RNA-Seq Identification of RACGAP1 As a Metastatic Driver in Uterine Carcinosarcoma

Published OnlineFirst April 27, 2016; DOI: 10.1158/1078-0432.CCR-15-2116

Cancer Therapy: Preclinical Clinical Cancer Research RNA-seq Identiﬁcation of RACGAP1 as a Metastatic Driver in Uterine Carcinosarcoma Shijun Mi1,MingyanLin2, Jurriaan Brouwer-Visser1,JenniferHeim1,DavidSmotkin1, Tiffany Hebert3, Marc J. Gunter4, Gary L. Goldberg1,5,DeyouZheng2,6, and Gloria S. Huang1,5,7

Abstract

Purpose: Uterine carcinosarcoma is a rare aggressive malignan- with uterine carcinosarcoma from The Cancer Genome Atlas cy frequently presenting at advanced stage of disease with extra- (TCGA). uterine metastases. Median survival is less than 2 years due to high Results: Rac GTPase–activating protein 1 (RACGAP1) was iden- relapse rates after surgery and poor response to chemotherapy or tiﬁed to be highly upregulated in uterine carcinosarcoma. Func- radiotherapy. The goal of this study was to identify novel ther- tional assays showed that RACGAP1 mediates motility and inva- apeutic targets. sion via regulation of STAT3 phosphorylation and survivin expres- Experimental Design: We applied RNA-seq analysis to pro- sion. RACGAP1 depletion or survivin inhibition abrogated motil- spectively collected uterine carcinosarcoma tumor samples ity and invasiveness of carcinosarcoma cells, while RACGAP1 from patients undergoing primary surgical resection and for overexpression conferred invasiveness to endometrial adenocarci- comparison, normal endometrial tissues from postmenopaus- noma cells. In the TCGA cohort, RACGAP1 expression correlated al women undergoing hysterectomy for benign indications. with survivin expression and extrauterine spread of disease. Functional assays were done in primary carcinosarcoma cell Conclusions: The RACGAP1–STAT3–survivin signaling path- lines developed from patients and in established cell lines, way is required for the invasive phenotype of uterine carcinosar- as well as a cell line–derived xenograft model. Validation coma and is a newly identiﬁed therapeutic target in this lethal was done by analysis of an independent cohort of patients disease. Clin Cancer Res; 22(18); 4676–86. 2016 AACR.

Introduction metaplastic carcinoma that has undergone epithelial-to-mesen- chymal transition (3). Uterine carcinosarcoma, also known as malignant mixed These uncommon tumors, which represent < 5% of uterine Mullerian€ tumors, is a highly aggressive form of uterine cancer, corpus cancers, account for greater than 15% of uterine cancer– with a propensity for extrauterine metastases and a high case related deaths (1, 4). The disease usually affects postmenopausal fatality rate (1). The histologic diagnosis is based on the presence women, with a median age at diagnosis of 62 to 67 years. The of both malignant carcinomatous and sarcomatous elements (2). incidence is significantly higher in black women with an age- Prior studies support a monoclonal endometrial origin of both adjusted incidence rate of 4.3 per 100,000 compared with 1.7 per elements, supporting the view of uterine carcinosarcoma as a 100,000 in white women (5). Previously identified risk factors for the development of uterine carcinosarcoma include obesity, nulliparity, and exogenous estrogen or tamoxifen exposure (6). 1Division of Gynecologic Oncology, Department of Obstetrics and Treatment of uterine carcinosarcoma is primarily surgical. Gynecology, Albert Einstein College of Medicine and Montefiore Med- Approximately one-third of patients have disease spread beyond ical Center, Bronx, New York. 2Department of Genetics, Albert Einstein College of Medicine, Bronx, New York. 3Department of Pathology, the uterus at the time of diagnosis, and the recurrence rate after Albert Einstein College of Medicine and Montefiore Medical Center, surgery exceeds 50%. Combination chemotherapy may improve 4 Bronx, New York. Section of Nutrition and Metabolism, International overall and progression-free survival in patients with advanced Agency for Research on Cancer, Lyon, France. 5Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York. 6Depart- stage or recurrent carcinosarcoma (7, 8). Despite multimodal ments of Neurology and Neuroscience, Albert Einstein College of treatment approaches, the median overall survival is approxi- Medicine, Bronx, New York. 7Department of Molecular Pharmacology, mately 21 months, and in patients with advanced disease, less Albert Einstein College of Medicine, Bronx, New York. than one year (1). Note: Supplementary data for this article are available at Clinical Cancer The underlying molecular drivers of the aggressive phenotype Research Online (http://clincancerres.aacrjournals.org/). of uterine carcinosarcoma have not been identified. Poor clinical S. Mi and M. Lin contributed equally to this article. prognostic factors include higher FIGO stage of disease and serum Corresponding Author: Gloria S. Huang, Division of Gynecologic Oncology, CA125 elevation (6). In this study, we applied RNA-seq analysis to Department of Obstetrics & Gynecology and Women's Health, Montefiore prospectively collected uterine carcinosarcoma tumor samples Medical Center and Albert Einstein College of Medicine, 1695 Eastchester Road, from patients undergoing primary surgical resection and for Suite 601, Bronx, NY 10461. Phone: 718-405-8082; Fax: 718-405-8087; E-mail: comparison, normal endometrial tissues from postmenopausal ghuang@montefiore.org women undergoing hysterectomy for benign indications, with the doi: 10.1158/1078-0432.CCR-15-2116 goal of identifying novel oncogenic drivers and potential thera- 2016 American Association for Cancer Research. peutic targets.

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RACGAP1 Is a Metastatic Driver in Uterine Carcinosarcoma

Primary cell line isolation and tissue culture Translational Relevance For isolation of primary cell lines, under an IRB-approved Uterine carcinosarcoma is a highly aggressive endometrial protocol (#2011-404), fresh tumor tissue from patients under- malignancy that causes a disproportionate number of deaths going hysterectomy and staging surgery for histologically con- from uterine cancer. By comparing the gene expression of firmed uterine carcinosarcoma was transported to the laboratory uterine carcinosarcoma with benign endometrial tissue, Rac in sterile RPMI tissue culture media. Following mechanical dis- GTPase–activating protein 1 (RACGAP1) was identified to be sociation, enzymatic digestion with 3 mg/mL collagenase A highly upregulated, and functional studies showed that RAC- (Roche) and 150 mg/mL DNase 1 (Thermo Fisher Scientific), and GAP1 regulates motility and invasion via STAT3–survivin red blood cell lysis (eBioscience), cells were washed with RPMI signaling. Analyzing an independent cohort from The Cancer with FBS (10%), and resuspended in F-media supplemented as Genome Atlas showed that patients with more advanced described previously (9). Cells were seeded in tissue culture flasks uterine carcinosarcoma had significantly higher RACGAP1 and media replaced as needed. Cell line authentication was done expression in their tumors correlated with increased survivin by short tandem repeat (STR) profiling using the Genemarker 10 expression. Furthermore, RACGAP1 expression predicted sen- kit (Promega) and matching to the original carcinosarcoma sitivity to survivin-targeted therapy in primary cell lines patient samples (Supplementary Table S4). derived from patients. On the basis of these novel findings, CS99 cells and their derivatives were maintained as subcon- the RACGAP1–STAT3–survivin signaling pathway is identified fluent monolayer cultures in RPMI1640 containing 10% FBS at as a promising therapeutic target in uterine carcinosarcoma, a 37 C with 5% CO2. highly lethal disease of the female reproductive tract. All cells were routinely screened with MycoAlert (Lonza) and were negative for mycoplasma.

RNA isolation We found that RACGAP1 is a highly upregulated gene in To extract RNA, frozen tissues were pulverized in a tissueTUBE uterine carcinosarcoma, and its overexpression promotes the bag (Covaris) using a cryoPREP (Covaris) and then homogenized metastatic phenotype. We identified STAT3 and survivin as in Buffer RLT (Qiagen) using a Covaris adaptive focused acoustics bona fide downstream targets of RACGAP1, and showed that tissue disrupter. The Qiagen AllPrep kit was used following the RACGAP1 is a critical regulator of STAT3 phosphorylation and manufacturer's instructions. The RNA concentration and purity survivin expression in uterine carcinosarcoma. Targeting RAC- was measured using the Nanodrop spectrophotometer (Thermo GAP1 directly, or its downstream effectors, significantly dimin- Fisher Scientific), and RNA integrity was evaluated with the ished the invasive capacity of uterine carcinosarcoma cells. Agilent Bioanalyzer (Agilent). RNA quality was uniformly excel- Analysis of an independent cohort of 57 patients with uterine lent and met the following criteria; Nanodrop, 260/280 ratio >1.8; carcinosarcoma showed that high RACGAP1 predicted extra- Agilent Bioanalyzer, RIN > 7. uterine metastasis, validating its clinical significance as a metastatic driver. Thus, we have identified a novel molecular driver Paired-end library preparation and Illumina sequencing of the metastatic phenotype of uterine carcinosarcoma, sug- Twelve RNA-seq libraries (CS001, CS005, CS008, CS010, gesting new therapeutic approaches for targeting this and other CS011, CS718, NP002, NP006, NP011, NP013, NP024, and highly aggressive cancers. NP028) were prepared for paired-end sequencing using the Illumina HiSeq platform in the epigenetics core facility of the Materials and Methods Albert Einstein College of Medicine according to directional whole transcript seq protocol described on WASP (wiki-based Tissue acquisition automated sequence processor, http://wasp.einstein.yu.edu). In Under Institutional review board (IRB) approval, carcino- brief, the purified cDNA library products were evaluated using the sarcoma tumor samples (IRB# 2011-404) and normal endo- Agilent bioanalyzer and diluted to 10 nmol/L for cluster gener- metrial tissues (IRB#2009-406) were prospectively collected ation in situ on the HiSeq paired-end flow cell using the cBot from consenting patients undergoing surgery at Montefiore automated cluster generation system followed by massively par- Medical Center, and the corresponding clinical data recorded. allel sequencing (2100 bp) on HiSeq 2000. The study coordinator assigned a unique study ID to tissue samples, by which samples were identified in the laboratory. In RNA-seq analysis and enrichment analysis conjunction with surgical pathology, the standard operating We obtained 92-bp mate-paired reads from DNA fragments procedure was followed for prospective tissue collection of with an average size of 250-bp (SD for the distribution of inner leftover material not needed for diagnostic purposes: (i) distances between mate pairs is approximately 100 bp). RNA-seq immersion of tissue pieces in RNA Later (Life Technologies), reads were aligned to the human genome (GRCh37/hg19) using followed by storage at 80 C(forsubsequentRNA/DNA the software GSNAP version 2012-07-20 (PMID: 15728110). We assays); (ii) snap-freezing of tissue pieces in a cryovial partially counted the number of fragments mapped to each gene annotated immersed in liquid nitrogen (for subsequent protein assays); in the GENCODE database (version 18; PMID: 22955987) using (iii) immersion of fresh tissue in sterile RPMI1640 medium for HTSeq v0.5.3p3 (PMID: 25260700). The category of transcripts immediate transport to the laboratory (for preparation of used for our expression analysis is described at http://www. primary cell lines); (iv) frozen optimal cutting temperature gencodegenes.org/gencode_biotypes.html. We used DESeq2 to (OCT)-embedded tissue block (storage at 80 C); (v) forma- determine differential expression based on count values (10). lin-fixed, paraffin-embedded tissue block for sectioning (for Specifically, DESeq2 models the dispersion using empirical Bayes histopathology). shrinkage and tests whether, for a given gene, the fold change in

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expression strength between the two experimental conditions blocking in antibody diluent (DAKO #S3022). The primary significantly differs from zero using a Wald test. We also quan- antibody, mouse monoclonal anti-RACGAP from Abnova tified transcript abundances in Fragment Per Kilobase of Exon Per (M01), clone 1G6, was used at a final concentration of 2 mcg/mL Million (FPKM) by dividing the count by effective gene length incubated overnight at 4C. After washing, the anti-mouse sec- (derived from regions covered by reads). Only genes with average ondary antibody (DAKO Envisionþ Kit) was applied for 30 FPKM >1 across all samples were considered for differential minutes. Finally, slides were washed, and DAB detection was expression analysis. P values were corrected by FDR (11). Signif- done, followed by counterstaining with hematoxylin, dehydra- icant differences in gene expression between tumors and controls tion through graded alcohols and xylene, and mounting with were determined according to the following criteria: fold change > coverslip application. Concurrently, IHC staining of testis was 2 and FDR < 0.05. We performed overrepresentation analysis to used as a positive control, and omission of the primary antibody identify enriched pathways with the Ingenuity Pathway Analysis was used as a negative control. IHC staining was evaluated by the (IPA) and statistically significant gene ontology (GO) terms with study pathologist (T.M. Hebert), who evaluated and scored the DAVID (PMID: 19131956). We used all genes with expression cytoplasmic and nuclear staining intensity (0, 1, 2, or 3) and levels above FPKM >1 as the background list. A false discovery rate percentage of positive cells (0–100) for the entire tumor, for the (FDR) of 5% (q < 0.05) was used to interpret statistical carcinomatous component, and for the sarcomatous component. significance. An IHC H-score (product of staining intensity and percentage positive cells) was calculated separately for the cytoplasmic stain- Reverse transcriptase quantitative real-time PCR ing and for the nuclear staining of the entire tumor, and for each Reverse transcriptase quantitative real-time PCR was done individual component. similarly as described previously (12). In brief, complementary DNA was synthesized from 1 mg of total RNA using the SuperScript Cell proliferation and tumor growth assays VILO cDNA Synthesis Kit (Life Technologies). Quantitative real- Log-phase cells were seeded onto 6-well plates at 10,000 cells time PCR reactions were carried out using investigator-validated per well. Triplicate wells were collected and counted with a forward and reverse primers for the target genes (see Supplemen- Millipore Scepter at 24-hour intervals for 96 hours. Mean cell tary Table S6) and PowerSYBR Green (Life Technologies) detec- number at each time point was determined from at least two tion on a Realplex2 (Eppendorf). Target gene expression was independent experiments. To assess in vivo tumor growth, internally normalized to the mRNA expression of a housekeeping female athymic nude mice (Harlan) between 6 and 8 weeks gene, peptidylprolyl isomerase B (PPIB). Each qPCR reaction was run old were injected subcutaneously with 1 106 cells of the in triplicate on the same plate. Melting curve analysis was done to indicated cell lines (CS99-shRACGAP1 or CS99-shScramble). confirm a single amplicon corresponding to the PCR product size. Log-phase cells were collected, counted, and suspended in 100- Each assay plate included two reactions that omit either the mRNA mL Opti-MEM for injection. Tumor size was measured using template or the reverse transcriptase enzyme to exclude the digital calipers every 3 days and tumor volume calculated using possibility of contamination. Results were analyzed by the the formula: (length width2)/2. Animals were cared for as per DDC 2 t method to quantify the relative mRNA expression level. the Animal Welfare Act and the NIH "Guide for the Care and Use of Laboratory Animals." All animal experiments were done Immunoblotting with the approval of the Institutional Animal Care and Use Cell lysates were prepared from tissue samples by pulveri- Committee (Protocol 20130604) of the Albert Einstein College zation of snap-frozen tissue using a cryoPREP (Covaris), resus- of Medicine of Yeshiva University (Bronx, NY), under accred- pension in SDS lysis buffer, and protein quantitation by a itation by the Association for the Assessment and Accreditation modified Lowry method. Proteins were resolved by SDS-PAGE of Laboratory Animal Care. and transferred to nitrocellulose membranes (Bio-Rad). Block- ing was done for 30 minutes using 3% BSA in TBS with 0.1% Cytotoxicity assays Tween-20, prior to incubation with primary antibody overnight Cells were seeded into 96-well plates and treated with serial at 4C. The following primary antibodies were used: RACGAP1 dilutions of YM155 for 5 days (for primary cell lines) or 3 days (for mAb (M01), clone 1G6 (Abnova); survivin (FL-142) polyclonal CS99 cell lines) to approximate at least three doubling times. The antibody (Santa Cruz Biotechnology); GAPDH antibody (FL- Sulforhodamine B colorimetric assay was used to quantify cell 335; Santa Cruz Biotechnology); phospho-Stat3 Tyr 705 poly- number. IC50 (inhibitory concentration 50) values for YM155 clonal antibody (Cell Signaling Technology); Stat3 polyclonal were calculated for each cell line as the drug concentration (mean antibody (Cell Signaling Technology); and anti-a-Tubulin of at least two independent experiments) that decreases viable cell (DM1A) antibody (Sigma-Aldrich). The appropriate horserad- number by 50% compared with vehicle alone. ish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) was used, followedbyenhancedchemilumi- Cell-cycle analysis using propidium iodide and flow cytometry nescence detection (GE Healthcare). All films were scanned and Cells were harvested from the plate and single-cell suspen- saved in unmodified TIFF format. Densitometry was done using sions were made by passing cells through a polystyrene round- ImageJ software. bottom tube with cell strainer cap (BD Biosciences) three times. For cell-cycle analysis, cells were fixed with 70% cold ethanol RACGAP1 IHC for 1 hour, then stained with 20 mg/mL propidium iodide Formalin-fixed paraffin-embedded tissue sections were depar- (Sigma-Aldrich) and 100 mg/mL RNase (Thermo Fisher Scien- affinized and rehydrated. Antigen retrieval was done in DAKO tific) in PBS. Cell data was acquired on a FACSCanto (BD antigen retrieval solution at 95C for 30 minutes. Endogenous Biosciences) and analyzed using the cell-cycle module in Flow peroxidases were blocked with hydrogen peroxide, followed by Jo 9 (Tree Star).

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RACGAP1 Is a Metastatic Driver in Uterine Carcinosarcoma

Immunofluorescence Results Cells were fixed in in 4% paraformaldehyde for 20 minutes at Clinical characteristics of subjects and controls room temperature and permeabilized with 0.2% Triton X-100 for After IRB approval, 19 patients with suspected uterine carcino- 10 minutes and incubated in 1% BSA/PBS for 45 minutes at room sarcoma enrolled in this prospective study and underwent hys- temperature. The cells were incubated with anti-a-tubulin anti- terectomy and surgical staging from September 2011 to Novem- body for 45 minutes and then incubated with secondary antibody ber 2013. Of these, 13 patients had confirmed carcinosarcoma on for 30 minutes. After washing with 1% BSA/PBS, nuclei were final pathology; the other 6 patients had either high-grade carci- stained with DAPI. Images were acquired on a Zeiss AxioObserver noma or high-grade sarcoma on final pathology and were not CLEM microscope. included in the analysis. An additional 14th patient with con- fi Migration and invasion assays rmed uterine carcinosarcoma had tumor tissue available for To evaluate the ability of cells to migrate, the in vitro scratch analysis through her participation in the IRB-approved GYN tissue assay was done as described previously (13). Cells were photo- repository protocol. For 6 of the 14 patients with carcinosarcoma, graphed at 10 magnification on a phase-contrast microscope at nontumor tissue from histologically benign endometrium was 0, 24, and 48 hours, and the mean % wound closure at each time available and collected concurrently with the carcinosarcoma point was determined from three independent experiments. To tumor tissue. The 12 control patients were postmenopausal determine the ability of cells to invade, a modified Boyden women undergoing hysterectomy for benign indications and who chamber assay was used. Cell lines were starved overnight, consented for tissue collection of normal endometrial tissue counted, and suspended in assay media (serum-free RPMI). under an IRB-approved protocol. The clinical characteristics of Inserts with 8-mm pores (BD Biosciences) were placed into 24- the 14 carcinosarcoma cases and the 12 control normal postmen- well plates and covered with 0.1 mL of 200 mg/mL Matrigel Matrix opausal (NP) patients are described in Table 1. The cases and Growth Factor Reduced (BD Biosciences). Suspended cells (1 controls were similar in age and racial distribution. 104 cells) in 200 mL of serum-free RPMI were added to the top chamber. The bottom chamber contained RPMI with 10% FBS. Transcriptome analysis of uterine carcinosarcoma and normal After incubation for 18 hours at 37 C incubator with 5% CO2, endometrial samples noninvaded cells were removed with a cotton swab, and the cells Six tissue samples from each group were subjected to RNA-seq were fixed with 3.7% formaldehyde and stained with 0.5% crystal analysis. RNA-seq statistics were similar for carcinosarcoma cases violet, and counted. The percentage of invaded cells was normal- and NP controls (Table S1). The coefficient of variance (CV) was ized to the total cell number. low for the 6 NP control samples (0.17), indicating high repro- ducibility of the RNA-seq data. Despite heterogeneity among Statistical analysis carcinosarcoma tumor samples (CV ¼ 0.5 for 6 tumor samples), The number of biologically independent experiments is indicated unsupervised cluster analysis of transcriptomic expression in the figure legends. All statistical analyses were performed using resulted in two groups in which carcinosarcoma tumor samples GraphPad Prism 6. Means were compared using a two-tailed t test, or were clearly separated from NP controls (Fig. 1A). Shown in Fig. using a one-way ANOVA test with Tukey test when performing 1B, 3,425 genes were significantly differentially expressed with 2 multiple comparisons. c analysis was performed for comparison of FDR < 0.05 and fold change > 2. Of these, 2,005 genes were categorical variables between groups. Correlation analysis was done increased and 1,420 genes were decreased in carcinosarcoma using Pearson's product moment correlation. Statistical methods for relative to NP. The highest ranking up- and downregulated RNA-seq analysis are described separately; see above section "RNA canonical pathways in carcinosarcoma as identified by IPA are sequencing analysis and statistical analysis" for details. shown in Supplementary Table S2. Enriched GO terms were also

Table 1. Clinical and pathologic characteristics Carcinosarcoma NP control cases (N ¼ 14) (%) (N ¼ 11) (%) Age, mean (range) 67 (46-84) 62 (51–71) P ¼ 0.15, Unpaired t test Race, n (%) P ¼ 0.12, c2 test Black 7 (50.0) 2 (16.7) White, not Hispanic 2 (14.3) 5 (41.7) Hispanic 1 (7.1) 4 (33.3) Asian 1 (7.1) 0 (0.0) Multiracial 2 (14.3) 0 (0.0) Declined to identify 1 (7.1) 1 (8.3) Carcinosarcoma-FIGO Stage, n (%) I 5 (35.7) II 0 (0.0) III 5 (35.7) IV 4 (28.6) Carcinosarcoma-Sarcomatous component, n (%) Homologous 9 (64.3) Heterologous 5 (35.7) NP-Indication for surgery, n (%) Pelvic organ prolapse 9 (75.0) Other benign disease 3 (25.0)

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A B C Color key DEGs|FDR<0.05|FC>2

0.4 RACGAP1 -3 -2 -1 0123 1,000 Row Z-score *** 800

Height 600 400

0.0 0.2 200 RACGAP1 mRNA score Expression 0 Normal CS CS001 CS718 CS008 CS011 CS005 CS010 NP024 NP011 NP013 NP028 NP002 NP006

D Normal endometrial ssues Uterine CS ssues E Paired nontumor and CS tumor Cell line CS018 CS012 CS013 NT019 NT012 NT013 NT014 CS014 NT018 CS019 NT008 CS008 NP008 NP013 NP014 NP018 NP019 NP021 NP030 CS005 CS008 CS010 CS011 CS718 CS099 NP006 CS001 CS006

An-RACGAP1

An-GAPDH

FGH I

100

1.2 3 8 80 1.0 6 0.8 * * Scramble shRNA shRACGAP1-1 60 0.6 4 ** Level 0.4 *** *** *** 2 40 0.2

RACGAP1 mRNA 0.0 0 20 Cell number × 10 NA R h Normalized to mode 0 shRACGAP1-1shRACGAP1-2shRACGAP1-3 shRACGAP1-1shRACGAP1-2shRACGAP1-3 0 50K 100K 150K 200K 250K Scramble shRNA Scramble s shRACGAP1-2 shRACGAP1-3 PE-A

Figure 1. RACGAP1 expression is increased in uterine carcinosarcoma (CS) tissues. A, Cluster analysis of samples based on the transcriptomic expression in normal endometrial tissues and uterine carcinosarcomas. The bar indicates the average difference of correlation coefﬁcient between the samples. B, heatmap showing relative expression of genes that exhibited signiﬁcant change in gene expression between controls and cases at FDR < 0.05 and absolute fold change type¼"Other"> 2. C, RACGAP1 mRNA levels in carcinosarcoma tissue (n ¼ 9) and normal endometrial tissues (n ¼ 9), as determined by qRT-PCR. The horizontal bar depicts the mean RACGAP1 expression score in the normal and carcinosarcoma groups, respectively. , P < 0.001; two-tailed t test. D, RACGAP1 protein expression was high in most carcinosarcoma tissues and a cell line compared with normal endometrial tissues. E, RACGAP1 protein levels were increased in paired carcinosarcoma tumor tissues compared with adjacent nontumor tissues (NT). Cell growth and cell cycle analysis were performed after treating carcinosarcoma cells with RACGAP1 shRNA. F, RACGAP1 mRNA levels are shown 48 hours after transient RACGAP1 knockdown with three different shRNAs. Bars, mean SD of three independent experiments. shRACGAP1-1, -2, and -3 versus Scramble; , P < 0.001 by one-way ANOVA with Tukey test. G, cell viability was measured by counting the adherent cells 48 hours after transient RACGAP1 knockdown. The bars represent cell number 103 mean SD for three independent experiments. shRACGAP1-1, -3 versus Scramble: , P < 0.05, shRACGAP1-2 versus Scramble: , P < 0.01 by one-way ANOVA with Tukey test. H, following RACGAP1 knockdown, an increased number of binucleated cells are observed at 48 hours compared with Scramble, as shown by immunocytochemistry using an anti-a-tubulin antibody (red) to detect cytoplasm and DAPI stain (blue) to show nuclei. White arrows indicate binucleated cells. One representative experiment of two independent experiments is shown. I, compared with Scramble, RACGAP1 knockdown leads to cell-cycle accumulation in G2–M. After 48 hours, the percentage of cells in G2–M was 36.3% for Scramble cells compared with 50.3% and 47.7% for shRACGAP1-1 and -2, respectively. One representative experiment of two independent experiments is shown.

examined for differentially expressed genes using DAVID Bioin- expression in uterine carcinosarcoma compared with normal formatics. The highest ranking GO terms are shown in Supple- endometrium at the mRNA (Fig. 1C) and protein level (Fig. mentary Table S3. 1D), as determined by qRT-PCR and immunoblotting, using snap-frozen tissues from carcinosarcoma cases and NP endome- Exclusive overexpression of RACGAP1 in uterine trial control tissues. Analysis done for the 6 carcinosarcoma carcinosarcoma tissues patients with nontumor tissue (NT) available for comparison Among the genes with significantly increased expression in showed higher RACGAP1 protein expression in the carcinosar- uterine carcinosarcoma identified by RNA-seq, RACGAP1 over- coma tumor tissue relative to adjacent histologically benign expression was exclusively restricted to carcinosarcoma and was endometrium (Fig. 1E). IHC using a specific antibody to RAC- selected as the initial overexpressed gene for further study. We GAP1 showed strong nuclear localization of RACGAP1 in carci- confirmed increased RACGAP1 (also known as mgcracgap) nosarcoma cells (Supplementary Fig. S1). The RACGAP1 IHC

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H-score showed a strong linear correlation with the mRNA efficient RACGAP1 knockdown at the protein level in CS99- expression score, with a Pearson correlation coefficient >0.95 shRACGAP1 cells compared with CS99-shScramble cells (Fig. (Supplementary Fig. S1 C). 2A). The CS99-shRACGAP1 cells and CS99-shScramble cells showed similar cellular proliferation (Fig. 2B). However, in vivo Induction of cell-cycle arrest by RACGAP1 depletion tumor growth was significantly impaired in CS99-shRACGAP1 To interrogate the functional significance of RACGAP1 over- compared with CS99-shScramble cells (Fig. 2C). The wound- expression, we used the CS99 cell line and transfected three healing assay showed that CS99-shRACGAP1 cells were signifi- different short hairpin RNA (shRNA) vectors to knockdown RAC- cantly impaired in their ability to migrate compared with CS99- GAP1 or a control vector (scramble shRNA vector). Knockdown shScramble (Fig. 2D and E). CS99-shRACGAP1 cells also showed efficiency of approximately 80% at 48 hours was achieved using significantly reduced invasive capacity compared with shScramble each of the shRNAs (Fig. 1F). RACGAP1 knockdown significantly cells, as determined by Matrigel-coated Boyden chamber assay. decreased the viable cell number compared with control transfec- (Fig. 2F and G). tion at 48 hours (Fig. 1G). This correlated with the appearance of binucleated cells (10%) and the accumulation of cells in G2–M RACGAP1 expression in primary carcinosarcoma cell lines phase, indicating defective cytokinesis and cell-cycle arrest follow- predicts invasive capacity ing RACGAP1 knockdown (Fig. 1H and I). These effects are Primary carcinosarcoma cell lines were propagated from fresh consistent with the previously described role of RACGAP1 in the carcinosarcoma tumor tissue obtained at the time of surgical central spindle complex required for cytokinesis (14). resection. The origin of the carcinosarcoma primary cell lines was confirmed by comparison of the STR profile with the snap-frozen RACGAP1 is required for migration and invasion of primary tumor tissue (Supplementary Table S4). The protein carcinosarcoma cells expression of RACGAP1 in each the primary cell lines was deter- CS99 cells selected for stable RACGAP1 knockdown were mined by immunoblotting (Fig. 3A). The carcinosarcoma cell compared with control (scramble shRNA) cells. We confirmed lines with high endogenous RACGAP1 expression (CS008,

A D shScramble shRACGAP1 F ------0 h Scramble shRACGAP1 ------An-RACGAP1 shRACGAP1-1 An-GAPDH ------24 h ------Scramble ------B ------2,500,000 Scramble shRACGAP1-2 2,000,000 ShRACGAP1 ------1,500,000 48 h ------1,000,000 ------

Cell number 500,000 ------0 0 24 48 72 96 Hours C E * G

) CS99-shRACGAP1 3 ** ShRACGAP1 4 800 CS99-shScrambled 100 Scramble 600 75 3 ** 400 50 2 ** 1 200 25 0 Wound (%) closure 0 Tumor volume (mm 0 46810 13 15 17 0 24 48 Time (h) Days aer cell injecon Scramble Invading cells normalized to total (%) ShRACGAP1-1 ShRACGAP1-2

Figure 2. RACGAP1 knockdown in CS99 uterine carcinosarcoma cells. A, stable transfection of shRACGAP1 in CS99 cells decreased protein levels of RACGAP1, as shown by Western blotting. One Western blot of two independent experiments is shown. Representative data from shScramble and shRACGAP1 is shown in panels B–G. B, the cell growth curves of CS99-shScramble cells and CS99-shRACGAP1 cells are similar. Each data point shows the cell number (mean SD of two experiments). C, combined data from two independent experiments are shown depicting xenograft growth following subcutaneous injection of the indicated cell lines into athymic nude mice, N ¼ 14 per group. CS99-shRACGAP1 xenograft growth was signiﬁcantly reduced compared with CS99-shScrambled xenograft growth. , P < 0.01; , P < 0.001, by two-tailed t test. D, a uniform scratch was made in 95%–100% conﬂuent monolayer cultures of CS99-shScramble and CS99-shRACGAP1. Wound closure was monitored under phase-contrast microscopy and photographed (10) at 0, 24, and 48 hours. Representative images of three independent experiments are shown. E, CS99-shRACGAP1 cells show retardation of wound closure. The mean SD of three independent experiments is shown. , P < 0.05; , P < 0.01 by two-tailed t test. F, representative images of crystal violet stained CS99-shScramble and CS99-shRACGAP1 invading into Matrigel-coated inserts of the Boyden chambers. G, the graph shows the percentage of invading cells normalized to the total cell number. The bars represent the mean SD of three independent experiments. , P < 0.01, one-way ANOVA with Tukey test.

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A C CS008 CS013 CS017 CS019

An-RACGAP1 2.5 ns Figure 3. RACGAP1 expression correlates with invasive 2.0 An-GAPDH ability in primary carcinosarcoma cell lines. 1.5 A, RACGAP1 protein expression in four unique patient-derived primary cell lines is 1.0 ** ** shown by immunoblotting. B, representative 0.5 images of crystal violet–stained primary carcinosarcoma cells that have invaded into B 0.0 CS008 CS013 CS017 CS019 Matrigel-coated inserts of the Boyden chambers. C, the percentage of invading cells normalized to total cell number is shown for each cell line. Each bar represents the mean Percentage of invasive cells normalized total (%) CS008 CS013 SEM of two independent experiments. , P < 0.01; , P < 0.001; one-way ANOVA with Tukey multiple comparisons test. ns, not signiﬁcant.

CS017 CS019

CS019) showed significantly increased invasiveness compared RACGAP1 overexpression promotes invasiveness of with carcinosarcoma cell lines with low RACGAP1 expression endometrial cells (CS013, CS017), as determined using a Matrigel-coated Bowden Next, the effect of RACGAP1 overexpression was evaluated in chamber assay (Figs. 3B and C). the endometrioid adenocarcinoma cell line Hec1b. Expression of full-length RACGAP1 following transfection was confirmed by RACGAP1 regulates STAT3 phosphorylation and survivin immunoblotting (Fig. 5A). The invasive capacity of Hec1b/RAC- expression in uterine carcinosarcoma GAP1–transfected cells was significantly increased compared with On the basis of a prior report that RACGAP1 facilitates the Hec1b/empty vector–transfected cells (Fig. 5B). These data sug- Tyr705 phosphorylation of STAT3 and promotes its translocation gest that increased RACGAP1 expression is sufficient to promote to the nucleus (15), we evaluated the relationship of RACGAP1, invasive ability in this cell line model of endometrioid endome- phospho-STAT3 (Y705), and total STAT3 in uterine carcinosar- trial cancer, which is the more common and less aggressive BIRC5 coma (Fig. 4A). The expression of survivin (encoded by the histologic type of uterine corpus cancer. gene, baculoviral inhibitor of apoptosis repeat containing 5), a transcriptional target of STAT3, was also evaluated by immuno- Correlation of RACGAP1 and survivin expression in uterine blotting and qRT-PCR. In carcinosarcoma tumor tissues, RACGAP1 carcinosarcoma expression was significantly correlated with levels of phospho- To validate our findings in an independent cohort, we obtained STAT3 (Fig. 4B) and with survivin expression (Fig. 4C). Further- deidentified clinical data and gene expression data from the TCGA more, we found that RACGAP1 knockdown decreased phosphor- uterine carcinosarcoma cohort (N ¼ 57 patients). First, we deter- ylation of STAT3 and reduced survivin expression in CS99 cells mined the correlation of RACGAP1 and survivin (BIRC5) in these (Fig. 4D). On the basis of these data, STAT3 and survivin are bona patients. As in our institutional cohort, RACGAP1 and survivin fide downstream targets of RACGAP1 in uterine carcinosarcoma. mRNA levels were significantly positively correlated in the TCGA cohort (Fig. 5C). The difference in the absolute correlation coef- fi RACGAP1 expression is a biomarker of sensitivity to anti- cient of RACGAP1 and survivin in our institutional cohort versus survivin therapy the multi-institutional TCGA cohort could be related to the YM155 is a first-in-class small molecule that selectively sup- different method of measuring mRNA levels. In the TCGA cohort, presses survivin at the mRNA and protein level (16, 17). Results of mRNA expression levels of RACGAP1 and survivin were deter- cytotoxicity assays showed that YM155 potently inhibited the mined by RNA-seq on an Illumina HiSeq platform. In the corre- proliferation of primary and established carcinosarcoma cell lation analysis for our institutional cohort, mRNA levels were fi lines, with IC concentrations ranging from 1.9 nmol/L (for quanti ed by qRT-PCR, using optimized primers and linear 50 fi CS99) to 24.3 nmol/L (for CS017; Supplementary Table S5). ampli cation conditions, as well as internal normalization. Target inhibition was confirmed by immunoblotting that showed reduced survivin protein expression at these YM155 concentra- Increased risk of extrauterine disease in patients with high tions (Supplementary Fig. S2). The IC50 concentrations inversely RACGAP1-expressing carcinosarcoma correlated with RACGAP1 and survivin mRNA expression levels As our data showed RACGAP1 regulates the migratory and inva- (Fig. 4E and F). On the basis of these data, high RACGAP1 sive behavior of carcinosarcoma cells, we hypothesized that patients expression predicts sensitivity to anti-survivin therapy. The effect with high RACGAP1-expressing tumors would be more likely to of YM155 on the invasiveness of CS99 cells was determined using present with higher stage of disease (indicating metastatic spread) the Matrigel-coated Boyden chamber assay (Fig. 4G). In a dose- compared with patients with low RACGAP1–expressing tumors. dependent fashion, YM155 significantly reduced the invasive Analysis of data from an independent cohort of 57 carcinosarcoma capacity of CS99 cells (Fig. 4H). patients from the TCGA was done to evaluate the potential

4682 Clin Cancer Res; 22(18) September 15, 2016 Clinical Cancer Research

RACGAP1 Is a Metastatic Driver in Uterine Carcinosarcoma

ABC D

Carcinosarcoma ssues r = 0.91** 2.0 r = 0.71* 100 80 1.5 Scramble 60 ShRACGAP1 CS001 CS005 CS006 CS008 CS010 CS011 CS718 line CS099 Cell 1.0 40 An-RACGAP1 RACGAP1 0.5 20 An-p-STAT3 (Y705) Survivin expression p-STAT3 (Y705) p-STAT3 Expression 0.0 0 0.0 0.5 1.0 1.5 020406080 RACGAP1 Expression RACGAP1 Expression An-STAT3 (total) STAT3 (total) An-Survivin Survivin An-GAPDH

GAPDH G

YM155 0 nmol/L 1 nmol/L 5 nmol/L

EF 30 r = –0.76* 30 r = –0.75* H 20 20 (nmol/L) (nmol/L) 4 YM155 YM155 0 nmol/L 50 10 50 IC

IC 1 nmol/L

10 (%) 3 * 5 nmol/L 2 * 0 0 100 200 300 400 200 400 600 800 1,000 1

Survivin expression score cells to total RACGAP1 Expression score 0 Invading cells cells Invading normalized

0 nmol/L 1 nmol/L 5 nmol/L YM155

Figure 4. RACGAP1 expression correlates with p-STAT3 and Survivin expression. A, RACGAP1 protein expression was positively correlated to p-STAT3 and Survivin expression in carcinosarcoma clinical samples, as shown by immunoblotting of tumor lysates. B, positive correlation of RACGAP1 protein expression and phospho-STAT3(Y705) protein expression in clinical samples, as determined by ImageJ quantification of immunoblot images (shown in A). Internal normalization to GAPDH protein expression was done. The Pearson correlation coefficient, r ¼ 0.71; , P < 0.05. C, positive correlation of RACGAP1 and survivin mRNA expression, as determined by qRT-PCR using RNA isolated from the carcinosarcoma samples shown in A. Internal normalization for survivin mRNA expression to PPIB mRNA expression was done. The Pearson correlation coefficient, r ¼ 0.91; , P < 0.01. D, CS99-shRACGAP1 knockdown cells have decreased expression of phosphorylated STAT3 and survivin compared with CS99-shScrambled cells. One of two representative immunoblots is shown. E, for each carcinosarcoma primary cell line (n ¼ 7) and CS99, the IC50 of YM155 is plotted versus the RACGAP1 mRNA expression score determined by qRT-PCR (see Supplementary Table S5), showing that RACGAP1 mRNA expression negatively correlates with IC50 concentration; Pearson correlation coefficient, r ¼0.76; , P < 0.05. This indicates greater sensitivity to survivin inhibition in the higher RACGAP1-expressing cell lines. F, the IC50 of YM155 is plotted versus the Survivin mRNA expression score. A negative correlation was observed, similar to that observed in A; Pearson correlation coefficient, r¼0.75; , P < 0.05. G, effect of YM155 on CS99 cell invasion was evaluated by Boyden chamber assay; representative images of cells invading the Matrigel-coated inserts are shown. YM155 treatment decreased cell invasion. H, the quantification of invading cells into the Matrigel-coated inserts normalized to total cell number. The bars represent the mean SD of two independent experiments. , P < 0.05 versus untreated cells, One-way ANOVA with Tukey test. relationship of RACGAP1 and stage. As shown in Fig. 5D, patients carcinosarcoma cells, while RACGAP1 overexpression confers a with higher stage of disease (indicating cancer spread beyond the metastatic phenotype to endometrial adenocarcinoma cells. In uterus) had significantly higher RACGAP1 mRNA expression com- addition, we have found that high RACGAP1 expression is signif- pared with patients without spread of disease. These findings in an icantly correlated with extrauterine spread of disease in patients, independent cohort of patients withcarcinosarcoma support the role substantiating its clinical relevance in uterine carcinosarcoma. of RACGAP1 as a metastatic driver in this disease, consistent with the Our data show that RACGAP1 is a key regulator of STAT3 findings in our cell line models of carcinosarcoma. phosphorylation and survivin expression in uterine carcinosarcoma cells, consistent with a prior observation that RACGAP1 can function as a nuclear chaperone for STAT3 (15). In uterine Discussion carcinosarcoma cells, we found that RACGAP1 depletion reduces To our knowledge, this study is the first to apply RNA-seq STAT3 phosphorylation and survivin expression. Analysis of two technology to compare clinical tumor samples of uterine carcino- independent cohorts of patients with uterine carcinosarcoma sarcoma, a highly aggressive malignancy, with normal endometrial showed that RACGAP1 and survivin expression are significantly tissue.Usingthisapproach,wehaveidentifiedRACGAP1asacritical positively correlated in tumor samples. upregulated gene and driver of the metastatic phenotype in uterine On the basis of these novel findings, we evaluated the thera- carcinosarcoma. In functional assays, we show that RACGAP1 peutic potential of a first-in-class survivin inhibitor YM155 using depletion abolishes the migratory and invasive capacity of uterine primary carcinosarcoma cell lines developed from human

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Mi et al.

A Hec1B Hec1B B EV RACGAP1 4 Hec1B/EV ** Hec1B/RACGAP1 An-RACGAP1 3 2

An-GAPDH 1 Invading cells (%) 0

Hec1B/EV

Hec1B/RACGAP1

C D 3,000 6,000 * r = 0.31*

4,000 2,000 Birc5 racgap1 2,000 1,000

0 0 0 2,000 4,000 6,000 I II–IV racgap1

Figure 5. Constitutive expression of RACGAP1 increases invasive capacity of Hec1B endometrial carcinoma cells. A, RACGAP1 protein levels, detected by immunoblotting, are increased at 72 hours following transfection with full-length RACGAP1 compared with empty vector (EV) transfection. One of two independent experiments is shown. B, invasive capacity was measured by Matrigel-coated Bowden chamber assay done 48 hours after transfection with full-length RACGAP1 or EV. The percentage of invading cells, normalized to total cell number, is shown in the bar graph depicting mean (%)SEM of three independent experiments. , P < 0.01, paired two-tailed t test (RACGAP1 vs. EV). C, RACGAP1 expression in the TCGA cohort of uterine carcinosarcoma (N ¼ 57). RACGAP1 and BIRC5 (survivin) mRNA expression were positively correlated in this independent cohort of 57 patients with carcinosarcoma; Pearson correlation coefﬁcient, r ¼ 0.31; , P < 0.05. D, RACGAP1 expression and higher stage of disease in the TCGA cohort of uterine carcinosarcoma (n ¼ 57). Patients with cancer spread beyond the uterine corpus (FIGO stages II–IV; right side) had signiﬁcantly higher RACGAP1 expression compared with patients with non-metastatic disease (FIGO stage I; left side). The box and whiskers plot depicts the median, quartiles, and 10th–90th percentile of RACGAP1 expression for each group. , P < 0.05, unpaired two-tailed t test (stage II–IV vs. stage I).

carcinosarcoma tumors. YM155 is a first-in-class inhibitor of (21, 22). The antitumor activity of YM155 in patients with uterine survivin that shows promising therapeutic activity in preclinical carcinosarcoma awaits future investigation. and clinical trials. For many targeted therapies, overexpression of RACGAP1, also known as MgcRacGAP, was initially identified the target (in this case, survivin) correlates with increased sensitivity as a GTPase-activating protein (GAP) expressed in testis and male to the therapeutic agent. Recently, survivin overexpression was germ cells (23). Unlike other GAPs, RACGAP1 is an essential identified in anaplastic thyroid cancer; the authors performed a component of the centralspindlin complex (with the kinesin high-throughput screen of >3,000 drugs and identified the survivin KlF23), where its GAP activity is required for cytokinesis and inhibitor YM155 as one of the most active agents in that aggressive inactivation of Rac1 at the cleavage furrow (24–28). RACGAP1 disease (18). In our studies, we found that YM155 showed potent also regulates tethering of the mitotic spindle to the plasma antiproliferative activity in primary and established uterine carci- membrane during cytokinesis (29). Apart from its role in cyto- nosarcoma lines and moreover, abolished the invasive capacity of kinesis, RACGAP1 was shown to regulate STAT3 activation in carcinosarcoma cells. Furthermore, the expression levels of RAC- leukemia cells (30), and may also play a role in regulating GAP1 and survivin predicted sensitivity to YM155 in primary endothelial permeability (31). Recently, RACGAP1 expression carcinosarcoma cell lines. Phase I trials have shown that YM155 has been linked with aggressive clinical behavior in several can- is well-tolerated with a very favorable safety profile (19, 20). Phase cers, including colorectal cancer (32), hepatocellular carcinoma II trials in several disease sites have demonstrated single-agent (33, 34), gastric cancer (35), meningiomas (36), and breast cancer activity, for example, in patients with advanced, refractory non– (37). Thus, our findings may have broader relevance for targeting small cell lung cancer and castration-resistant prostate cancer the metastatic phenotype in diverse tumor types.

4684 Clin Cancer Res; 22(18) September 15, 2016 Clinical Cancer Research

RACGAP1 Is a Metastatic Driver in Uterine Carcinosarcoma

Very little has been previously published regarding underlying Acquisition of data (provided animals, acquired and managed patients, drivers of the development and progression of this disease, and provided facilities, etc.): S. Mi, J. Brouwer-Visser, J. Heim, T.M. Hebert, there is a dire lack of effective therapies for this highly lethal M.J. Gunter, G.S. Huang Analysis and interpretation of data (e.g., statistical analysis, biostatistics, disease. This knowledge gap is one of the motivators for the computational analysis): S. Mi, M. Lin, J. Brouwer-Visser, D. Smotkin, current study, which has led to our discovery of a key metastatic M.J. Gunter, G.L. Goldberg, D. Zheng, G.S. Huang driver and identification of a promising, novel targeted therapy. In Writing, review, and/or revision of the manuscript: S. Mi, M. Lin, J. Heim, summary, RACGAP1 promotes the metastatic phenotype in uter- D. Smotkin, M.J. Gunter, G.L. Goldberg, D. Zheng, G.S. Huang ine carcinosarcoma via a STAT3/survivin signaling pathway. The Administrative, technical, or material support (i.e., reporting or organizing survivin inhibitor YM155 potently suppresses carcinosarcoma cell data, constructing databases): M. Lin, G.S. Huang Study supervision: G.S. Huang proliferation and abrogates the invasive capacity of carcinosar- – coma cells. We suggest that inhibition of the RACGAP1 STAT3 Acknowledgments survivin pathway should be investigated as a novel therapeutic The results shown are in part based upon data generated by the TCGA strategy for this lethal malignancy, as well as for other high Research Network: http://cancergenome.nih.gov/. RACGAP1–expressing cancers. Grant Support Disclosure of Potential Conflicts of Interest This work was supported by the Albert Einstein Cancer Center Support Grant fl No potential con icts of interest were disclosed. of the NIH under award number P30CA013330 and Albert Einstein Cancer Disclaimer Center Pilot Award (to G.S. Huang). The costs of publication of this article were defrayed in part by the The content is solely the responsibility of the authors and does not neces- payment of page charges. This article must therefore be hereby marked fi sarily represent the of cial views of the National Institutes of Health. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: S. Mi, J. Heim, D. Smotkin, G.L. Goldberg, G.S. Huang Development of methodology: S. Mi, M. Lin, J. Brouwer-Visser, D. Zheng, Received August 29, 2015; revised March 9, 2016; accepted April 4, 2016; G.S. Huang published OnlineFirst April 27, 2016.

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4686 Clin Cancer Res; 22(18) September 15, 2016 Clinical Cancer Research

RNA-seq Identification of RACGAP1 as a Metastatic Driver in Uterine Carcinosarcoma

Shijun Mi, Mingyan Lin, Jurriaan Brouwer-Visser, et al.

Clin Cancer Res 2016;22:4676-4686. Published OnlineFirst April 27, 2016.

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