rna-seq identification of racgap1 as a metastatic driver in … · twelve rna-seq libraries (cs001,...

Cancer Therapy: Preclinical

RNA-seq Identification of RACGAP1 as aMetastatic Driver in Uterine CarcinosarcomaShijun Mi1, Mingyan Lin2, Jurriaan Brouwer-Visser1, Jennifer Heim1, David Smotkin1,TiffanyHebert3,Marc J.Gunter4,Gary L.Goldberg1,5, DeyouZheng2,6, andGloria S. Huang1,5,7

Abstract

Purpose:Uterine carcinosarcoma is a rare aggressivemalignan-cy frequently presenting at advanced stage of disease with extra-uterinemetastases.Median survival is less than 2 years due to highrelapse rates after surgery and poor response to chemotherapy orradiotherapy. The goal of this study was to identify novel ther-apeutic targets.

Experimental Design: We applied RNA-seq analysis to pro-spectively collected uterine carcinosarcoma tumor samplesfrom patients undergoing primary surgical resection and forcomparison, normal endometrial tissues from postmenopaus-al women undergoing hysterectomy for benign indications.Functional assays were done in primary carcinosarcoma celllines developed from patients and in established cell lines,as well as a cell line–derived xenograft model. Validationwas done by analysis of an independent cohort of patients

with uterine carcinosarcoma from The Cancer Genome Atlas(TCGA).

Results: RacGTPase–activating protein 1 (RACGAP1)was iden-tified to be highly upregulated in uterine carcinosarcoma. Func-tional assays showed that RACGAP1 mediates motility and inva-sion via regulation of STAT3 phosphorylation and survivin expres-sion. RACGAP1 depletion or survivin inhibition abrogated motil-ity and invasiveness of carcinosarcoma cells, while RACGAP1overexpression conferred invasiveness to endometrial adenocarci-noma cells. In the TCGA cohort, RACGAP1 expression correlatedwith survivin expression and extrauterine spread of disease.

Conclusions: The RACGAP1–STAT3–survivin signaling path-way is required for the invasive phenotype of uterine carcinosar-coma and is a newly identified therapeutic target in this lethaldisease. Clin Cancer Res; 1–11. �2016 AACR.

IntroductionUterine carcinosarcoma, also known as malignant mixed

M€ullerian tumors, is a highly aggressive form of uterine cancer,with a propensity for extrauterine metastases and a high casefatality rate (1). The histologic diagnosis is based on the presenceof bothmalignant carcinomatous and sarcomatous elements (2).Prior studies support a monoclonal endometrial origin of bothelements, supporting the view of uterine carcinosarcoma as a

metaplastic carcinoma that has undergone epithelial-to-mesen-chymal transition (3).

These uncommon tumors, which represent < 5% of uterinecorpus cancers, account for greater than 15% of uterine cancer–related deaths (1, 4). The disease usually affects postmenopausalwomen, with a median age at diagnosis of 62 to 67 years. Theincidence is significantly higher in black women with an age-adjusted incidence rate of 4.3 per 100,000 compared with 1.7 per100,000 in white women (5). Previously identified risk factors forthe development of uterine carcinosarcoma include obesity,nulliparity, and exogenous estrogen or tamoxifen exposure (6).

Treatment of uterine carcinosarcoma is primarily surgical.Approximately one-third of patients have disease spread beyondthe uterus at the time of diagnosis, and the recurrence rate aftersurgery exceeds 50%. Combination chemotherapy may improveoverall and progression-free survival in patients with advancedstage or recurrent carcinosarcoma (7, 8). Despite multimodaltreatment approaches, the median overall survival is approxi-mately 21 months, and in patients with advanced disease, lessthan one year (1).

The underlying molecular drivers of the aggressive phenotypeof uterine carcinosarcoma have not been identified. Poor clinicalprognostic factors include higher FIGO stage of disease and serumCA125 elevation (6). In this study,we appliedRNA-seq analysis toprospectively collected uterine carcinosarcoma tumor samplesfrom patients undergoing primary surgical resection and forcomparison, normal endometrial tissues from postmenopausalwomenundergoing hysterectomy for benign indications, with thegoal of identifying novel oncogenic drivers and potential thera-peutic targets.

1Division of Gynecologic Oncology, Department of Obstetrics andGynecology, Albert Einstein College ofMedicine andMontefioreMed-ical Center, Bronx, NewYork. 2Department ofGenetics, Albert EinsteinCollege of Medicine, Bronx, New York. 3Department of Pathology,Albert Einstein College of Medicine and Montefiore Medical Center,Bronx, New York. 4Section of Nutrition and Metabolism, InternationalAgency for Research on Cancer, Lyon, France. 5Albert Einstein CancerCenter, Albert EinsteinCollegeofMedicine, Bronx, NewYork. 6Depart-ments of Neurology and Neuroscience, Albert Einstein College ofMedicine, Bronx, NewYork. 7Department of Molecular Pharmacology,Albert Einstein College of Medicine, Bronx, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

S. Mi and M. Lin contributed equally to this article.

Corresponding Author: Gloria S. Huang, Division of Gynecologic Oncology,Department of Obstetrics & Gynecology and Women's Health, MontefioreMedical Center and Albert Einstein College of Medicine, 1695 Eastchester Road,Suite 601, Bronx, NY 10461. Phone: 718-405-8082; Fax: 718-405-8087; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-15-2116

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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We found that RACGAP1 is a highly upregulated gene inuterine carcinosarcoma, and its overexpression promotes themetastatic phenotype. We identified STAT3 and survivin asbona fide downstream targets of RACGAP1, and showed thatRACGAP1 is a critical regulator of STAT3 phosphorylation andsurvivin expression in uterine carcinosarcoma. Targeting RAC-GAP1 directly, or its downstream effectors, significantly dimin-ished the invasive capacity of uterine carcinosarcoma cells.Analysis of an independent cohort of 57 patients with uterinecarcinosarcoma showed that high RACGAP1 predicted extra-uterine metastasis, validating its clinical significance as a met-astatic driver. Thus, we have identified a novel molecular driverof the metastatic phenotype of uterine carcinosarcoma, sug-gesting new therapeutic approaches for targeting this and otherhighly aggressive cancers.

Materials and MethodsTissue acquisition

Under Institutional review board (IRB) approval, carcino-sarcoma tumor samples (IRB# 2011-404) and normal endo-metrial tissues (IRB#2009-406) were prospectively collectedfrom consenting patients undergoing surgery at MontefioreMedical Center, and the corresponding clinical data recorded.The study coordinator assigned a unique study ID to tissuesamples, by which samples were identified in the laboratory. Inconjunction with surgical pathology, the standard operatingprocedure was followed for prospective tissue collection ofleftover material not needed for diagnostic purposes: (i)immersion of tissue pieces in RNA Later (Life Technologies),followed by storage at �80�C (for subsequent RNA/DNAassays); (ii) snap-freezing of tissue pieces in a cryovial partiallyimmersed in liquid nitrogen (for subsequent protein assays);(iii) immersion of fresh tissue in sterile RPMI1640 medium forimmediate transport to the laboratory (for preparation ofprimary cell lines); (iv) frozen optimal cutting temperature(OCT)-embedded tissue block (storage at �80�C); (v) forma-lin-fixed, paraffin-embedded tissue block for sectioning (forhistopathology).

Primary cell line isolation and tissue cultureFor isolation of primary cell lines, under an IRB-approved

protocol (#2011-404), fresh tumor tissue from patients under-going hysterectomy and staging surgery for histologically con-firmed uterine carcinosarcoma was transported to the laboratoryin sterile RPMI tissue culture media. Following mechanical dis-sociation, enzymatic digestion with 3 mg/mL collagenase A(Roche) and 150 mg/mL DNase 1 (Thermo Fisher Scientific), andred blood cell lysis (eBioscience), cells were washed with RPMIwith FBS (10%), and resuspended in F-media supplemented asdescribed previously (9). Cells were seeded in tissue culture flasksand media replaced as needed. Cell line authentication was doneby short tandem repeat (STR) profiling using the Genemarker 10kit (Promega) and matching to the original carcinosarcomapatient samples (Supplementary Table S4).

CS99 cells and their derivatives were maintained as subcon-fluent monolayer cultures in RPMI1640 containing 10% FBS at37�C with 5% CO2.

All cells were routinely screened with MycoAlert (Lonza) andwere negative for mycoplasma.

RNA isolationTo extract RNA, frozen tissues were pulverized in a tissueTUBE

bag (Covaris) using a cryoPREP (Covaris) and then homogenizedin Buffer RLT (Qiagen) using a Covaris adaptive focused acousticstissue disrupter. The Qiagen AllPrep kit was used following themanufacturer's instructions. The RNA concentration and puritywas measured using the Nanodrop spectrophotometer (ThermoFisher Scientific), and RNA integrity was evaluated with theAgilent Bioanalyzer (Agilent). RNA quality was uniformly excel-lent andmet the following criteria;Nanodrop, 260/280 ratio>1.8;Agilent Bioanalyzer, RIN > 7.

Paired-end library preparation and Illumina sequencingTwelve RNA-seq libraries (CS001, CS005, CS008, CS010,

CS011, CS718, NP002, NP006, NP011, NP013, NP024, andNP028) were prepared for paired-end sequencing using theIllumina HiSeq platform in the epigenetics core facility of theAlbert Einstein College of Medicine according to directionalwhole transcript seq protocol described on WASP (wiki-basedautomated sequence processor, http://wasp.einstein.yu.edu). Inbrief, the purified cDNA library products were evaluated using theAgilent bioanalyzer and diluted to 10 nmol/L for cluster gener-ation in situ on the HiSeq paired-end flow cell using the cBotautomated cluster generation system followed by massively par-allel sequencing (2�100 bp) on HiSeq 2000.

RNA-seq analysis and enrichment analysisWe obtained 92-bp mate-paired reads from DNA fragments

with an average size of 250-bp (SD for the distribution of innerdistances between mate pairs is approximately 100 bp). RNA-seqreads were aligned to the human genome (GRCh37/hg19) usingthe software GSNAP version 2012-07-20 (PMID: 15728110). Wecounted the number of fragmentsmapped to each gene annotatedin the GENCODE database (version 18; PMID: 22955987) usingHTSeq v0.5.3p3 (PMID: 25260700). The category of transcriptsused for our expression analysis is described at http://www.gencodegenes.org/gencode_biotypes.html. We used DESeq2 todetermine differential expression based on count values (10).Specifically, DESeq2models the dispersion using empirical Bayesshrinkage and tests whether, for a given gene, the fold change in

Translational Relevance

Uterine carcinosarcoma is a highly aggressive endometrialmalignancy that causes a disproportionate number of deathsfrom uterine cancer. By comparing the gene expression ofuterine carcinosarcoma with benign endometrial tissue, RacGTPase–activating protein 1 (RACGAP1) was identified to behighly upregulated, and functional studies showed that RAC-GAP1 regulates motility and invasion via STAT3–survivinsignaling. Analyzing an independent cohort from The CancerGenome Atlas showed that patients with more advanceduterine carcinosarcoma had significantly higher RACGAP1expression in their tumors correlated with increased survivinexpression. Furthermore, RACGAP1 expression predicted sen-sitivity to survivin-targeted therapy in primary cell linesderived from patients. On the basis of these novel findings,the RACGAP1–STAT3–survivin signaling pathway is identifiedas a promising therapeutic target in uterine carcinosarcoma, ahighly lethal disease of the female reproductive tract.

Mi et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF2




expression strength between the two experimental conditionssignificantly differs from zero using a Wald test. We also quan-tified transcript abundances in Fragment Per Kilobase of Exon PerMillion (FPKM) by dividing the count by effective gene length(derived from regions covered by reads). Only genes with averageFPKM >1 across all samples were considered for differentialexpression analysis. P values were corrected by FDR (11). Signif-icant differences in gene expression between tumors and controlswere determined according to the following criteria: fold change >2 and FDR < 0.05. We performed overrepresentation analysis toidentify enriched pathways with the Ingenuity Pathway Analysis(IPA) and statistically significant gene ontology (GO) terms withDAVID (PMID: 19131956). We used all genes with expressionlevels above FPKM >1 as the background list. A false discovery rate(FDR) of 5% (q < 0.05) was used to interpret statisticalsignificance.

Reverse transcriptase quantitative real-time PCRReverse transcriptase quantitative real-time PCR was done

similarly as described previously (12). In brief, complementaryDNAwas synthesized from1mgof total RNAusing the SuperScriptVILO cDNA Synthesis Kit (Life Technologies). Quantitative real-time PCR reactions were carried out using investigator-validatedforward and reverse primers for the target genes (see Supplemen-tary Table S6) and PowerSYBR Green (Life Technologies) detec-tion on a Realplex2 (Eppendorf). Target gene expression wasinternally normalized to themRNA expression of a housekeepinggene, peptidylprolyl isomerase B (PPIB). Each qPCR reaction was runin triplicate on the same plate. Melting curve analysis was done toconfirm a single amplicon corresponding to the PCRproduct size.Each assayplate included two reactions that omit either themRNAtemplate or the reverse transcriptase enzyme to exclude thepossibility of contamination. Results were analyzed by the2�DDCt method to quantify the relative mRNA expression level.

ImmunoblottingCell lysates were prepared from tissue samples by pulveri-

zation of snap-frozen tissue using a cryoPREP (Covaris), resus-pension in SDS lysis buffer, and protein quantitation by amodified Lowry method. Proteins were resolved by SDS-PAGEand transferred to nitrocellulose membranes (Bio-Rad). Block-ing was done for 30 minutes using 3% BSA in TBS with 0.1%Tween-20, prior to incubation with primary antibody overnightat 4�C. The following primary antibodies were used: RACGAP1mAb (M01), clone 1G6 (Abnova); survivin (FL-142) polyclonalantibody (Santa Cruz Biotechnology); GAPDH antibody (FL-335; Santa Cruz Biotechnology); phospho-Stat3 Tyr 705 poly-clonal antibody (Cell Signaling Technology); Stat3 polyclonalantibody (Cell Signaling Technology); and anti-a-Tubulin(DM1A) antibody (Sigma-Aldrich). The appropriate horserad-ish peroxidase–conjugated secondary antibody (Santa CruzBiotechnology) was used, followed by enhanced chemilumi-nescence detection (GE Healthcare). All films were scanned andsaved in unmodified TIFF format. Densitometry was done usingImageJ software.

RACGAP1 IHCFormalin-fixed paraffin-embedded tissue sections were depar-

affinized and rehydrated. Antigen retrieval was done in DAKOantigen retrieval solution at 95�C for 30 minutes. Endogenousperoxidases were blocked with hydrogen peroxide, followed by

blocking in antibody diluent (DAKO #S3022). The primaryantibody, mouse monoclonal anti-RACGAP from Abnova(M01), clone 1G6, was used at a final concentration of 2 mcg/mLincubated overnight at 4�C. After washing, the anti-mouse sec-ondary antibody (DAKO Envisionþ Kit) was applied for 30minutes. Finally, slides were washed, and DAB detection wasdone, followed by counterstaining with hematoxylin, dehydra-tion through graded alcohols and xylene, and mounting withcoverslip application. Concurrently, IHC staining of testis wasused as a positive control, and omission of the primary antibodywas used as a negative control. IHC staining was evaluated by thestudy pathologist (T.M. Hebert), who evaluated and scored thecytoplasmic and nuclear staining intensity (0, 1, 2, or 3) andpercentage of positive cells (0–100) for the entire tumor, for thecarcinomatous component, and for the sarcomatous component.An IHC H-score (product of staining intensity and percentagepositive cells) was calculated separately for the cytoplasmic stain-ing and for the nuclear staining of the entire tumor, and for eachindividual component.

Cell proliferation and tumor growth assaysLog-phase cells were seeded onto 6-well plates at 10,000 cells

per well. Triplicate wells were collected and counted with aMillipore Scepter at 24-hour intervals for 96 hours. Mean cellnumber at each time point was determined from at least twoindependent experiments. To assess in vivo tumor growth,female athymic nude mice (Harlan) between 6 and 8 weeksold were injected subcutaneously with 1 � 106 cells of theindicated cell lines (CS99-shRACGAP1 or CS99-shScramble).Log-phase cells were collected, counted, and suspended in 100-mL Opti-MEM for injection. Tumor size was measured usingdigital calipers every 3 days and tumor volume calculated usingthe formula: (length �width2)/2. Animals were cared for as perthe Animal Welfare Act and the NIH "Guide for the Care andUse of Laboratory Animals." All animal experiments were donewith the approval of the Institutional Animal Care and UseCommittee (Protocol 20130604) of the Albert Einstein Collegeof Medicine of Yeshiva University (Bronx, NY), under accred-itation by the Association for the Assessment and Accreditationof Laboratory Animal Care.

Cytotoxicity assaysCells were seeded into 96-well plates and treated with serial

dilutions of YM155 for 5days (for primary cell lines) or 3days (forCS99 cell lines) to approximate at least three doubling times. TheSulforhodamine B colorimetric assay was used to quantify cellnumber. IC50 (inhibitory concentration 50) values for YM155were calculated for each cell line as the drug concentration (meanof at least two independent experiments) that decreases viable cellnumber by 50% compared with vehicle alone.

Cell-cycle analysis using propidium iodide and flow cytometryCells were harvested from the plate and single-cell suspen-

sions were made by passing cells through a polystyrene round-bottom tube with cell strainer cap (BD Biosciences) three times.For cell-cycle analysis, cells were fixed with 70% cold ethanolfor 1 hour, then stained with 20 mg/mL propidium iodide(Sigma-Aldrich) and 100 mg/mL RNase (Thermo Fisher Scien-tific) in PBS. Cell data was acquired on a FACSCanto (BDBiosciences) and analyzed using the cell-cycle module in FlowJo 9 (Tree Star).

RACGAP1 Is a Metastatic Driver in Uterine Carcinosarcoma

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ImmunofluorescenceCells were fixed in in 4% paraformaldehyde for 20 minutes at

room temperature and permeabilized with 0.2% Triton X-100 for10minutes and incubated in 1%BSA/PBS for 45minutes at roomtemperature. The cells were incubated with anti-a-tubulin anti-body for 45minutes and then incubatedwith secondary antibodyfor 30 minutes. After washing with 1% BSA/PBS, nuclei werestained with DAPI. Images were acquired on a Zeiss AxioObserverCLEM microscope.

Migration and invasion assaysTo evaluate the ability of cells to migrate, the in vitro scratch

assay was done as described previously (13). Cells were photo-graphed at 10�magnification on a phase-contrast microscope at0, 24, and 48 hours, and the mean % wound closure at each timepoint was determined from three independent experiments. Todetermine the ability of cells to invade, a modified Boydenchamber assay was used. Cell lines were starved overnight,counted, and suspended in assay media (serum-free RPMI).Inserts with 8-mm pores (BD Biosciences) were placed into 24-well plates and coveredwith 0.1mL of 200 mg/mLMatrigelMatrixGrowth Factor Reduced (BD Biosciences). Suspended cells (1 �104 cells) in 200 mL of serum-free RPMI were added to the topchamber. The bottom chamber contained RPMI with 10% FBS.After incubation for 18 hours at 37�C incubator with 5% CO2,noninvaded cells were removed with a cotton swab, and the cellswere fixed with 3.7% formaldehyde and stained with 0.5% crystalviolet, and counted. The percentage of invaded cells was normal-ized to the total cell number.

Statistical analysisThenumber of biologically independent experiments is indicated

in the figure legends. All statistical analyses were performed usingGraphPadPrism6.Meanswere comparedusinga two-tailed t test, orusing a one-way ANOVA test with Tukey test when performingmultiple comparisons.c2 analysiswas performed for comparisonofcategorical variables between groups. Correlation analysis was doneusingPearson's productmoment correlation. Statisticalmethods forRNA-seq analysis are described separately; see above section "RNAsequencing analysis and statistical analysis" for details.

ResultsClinical characteristics of subjects and controls

After IRB approval, 19 patients with suspected uterine carcino-sarcoma enrolled in this prospective study and underwent hys-terectomy and surgical staging from September 2011 to Novem-ber 2013. Of these, 13 patients had confirmed carcinosarcoma onfinal pathology; the other 6 patients had either high-grade carci-noma or high-grade sarcoma on final pathology and were notincluded in the analysis. An additional 14th patient with con-firmed uterine carcinosarcoma had tumor tissue available foranalysis throughher participation in the IRB-approvedGYN tissuerepository protocol. For 6 of the 14 patients with carcinosarcoma,nontumor tissue from histologically benign endometrium wasavailable and collected concurrently with the carcinosarcomatumor tissue. The 12 control patients were postmenopausalwomenundergoing hysterectomy for benign indications andwhoconsented for tissue collection of normal endometrial tissueunder an IRB-approved protocol. The clinical characteristics ofthe 14 carcinosarcoma cases and the 12 control normal postmen-opausal (NP) patients are described in Table 1. The cases andcontrols were similar in age and racial distribution.

Transcriptome analysis of uterine carcinosarcoma and normalendometrial samples

Six tissue samples from each group were subjected to RNA-seqanalysis. RNA-seq statistics were similar for carcinosarcoma casesand NP controls (Table S1). The coefficient of variance (CV) waslow for the 6 NP control samples (0.17), indicating high repro-ducibility of the RNA-seq data. Despite heterogeneity amongcarcinosarcoma tumor samples (CV ¼ 0.5 for 6 tumor samples),unsupervised cluster analysis of transcriptomic expressionresulted in two groups in which carcinosarcoma tumor sampleswere clearly separated from NP controls (Fig. 1A). Shown in Fig.1B, 3,425 genes were significantly differentially expressed withFDR < 0.05 and fold change > 2. Of these, 2,005 genes wereincreased and 1,420 genes were decreased in carcinosarcomarelative to NP. The highest ranking up- and downregulatedcanonical pathways in carcinosarcoma as identified by IPA areshown in Supplementary Table S2. Enriched GO terms were also

Table 1. Clinical and pathologic characteristics

Carcinosarcomacases (N ¼ 14) (%)

NP control(N ¼ 11) (%)

Age, mean (range) 67 (46-84) 62 (51–71) P ¼ 0.15, Unpaired t testRace, n (%) P ¼ 0.12, c2 testBlack 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)

Mi et al.





examined for differentially expressed genes using DAVID Bioin-formatics. The highest ranking GO terms are shown in Supple-mentary Table S3.

Exclusive overexpression of RACGAP1 in uterinecarcinosarcoma tissues

Among the genes with significantly increased expression inuterine carcinosarcoma identified by RNA-seq, RACGAP1 over-expression was exclusively restricted to carcinosarcoma and wasselected as the initial overexpressed gene for further study. Weconfirmed increased RACGAP1 (also known as mgcracgap)

expression in uterine carcinosarcoma compared with normalendometrium at the mRNA (Fig. 1C) and protein level (Fig.1D), as determined by qRT-PCR and immunoblotting, usingsnap-frozen tissues from carcinosarcoma cases and NP endome-trial control tissues. Analysis done for the 6 carcinosarcomapatients with nontumor tissue (NT) available for comparisonshowed higher RACGAP1 protein expression in the carcinosar-coma tumor tissue relative to adjacent histologically benignendometrium (Fig. 1E). IHC using a specific antibody to RAC-GAP1 showed strong nuclear localization of RACGAP1 in carci-nosarcoma cells (Supplementary Fig. S1). The RACGAP1 IHC

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Figure 1.RACGAP1 expression is increased in uterine carcinosarcoma (CS) tissues.A,Cluster analysis of samples basedon the transcriptomic expression in normal endometrialtissues and uterine carcinosarcomas. The bar indicates the average difference of correlation coefficient between the samples. B, heatmap showing relativeexpression 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 depictsthemeanRACGAP1 expression score in thenormal and carcinosarcomagroups, respectively. �� ,P<0.001; two-tailed t test.D,RACGAP1 protein expressionwashighin most carcinosarcoma tissues and a cell line compared with normal endometrial tissues. E, RACGAP1 protein levels were increased in paired carcinosarcomatumor tissues compared with adjacent nontumor tissues (NT). Cell growth and cell cycle analysis were performed after treating carcinosarcoma cells withRACGAP1 shRNA. F, RACGAP1 mRNA levels are shown 48 hours after transient RACGAP1 knockdown with three different shRNAs. Bars, mean � SD of threeindependent experiments. shRACGAP1-1, -2, and -3 versus Scramble; ��, P < 0.001 by one-way ANOVA with Tukey test. G, cell viability was measured bycounting 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 RACGAP1knockdown, an increased number of binucleated cells are observed at 48 hours comparedwith Scramble, as shown by immunocytochemistry using an anti-a-tubulinantibody (red) to detect cytoplasm and DAPI stain (blue) to show nuclei. White arrows indicate binucleated cells. One representative experiment of twoindependent experiments is shown. I, compared with Scramble, RACGAP1 knockdown leads to cell-cycle accumulation in G2–M. After 48 hours, the percentage ofcells 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 twoindependent experiments is shown.






H-score showed a strong linear correlation with the mRNAexpression score, with a Pearson correlation coefficient >0.95(Supplementary Fig. S1 C).

Induction of cell-cycle arrest by RACGAP1 depletionTo interrogate the functional significance of RACGAP1 over-

expression, we used the CS99 cell line and transfected threedifferent short hairpin RNA (shRNA) vectors to knockdown RAC-GAP1 or a control vector (scramble shRNA vector). Knockdownefficiency of approximately 80% at 48 hours was achieved usingeach of the shRNAs (Fig. 1F). RACGAP1 knockdown significantlydecreased the viable cell number compared with control transfec-tion at 48 hours (Fig. 1G). This correlated with the appearance ofbinucleated cells (�10%) and the accumulation of cells in G2–Mphase, indicating defective cytokinesis and cell-cycle arrest follow-ing RACGAP1 knockdown (Fig. 1H and I). These effects areconsistent with the previously described role of RACGAP1 in thecentral spindle complex required for cytokinesis (14).

RACGAP1 is required for migration and invasion ofcarcinosarcoma cells

CS99 cells selected for stable RACGAP1 knockdown werecompared with control (scramble shRNA) cells. We confirmed

efficient RACGAP1 knockdown at the protein level in CS99-shRACGAP1 cells compared with CS99-shScramble cells (Fig.2A). The CS99-shRACGAP1 cells and CS99-shScramble cellsshowed similar cellular proliferation (Fig. 2B). However, in vivotumor growth was significantly impaired in CS99-shRACGAP1compared with CS99-shScramble cells (Fig. 2C). The wound-healing assay showed that CS99-shRACGAP1 cells were signifi-cantly impaired in their ability to migrate compared with CS99-shScramble (Fig. 2D and E). CS99-shRACGAP1 cells also showedsignificantly reduced invasive capacity comparedwith shScramblecells, as determined by Matrigel-coated Boyden chamber assay.(Fig. 2F and G).

RACGAP1 expression in primary carcinosarcoma cell linespredicts invasive capacity

Primary carcinosarcoma cell lines were propagated from freshcarcinosarcoma tumor tissue obtained at the time of surgicalresection. The origin of the carcinosarcoma primary cell lines wasconfirmed by comparison of the STR profile with the snap-frozenprimary tumor tissue (Supplementary Table S4). The proteinexpression of RACGAP1 in each the primary cell lines was deter-mined by immunoblotting (Fig. 3A). The carcinosarcoma celllines with high endogenous RACGAP1 expression (CS008,

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Figure 2.RACGAP1 knockdown in CS99 uterine carcinosarcoma cells. A, stable transfection of shRACGAP1 in CS99 cells decreased protein levels of RACGAP1, as shown byWestern blotting. One Western blot of two independent experiments is shown. Representative data from shScramble and shRACGAP1 is shown in panelsB–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 twoexperiments). C, combined data from two independent experiments are shown depicting xenograft growth following subcutaneous injection of the indicated celllines into athymic nude mice, N ¼ 14 per group. CS99-shRACGAP1 xenograft growth was significantly reduced compared with CS99-shScrambled xenograftgrowth. ��, 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-shScrambleand CS99-shRACGAP1. Wound closure wasmonitored under phase-contrast microscopy and photographed (10�) at 0, 24, and 48 hours. Representative images ofthree independent experiments are shown. E, CS99-shRACGAP1 cells show retardation of wound closure. The mean � SD of three independent experimentsis shown. � , P < 0.05; �� , P < 0.01 by two-tailed t test. F, representative images of crystal violet stained CS99-shScramble and CS99-shRACGAP1 invadinginto Matrigel-coated inserts of the Boyden chambers. G, the graph shows the percentage of invading cells normalized to the total cell number. The barsrepresent the mean � SD of three independent experiments. �� , P < 0.01, one-way ANOVA with Tukey test.

Mi et al.





CS019) showed significantly increased invasiveness comparedwith carcinosarcoma cell lines with low RACGAP1 expression(CS013, CS017), as determined using a Matrigel-coated Bowdenchamber assay (Figs. 3B and C).

RACGAP1 regulates STAT3 phosphorylation and survivinexpression in uterine carcinosarcoma

On the basis of a prior report that RACGAP1 facilitates theTyr705 phosphorylation of STAT3 and promotes its translocationto the nucleus (15), we evaluated the relationship of RACGAP1,phospho-STAT3 (Y705), and total STAT3 in uterine carcinosar-coma (Fig. 4A). The expression of survivin (encoded by the BIRC5gene, baculoviral inhibitor of apoptosis repeat containing 5), atranscriptional target of STAT3, was also evaluated by immuno-blotting andqRT-PCR. In carcinosarcoma tumor tissues, RACGAP1expression was significantly correlated with levels of phospho-STAT3 (Fig. 4B) and with survivin expression (Fig. 4C). Further-more, we found that RACGAP1 knockdown decreased phosphor-ylation of STAT3 and reduced survivin expression in CS99 cells(Fig. 4D). On the basis of these data, STAT3 and survivin are bonafide downstream targets of RACGAP1 in uterine carcinosarcoma.

RACGAP1 expression is a biomarker of sensitivity to anti-survivin therapy

YM155 is a first-in-class small molecule that selectively sup-presses survivin at themRNA and protein level (16, 17). Results ofcytotoxicity assays showed that YM155 potently inhibited theproliferation of primary and established carcinosarcoma celllines, with IC50 concentrations ranging from 1.9 nmol/L (forCS99) to 24.3 nmol/L (for CS017; Supplementary Table S5).Target inhibition was confirmed by immunoblotting that showedreduced survivin protein expression at these YM155 concentra-tions (Supplementary Fig. S2). The IC50 concentrations inverselycorrelated with RACGAP1 and survivin mRNA expression levels(Fig. 4E and F). On the basis of these data, high RACGAP1expression predicts sensitivity to anti-survivin therapy. The effectof YM155 on the invasiveness of CS99 cells was determined usingthe Matrigel-coated Boyden chamber assay (Fig. 4G). In a dose-

dependent fashion, YM155 significantly reduced the invasivecapacity of CS99 cells (Fig. 4H).

RACGAP1 overexpression promotes invasiveness ofendometrial cells

Next, the effect of RACGAP1 overexpression was evaluated inthe endometrioid adenocarcinoma cell line Hec1b. Expression offull-length RACGAP1 following transfection was confirmed byimmunoblotting (Fig. 5A). The invasive capacity of Hec1b/RAC-GAP1–transfected cells was significantly increased comparedwithHec1b/empty vector–transfected cells (Fig. 5B). These data sug-gest that increased RACGAP1 expression is sufficient to promoteinvasive ability in this cell line model of endometrioid endome-trial cancer, which is the more common and less aggressivehistologic type of uterine corpus cancer.

Correlation of RACGAP1 and survivin expression in uterinecarcinosarcoma

To validate our findings in an independent cohort, we obtaineddeidentified clinical data and gene expression data from the TCGAuterine carcinosarcoma cohort (N ¼ 57 patients). First, we deter-mined the correlation of RACGAP1 and survivin (BIRC5) in thesepatients. As in our institutional cohort, RACGAP1 and survivinmRNA levels were significantly positively correlated in the TCGAcohort (Fig. 5C). The difference in the absolute correlation coef-ficient of RACGAP1 and survivin in our institutional cohort versusthe multi-institutional TCGA cohort could be related to thedifferent method ofmeasuring mRNA levels. In the TCGA cohort,mRNA expression levels of RACGAP1 and survivin were deter-mined by RNA-seq on an Illumina HiSeq platform. In the corre-lation analysis for our institutional cohort, mRNA levels werequantified by qRT-PCR, using optimized primers and linearamplification conditions, as well as internal normalization.

Increased risk of extrauterine disease in patients with highRACGAP1-expressing carcinosarcoma

As our data showed RACGAP1 regulates the migratory and inva-sive behavior of carcinosarcoma cells, we hypothesized that patients

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Figure 3.RACGAP1 expression correlates with invasiveability in primary carcinosarcoma cell lines.A, RACGAP1 protein expression in fourunique patient-derived primary cell lines isshown by immunoblotting. B, representativeimages of crystal violet–stained primarycarcinosarcoma cells that have invaded intoMatrigel-coated inserts of the Boydenchambers. C, the percentage of invading cellsnormalized to total cell number is shown foreach cell line. Each bar represents the mean� SEM of two independent experiments.�� , P < 0.01; �� , P < 0.001; one-way ANOVAwith Tukey multiple comparisons test.ns, not significant.






with high RACGAP1-expressing tumors would be more likely topresent with higher stage of disease (indicating metastatic spread)compared with patients with low RACGAP1–expressing tumors.Analysis of data from an independent cohort of 57 carcinosarcomapatients from the TCGAwas done to evaluate the potential relation-shipofRACGAP1andstage.AsshowninFig.5D,patientswithhigherstage of disease (indicating cancer spread beyond the uterus) hadsignificantly higher RACGAP1 mRNA expression compared withpatientswithout spreadofdisease. Thesefindings in an independentcohortofpatientswithcarcinosarcomasupport theroleofRACGAP1as a metastatic driver in this disease, consistent with the findings inour cell line models of carcinosarcoma.

DiscussionTo our knowledge, this study is the first to apply RNA-seq

technology to compare clinical tumor samples of uterine carci-

nosarcoma, a highly aggressive malignancy, with normal endo-metrial tissue. Using this approach, we have identified RACGAP1as a critical upregulated gene and driver of the metastatic pheno-type in uterine carcinosarcoma. In functional assays, we show thatRACGAP1 depletion abolishes the migratory and invasive capac-ity of uterine carcinosarcoma cells, while RACGAP1 overexpres-sion confers a metastatic phenotype to endometrial adenocarci-noma cells. In addition, we have found that high RACGAP1expression is significantly correlated with extrauterine spread ofdisease in patients, substantiating its clinical relevance in uterinecarcinosarcoma.

Our data show that RACGAP1 is a key regulator of STAT3phosphorylation and survivin expression in uterine carcinosar-coma cells, consistent with a prior observation that RACGAP1 canfunction as a nuclear chaperone for STAT3 (15). In uterinecarcinosarcoma cells, we found that RACGAP1 depletion reducesSTAT3 phosphorylation and survivin expression. Analysis of two

r = 0.91**

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Figure 4.RACGAP1 expression correlateswith p-STAT3and Survivin expression.A,RACGAP1 protein expressionwas positively correlated to p-STAT3 and Survivin expressionin carcinosarcoma clinical samples, as shownby immunoblotting of tumor lysates.B,positive correlation of RACGAP1 protein expression andphospho-STAT3(Y705)protein expression in clinical samples, as determined by ImageJ quantification of immunoblot images (shown in A). Internal normalization to GAPDHprotein expression was done. The Pearson correlation coefficient, r ¼ 0.71; � , P < 0.05. C, positive correlation of RACGAP1 and survivin mRNA expression, asdetermined by qRT-PCR using RNA isolated from the carcinosarcoma samples shown in A. Internal normalization for survivin mRNA expression to PPIB mRNAexpression was done. The Pearson correlation coefficient, r ¼ 0.91; �� , P < 0.01. D, CS99-shRACGAP1 knockdown cells have decreased expression ofphosphorylated STAT3 and survivin compared with CS99-shScrambled cells. One of two representative immunoblots is shown. E, for each carcinosarcoma primarycell line (n¼ 7) and CS99, the IC50 of YM155 is plotted versus the RACGAP1mRNA expression score determined by qRT-PCR (see Supplementary Table S5), showingthat RACGAP1 mRNA expression negatively correlates with IC50 concentration; Pearson correlation coefficient, r ¼ �0.76; � , P < 0.05. This indicates greatersensitivity to survivin inhibition in the higher RACGAP1-expressing cell lines. F, the IC50 of YM155 is plotted versus the Survivin mRNA expression score. A negativecorrelation was observed, similar to that observed in A; Pearson correlation coefficient, r¼�0.75; � , P < 0.05. G, effect of YM155 on CS99 cell invasion wasevaluated by Boyden chamber assay; representative images of cells invading theMatrigel-coated inserts are shown. YM155 treatment decreased cell invasion.H, thequantification of invading cells into the Matrigel-coated inserts normalized to total cell number. The bars represent the mean � SD of two independentexperiments. � , P < 0.05 versus untreated cells, One-way ANOVA with Tukey test.

Mi et al.





independent cohorts of patients with uterine carcinosarcomashowed that RACGAP1 and survivin expression are significantlypositively correlated in tumor samples.

On the basis of these novel findings, we evaluated the thera-peutic potential of a first-in-class survivin inhibitor YM155 usingprimary carcinosarcoma cell lines developed from human carci-nosarcoma tumors. YM155 is a first-in-class inhibitor of survivinthat shows promising therapeutic activity in preclinical andclinical trials. For many targeted therapies, overexpression of thetarget (in this case, survivin) correlates with increased sensitivityto the therapeutic agent. Recently, survivin overexpression wasidentified in anaplastic thyroid cancer; the authors performed ahigh-throughput screen of >3,000 drugs and identified the survi-vin inhibitor YM155 as one of the most active agents in thataggressive disease (18). In our studies, we found that YM155showed potent antiproliferative activity in primary and estab-lished uterine carcinosarcoma lines and moreover, abolished theinvasive capacity of carcinosarcoma cells. Furthermore, the

expression levels of RACGAP1 and survivin predicted sensitivityto YM155 in primary carcinosarcoma cell lines. Phase I trials haveshown that YM155 is well-tolerated with a very favorable safetyprofile (19, 20). Phase II trials in several disease sites havedemonstrated single-agent activity, for example, in patients withadvanced, refractory non–small cell lung cancer and castration-resistant prostate cancer (21, 22). The antitumor activity ofYM155 in patients with uterine carcinosarcoma awaits futureinvestigation.

RACGAP1, also known as MgcRacGAP, was initially identifiedas a GTPase-activating protein (GAP) expressed in testis andmalegerm cells (23). Unlike other GAPs, RACGAP1 is an essentialcomponent of the centralspindlin complex (with the kinesinKlF23), where its GAP activity is required for cytokinesis andinactivation of Rac1 at the cleavage furrow (24–28). RACGAP1also regulates tethering of the mitotic spindle to the plasmamembrane during cytokinesis (29). Apart from its role in cyto-kinesis, RACGAP1 was shown to regulate STAT3 activation in

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Figure 5.Constitutive expression of RACGAP1 increases invasive capacity of Hec1B endometrial carcinoma cells.A, RACGAP1 protein levels, detected by immunoblotting, areincreased at 72 hours following transfection with full-length RACGAP1 compared with empty vector (EV) transfection. One of two independent experimentsis shown. B, invasive capacity was measured by Matrigel-coated Bowden chamber assay done 48 hours after transfection with full-length RACGAP1 or EV. Thepercentage 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 (N¼ 57). RACGAP1 andBIRC5 (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 uterinecorpus (FIGO stages II–IV; right side) had significantly higher RACGAP1 expression compared with patients with non-metastatic disease (FIGO stage I; left side). Thebox 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).






leukemia cells (30), and may also play a role in regulatingendothelial permeability (31). Recently, RACGAP1 expressionhas been linked with aggressive clinical behavior in several can-cers, including colorectal cancer (32), hepatocellular carcinoma(33, 34), gastric cancer (35),meningiomas (36), and breast cancer(37). Thus, our findings may have broader relevance for targetingthe metastatic phenotype in diverse tumor types.

Very little has been previously published regarding underly-ing drivers of the development and progression of this disease,and there is a dire lack of effective therapies for this highlylethal disease. This knowledge gap is one of the motivators forthe current study, which has led to our discovery of a keymetastatic driver and identification of a promising, noveltargeted therapy. In summary, RACGAP1 promotes the meta-static phenotype in uterine carcinosarcoma via a STAT3/survi-vin signaling pathway. The survivin inhibitor YM155 potentlysuppresses carcinosarcoma cell proliferation and abrogates theinvasive capacity of carcinosarcoma cells. We suggest thatinhibition of the RACGAP1–STAT3 survivin pathway shouldbe investigated as a novel therapeutic strategy for this lethalmalignancy, as well as for other high RACGAP1–expressingcancers.

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

DisclaimerThe content is solely the responsibility of the authors and does not neces-

sarily represent the official views of the National Institutes of Health.

Authors' ContributionsConception and design: S. Mi, J. Heim, D. Smotkin, G.L. Goldberg, G.S. HuangDevelopment of methodology: S. Mi, M. Lin, J. Brouwer-Visser, D. Zheng,G.S. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Mi, J. Brouwer-Visser, J. Heim, T.M. Hebert,M.J. Gunter, G.S. HuangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Mi, M. Lin, J. Brouwer-Visser, D. Smotkin,M.J. Gunter, G.L. Goldberg, D. Zheng, G.S. HuangWriting, review, and/or revision of the manuscript: S. Mi, M. Lin, J. Heim,D. Smotkin, M.J. Gunter, G.L. Goldberg, D. Zheng, G.S. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Lin, G.S. HuangStudy supervision: G.S. Huang

AcknowledgmentsThe results shown are in part based upon data generated by the TCGA

Research Network: http://cancergenome.nih.gov/.

Grant SupportThis work was supported by the Albert Einstein Cancer Center Support Grant

of the NIH under award number P30CA013330 and Albert Einstein CancerCenter Pilot Award (to G.S. Huang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 29, 2015; revised March 9, 2016; accepted April 4, 2016;published OnlineFirst April 4, 2016.

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Published OnlineFirst April 27, 2016.Clin Cancer Res Shijun Mi, Mingyan Lin, Jurriaan Brouwer-Visser, et al. Uterine CarcinosarcomaRNA-seq Identification of RACGAP1 as a Metastatic Driver in

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