell lines and RNAi reagents, and G. Cowley, D. Root, J. Scott, L. Ali, P. Lizotte, G. Jiang, J. Hsiao for performing the genome-wide RNAi screen in Project Achilles. Actin The authors thank members of the Hahn laboratory for excellent technical assistance and valuable advice on experiments. L. Luo was supported by Howard CAL120 Hughes Medical Institute Medical Research Fellowship. E. Kim is supported by a Samsung Scholarship. Figure 4. FRS2 promotes tumorigenesis via activation of the MAPK pathway. A, FRS2 functions as an adaptor protein in the FGFR signaling pathway, Grant Support adapted from Turner and Grose (40). B, effect of FRS2 overexpression on This work was supported in part by grants from the U.S. NIH (U01 phosphorylation of ERK in 293T cells and ovarian epithelial cells. C, effect CA176058), the Starr Foundation (I5-A533), and the H.L. Snyder Medical of FRS2 suppression on phosphorylation of ERK in the cancer cell line with Research Foundation. 12q15 amplification. 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 in accordance with 18 U.S.C. Section 1734 solely to indicate Here, we show a new functional class of adaptor proteins as this fact. driver oncogene in ovarian cancer. The adaptor proteins lack intrinsic enzymatic activities, but mediate protein–protein inter- Received July 22, 2014; revised October 23, 2014; accepted October 24, 2014; actions that drive protein complex formation. Classic examples of published OnlineFirst November 3, 2014.

508 Mol Cancer Res; 13(3) March 2015 Molecular Cancer Research

FRS2 Is an Ovarian Cancer Oncogene

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www.aacrjournals.org Mol Cancer Res; 13(3) March 2015 509

The Tyrosine Kinase Adaptor Protein FRS2 Is Oncogenic and Amplified in High-Grade Serous Ovarian Cancer

Leo Y. Luo, Eejung Kim, Hiu Wing Cheung, et al.

Mol Cancer Res 2015;13:502-509. Published OnlineFirst November 3, 2014.

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