Purpose: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets.

Experimental Design: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line–derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA).

Results: Rac GTPase–activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease.

Conclusions: The RACGAP1–STAT3–survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676–86. ©2016 AACR.

Translational Relevance

Uterine carcinosarcoma is a highly aggressive endometrial malignancy that causes a disproportionate number of deaths from uterine cancer. By comparing the gene expression of uterine carcinosarcoma with benign endometrial tissue, Rac GTPase–activating protein 1 (RACGAP1) was identified to be highly upregulated, and functional studies showed that RACGAP1 regulates motility and invasion via STAT3–survivin signaling. Analyzing an independent cohort from The Cancer Genome Atlas showed that patients with more advanced uterine carcinosarcoma had significantly higher RACGAP1 expression in their tumors correlated with increased survivin expression. Furthermore, RACGAP1 expression predicted sensitivity to survivin-targeted therapy in primary cell lines derived from patients. On the basis of these novel findings, the RACGAP1–STAT3–survivin signaling pathway is identified as a promising therapeutic target in uterine carcinosarcoma, a highly lethal disease of the female reproductive tract.

Uterine carcinosarcoma, also known as malignant mixed Müllerian tumors, is a highly aggressive form of uterine cancer, with a propensity for extrauterine metastases and a high case fatality rate (1). The histologic diagnosis is based on the presence of both malignant carcinomatous and sarcomatous elements (2). Prior studies support a monoclonal endometrial origin of both elements, supporting the view of uterine carcinosarcoma as a metaplastic carcinoma that has undergone epithelial-to-mesenchymal transition (3).

These uncommon tumors, which represent < 5% of uterine corpus cancers, account for greater than 15% of uterine cancer–related deaths (1, 4). The disease usually affects postmenopausal women, with a median age at diagnosis of 62 to 67 years. The incidence is significantly higher in black women with an age-adjusted incidence rate of 4.3 per 100,000 compared with 1.7 per 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).

Treatment of uterine carcinosarcoma is primarily surgical. Approximately one-third of patients have disease spread beyond the uterus at the time of diagnosis, and the recurrence rate after surgery exceeds 50%. Combination chemotherapy may improve overall and progression-free survival in patients with advanced stage or recurrent carcinosarcoma (7, 8). Despite multimodal treatment approaches, the median overall survival is approximately 21 months, and in patients with advanced disease, less than one year (1).

The underlying molecular drivers of the aggressive phenotype of uterine carcinosarcoma have not been identified. Poor clinical prognostic factors include higher FIGO stage of disease and serum CA125 elevation (6). In this study, we applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications, with the goal of identifying novel oncogenic drivers and potential therapeutic targets.

We found that RACGAP1 is a highly upregulated gene in uterine carcinosarcoma, and its overexpression promotes the metastatic phenotype. We identified STAT3 and survivin as bona fide downstream targets of RACGAP1, and showed that RACGAP1 is a critical regulator of STAT3 phosphorylation and survivin expression in uterine carcinosarcoma. Targeting RACGAP1 directly, or its downstream effectors, significantly diminished the invasive capacity of uterine carcinosarcoma cells. Analysis of an independent cohort of 57 patients with uterine carcinosarcoma showed that high RACGAP1 predicted extrauterine metastasis, validating its clinical significance as a metastatic driver. Thus, we have identified a novel molecular driver of the metastatic phenotype of uterine carcinosarcoma, suggesting new therapeutic approaches for targeting this and other highly aggressive cancers.

Tissue acquisition

Under Institutional review board (IRB) approval, carcinosarcoma tumor samples (IRB# 2011-404) and normal endometrial tissues (IRB#2009-406) were prospectively collected from consenting patients undergoing surgery at Montefiore Medical Center, and the corresponding clinical data recorded. The study coordinator assigned a unique study ID to tissue samples, by which samples were identified in the laboratory. In conjunction with surgical pathology, the standard operating procedure was followed for prospective tissue collection of leftover 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/DNA assays); (ii) snap-freezing of tissue pieces in a cryovial partially immersed in liquid nitrogen (for subsequent protein assays); (iii) immersion of fresh tissue in sterile RPMI1640 medium for immediate transport to the laboratory (for preparation of primary cell lines); (iv) frozen optimal cutting temperature (OCT)-embedded tissue block (storage at −80°C); (v) formalin-fixed, paraffin-embedded tissue block for sectioning (for histopathology).

Primary cell line isolation and tissue culture

For isolation of primary cell lines, under an IRB-approved protocol (#2011-404), fresh tumor tissue from patients undergoing hysterectomy and staging surgery for histologically confirmed uterine carcinosarcoma was transported to the laboratory in sterile RPMI tissue culture media. Following mechanical dissociation, enzymatic digestion with 3 mg/mL collagenase A (Roche) and 150 μg/mL DNase 1 (Thermo Fisher Scientific), and red blood cell lysis (eBioscience), cells were washed with RPMI with FBS (10%), and resuspended in F-media supplemented as described previously (9). Cells were seeded in tissue culture flasks and media replaced as needed. Cell line authentication was done by short tandem repeat (STR) profiling using the Genemarker 10 kit (Promega) and matching to the original carcinosarcoma patient samples (Supplementary Table S4).

CS99 cells and their derivatives were maintained as subconfluent monolayer cultures in RPMI1640 containing 10% FBS at 37°C with 5% CO2.

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

RNA isolation

To extract RNA, frozen tissues were pulverized in a tissueTUBE bag (Covaris) using a cryoPREP (Covaris) and then homogenized in Buffer RLT (Qiagen) using a Covaris adaptive focused acoustics tissue disrupter. The Qiagen AllPrep kit was used following the manufacturer's instructions. The RNA concentration and purity was measured using the Nanodrop spectrophotometer (Thermo Fisher Scientific), and RNA integrity was evaluated with the Agilent Bioanalyzer (Agilent). RNA quality was uniformly excellent and met the following criteria; Nanodrop, 260/280 ratio >1.8; Agilent Bioanalyzer, RIN > 7.

Paired-end library preparation and Illumina sequencing

Twelve RNA-seq libraries (CS001, CS005, CS008, CS010, CS011, CS718, NP002, NP006, NP011, NP013, NP024, and NP028) were prepared for paired-end sequencing using the Illumina HiSeq platform in the epigenetics core facility of the Albert Einstein College of Medicine according to directional whole transcript seq protocol described on WASP (wiki-based automated sequence processor, http://wasp.einstein.yu.edu). In brief, the purified cDNA library products were evaluated using the Agilent bioanalyzer and diluted to 10 nmol/L for cluster generation in situ on the HiSeq paired-end flow cell using the cBot automated cluster generation system followed by massively parallel sequencing (2 × 100 bp) on HiSeq 2000.

RNA-seq analysis and enrichment analysis

We obtained 92-bp mate-paired reads from DNA fragments with an average size of 250-bp (SD for the distribution of inner distances between mate pairs is approximately 100 bp). RNA-seq reads were aligned to the human genome (GRCh37/hg19) using the software GSNAP version 2012-07-20 (PMID: 15728110). We counted the number of fragments mapped to each gene annotated in the GENCODE database (version 18; PMID: 22955987) using HTSeq v0.5.3p3 (PMID: 25260700). The category of transcripts used for our expression analysis is described at http://www.gencodegenes.org/gencode_biotypes.html. We used DESeq2 to determine differential expression based on count values (10). Specifically, DESeq2 models the dispersion using empirical Bayes shrinkage and tests whether, for a given gene, the fold change in expression strength between the two experimental conditions significantly differs from zero using a Wald test. We also quantified transcript abundances in Fragment Per Kilobase of Exon Per Million (FPKM) by dividing the count by effective gene length (derived from regions covered by reads). Only genes with average FPKM >1 across all samples were considered for differential expression analysis. P values were corrected by FDR (11). Significant differences in gene expression between tumors and controls were determined according to the following criteria: fold change > 2 and FDR < 0.05. We performed overrepresentation analysis to identify enriched pathways with the Ingenuity Pathway Analysis (IPA) and statistically significant gene ontology (GO) terms with DAVID (PMID: 19131956). We used all genes with expression levels above FPKM >1 as the background list. A false discovery rate (FDR) of 5% (q < 0.05) was used to interpret statistical significance.

Reverse transcriptase quantitative real-time PCR

Reverse transcriptase quantitative real-time PCR was done similarly as described previously (12). In brief, complementary DNA was synthesized from 1 μg of total RNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies). Quantitative real-time PCR reactions were carried out using investigator-validated forward and reverse primers for the target genes (see Supplementary Table S6) and PowerSYBR Green (Life Technologies) detection on a Realplex2 (Eppendorf). Target gene expression was internally normalized to the mRNA expression of a housekeeping gene, peptidylprolyl isomerase B (PPIB). Each qPCR reaction was run in triplicate on the same plate. Melting curve analysis was done to confirm a single amplicon corresponding to the PCR product size. Each assay plate included two reactions that omit either the mRNA template or the reverse transcriptase enzyme to exclude the possibility of contamination. Results were analyzed by the 2−ΔΔCt method to quantify the relative mRNA expression level.

Immunoblotting

Cell lysates were prepared from tissue samples by pulverization of snap-frozen tissue using a cryoPREP (Covaris), resuspension in SDS lysis buffer, and protein quantitation by a modified Lowry method. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Blocking was done for 30 minutes using 3% BSA in TBS with 0.1% Tween-20, prior to incubation with primary antibody overnight at 4°C. The following primary antibodies were used: RACGAP1 mAb (M01), clone 1G6 (Abnova); survivin (FL-142) polyclonal antibody (Santa Cruz Biotechnology); GAPDH antibody (FL-335; Santa Cruz Biotechnology); phospho-Stat3 Tyr 705 polyclonal antibody (Cell Signaling Technology); Stat3 polyclonal antibody (Cell Signaling Technology); and anti-α-Tubulin (DM1A) antibody (Sigma-Aldrich). The appropriate horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) was used, followed by enhanced chemiluminescence detection (GE Healthcare). All films were scanned and saved in unmodified TIFF format. Densitometry was done using ImageJ software.

RACGAP1 IHC

Formalin-fixed paraffin-embedded tissue sections were deparaffinized and rehydrated. Antigen retrieval was done in DAKO antigen retrieval solution at 95°C for 30 minutes. Endogenous peroxidases were blocked with hydrogen peroxide, followed by blocking in antibody diluent (DAKO #S3022). The primary antibody, mouse monoclonal anti-RACGAP from Abnova (M01), clone 1G6, was used at a final concentration of 2 mcg/mL incubated overnight at 4°C. After washing, the anti-mouse secondary antibody (DAKO Envision+ Kit) was applied for 30 minutes. Finally, slides were washed, and DAB detection was done, followed by counterstaining with hematoxylin, dehydration through graded alcohols and xylene, and mounting with coverslip application. Concurrently, IHC staining of testis was used as a positive control, and omission of the primary antibody was used as a negative control. IHC staining was evaluated by the study pathologist (T.M. Hebert), who evaluated and scored the cytoplasmic and nuclear staining intensity (0, 1, 2, or 3) and percentage of positive cells (0–100) for the entire tumor, for the carcinomatous component, and for the sarcomatous component. An IHC H-score (product of staining intensity and percentage positive cells) was calculated separately for the cytoplasmic staining and for the nuclear staining of the entire tumor, and for each individual component.

Cell proliferation and tumor growth assays

Log-phase cells were seeded onto 6-well plates at 10,000 cells per well. Triplicate wells were collected and counted with a Millipore Scepter at 24-hour intervals for 96 hours. Mean cell number at each time point was determined from at least two independent experiments. To assess in vivo tumor growth, female athymic nude mice (Harlan) between 6 and 8 weeks old were injected subcutaneously with 1 × 106 cells of the indicated cell lines (CS99-shRACGAP1 or CS99-shScramble). Log-phase cells were collected, counted, and suspended in 100-μL Opti-MEM for injection. Tumor size was measured using digital calipers every 3 days and tumor volume calculated using the formula: (length × width2)/2. Animals were cared for as per the Animal Welfare Act and the NIH “Guide for the Care and Use of Laboratory Animals.” All animal experiments were done with the approval of the Institutional Animal Care and Use Committee (Protocol 20130604) of the Albert Einstein College of Medicine of Yeshiva University (Bronx, NY), under accreditation by the Association for the Assessment and Accreditation of Laboratory Animal Care.

Cytotoxicity assays

Cells were seeded into 96-well plates and treated with serial dilutions of YM155 for 5 days (for primary cell lines) or 3 days (for CS99 cell lines) to approximate at least three doubling times. The Sulforhodamine B colorimetric assay was used to quantify cell number. IC50 (inhibitory concentration 50) values for YM155 were calculated for each cell line as the drug concentration (mean of at least two independent experiments) that decreases viable cell number by 50% compared with vehicle alone.

Cell-cycle analysis using propidium iodide and flow cytometry

Cells were harvested from the plate and single-cell suspensions 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 ethanol for 1 hour, then stained with 20 μg/mL propidium iodide (Sigma-Aldrich) and 100 μg/mL RNase (Thermo Fisher Scientific) in PBS. Cell data was acquired on a FACSCanto (BD Biosciences) and analyzed using the cell-cycle module in Flow Jo 9 (Tree Star).

Immunofluorescence

Cells were fixed in in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with 0.2% Triton X-100 for 10 minutes and incubated in 1% BSA/PBS for 45 minutes at room temperature. The cells were incubated with anti-α-tubulin antibody for 45 minutes and then incubated with secondary antibody for 30 minutes. After washing with 1% BSA/PBS, nuclei were stained with DAPI. Images were acquired on a Zeiss AxioObserver CLEM microscope.

Migration and invasion assays

To evaluate the ability of cells to migrate, the in vitro scratch assay was done as described previously (13). Cells were photographed at 10× magnification on a phase-contrast microscope at 0, 24, and 48 hours, and the mean % wound closure at each time point was determined from three independent experiments. To determine the ability of cells to invade, a modified Boyden chamber assay was used. Cell lines were starved overnight, counted, and suspended in assay media (serum-free RPMI). Inserts with 8-μm pores (BD Biosciences) were placed into 24-well plates and covered with 0.1 mL of 200 μg/mL Matrigel Matrix Growth Factor Reduced (BD Biosciences). Suspended cells (1 × 104 cells) in 200 μL of serum-free RPMI were added to the top chamber. 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 cells were fixed with 3.7% formaldehyde and stained with 0.5% crystal violet, and counted. The percentage of invaded cells was normalized to the total cell number.

Statistical analysis

The number of biologically independent experiments is indicated in the figure legends. All statistical analyses were performed using GraphPad Prism 6. Means were compared using a two-tailed t test, or using a one-way ANOVA test with Tukey test when performing multiple comparisons. χ2 analysis was performed for comparison of categorical variables between groups. Correlation analysis was done using Pearson's product moment correlation. Statistical methods for RNA-seq analysis are described separately; see above section “RNA sequencing analysis and statistical analysis” for details.

Clinical characteristics of subjects and controls

After IRB approval, 19 patients with suspected uterine carcinosarcoma enrolled in this prospective study and underwent hysterectomy and surgical staging from September 2011 to November 2013. Of these, 13 patients had confirmed carcinosarcoma on final pathology; the other 6 patients had either high-grade carcinoma or high-grade sarcoma on final pathology and were not included in the analysis. An additional 14th patient with confirmed uterine carcinosarcoma had tumor tissue available for analysis through her participation in the IRB-approved GYN tissue repository protocol. For 6 of the 14 patients with carcinosarcoma, nontumor tissue from histologically benign endometrium was available and collected concurrently with the carcinosarcoma tumor tissue. The 12 control patients were postmenopausal women undergoing hysterectomy for benign indications and who consented for tissue collection of normal endometrial tissue under an IRB-approved protocol. The clinical characteristics of the 14 carcinosarcoma cases and the 12 control normal postmenopausal (NP) patients are described in Table 1. The cases and controls were similar in age and racial distribution.

Table 1.

Clinical and pathologic characteristics

Carcinosarcoma cases (N = 14) (%)NP control (N = 11) (%)
Age, mean (range) 67 (46-84) 62 (51–71) P = 0.15, Unpaired t test 
Race, n (%)   P = 0.12, χ2 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)  
Carcinosarcoma cases (N = 14) (%)NP control (N = 11) (%)
Age, mean (range) 67 (46-84) 62 (51–71) P = 0.15, Unpaired t test 
Race, n (%)   P = 0.12, χ2 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)  

Transcriptome analysis of uterine carcinosarcoma and normal endometrial samples

Six tissue samples from each group were subjected to RNA-seq analysis. RNA-seq statistics were similar for carcinosarcoma cases and NP controls (Table S1). The coefficient of variance (CV) was low for the 6 NP control samples (0.17), indicating high reproducibility of the RNA-seq data. Despite heterogeneity among carcinosarcoma tumor samples (CV = 0.5 for 6 tumor samples), unsupervised cluster analysis of transcriptomic expression resulted in two groups in which carcinosarcoma tumor samples were clearly separated from NP controls (Fig. 1A). Shown in Fig. 1B, 3,425 genes were significantly differentially expressed with FDR < 0.05 and fold change > 2. Of these, 2,005 genes were increased and 1,420 genes were decreased in carcinosarcoma relative to NP. The highest ranking up- and downregulated canonical pathways in carcinosarcoma as identified by IPA are shown in Supplementary Table S2. Enriched GO terms were also examined for differentially expressed genes using DAVID Bioinformatics. The highest ranking GO terms are shown in Supplementary Table S3.

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 type = “Other”> 2. C, RACGAP1 mRNA levels in carcinosarcoma tissue (n = 9) and normal endometrial tissues (n = 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-α-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.

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 type = “Other”> 2. C, RACGAP1 mRNA levels in carcinosarcoma tissue (n = 9) and normal endometrial tissues (n = 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-α-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.

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Exclusive overexpression of RACGAP1 in uterine carcinosarcoma tissues

Among the genes with significantly increased expression in uterine carcinosarcoma identified by RNA-seq, RACGAP1 overexpression was exclusively restricted to carcinosarcoma and was selected as the initial overexpressed gene for further study. We confirmed increased RACGAP1 (also known as mgcracgap) expression in uterine carcinosarcoma compared with normal endometrium at the mRNA (Fig. 1C) and protein level (Fig. 1D), as determined by qRT-PCR and immunoblotting, using snap-frozen tissues from carcinosarcoma cases and NP endometrial control tissues. Analysis done for the 6 carcinosarcoma patients with nontumor tissue (NT) available for comparison showed higher RACGAP1 protein expression in the carcinosarcoma tumor tissue relative to adjacent histologically benign endometrium (Fig. 1E). IHC using a specific antibody to RACGAP1 showed strong nuclear localization of RACGAP1 in carcinosarcoma cells (Supplementary Fig. S1). The RACGAP1 IHC H-score showed a strong linear correlation with the mRNA expression score, with a Pearson correlation coefficient >0.95 (Supplementary Fig. S1 C).

Induction of cell-cycle arrest by RACGAP1 depletion

To interrogate the functional significance of RACGAP1 overexpression, we used the CS99 cell line and transfected three different short hairpin RNA (shRNA) vectors to knockdown RACGAP1 or a control vector (scramble shRNA vector). Knockdown efficiency of approximately 80% at 48 hours was achieved using each of the shRNAs (Fig. 1F). RACGAP1 knockdown significantly decreased the viable cell number compared with control transfection at 48 hours (Fig. 1G). This correlated with the appearance of binucleated cells (∼10%) and the accumulation of cells in G2–M phase, indicating defective cytokinesis and cell-cycle arrest following RACGAP1 knockdown (Fig. 1H and I). These effects are consistent with the previously described role of RACGAP1 in the central spindle complex required for cytokinesis (14).

RACGAP1 is required for migration and invasion of carcinosarcoma cells

CS99 cells selected for stable RACGAP1 knockdown were compared 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 cells showed similar cellular proliferation (Fig. 2B). However, in vivo tumor growth was significantly impaired in CS99-shRACGAP1 compared with CS99-shScramble cells (Fig. 2C). The wound-healing assay showed that CS99-shRACGAP1 cells were significantly impaired in their ability to migrate compared with CS99-shScramble (Fig. 2D and E). CS99-shRACGAP1 cells also showed significantly reduced invasive capacity compared with shScramble cells, as determined by Matrigel-coated Boyden chamber assay. (Fig. 2F and G).

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 BG. 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 = 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.

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 BG. 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 = 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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RACGAP1 expression in primary carcinosarcoma cell lines predicts invasive capacity

Primary carcinosarcoma cell lines were propagated from fresh carcinosarcoma tumor tissue obtained at the time of surgical resection. The origin of the carcinosarcoma primary cell lines was confirmed by comparison of the STR profile with the snap-frozen primary tumor tissue (Supplementary Table S4). The protein expression of RACGAP1 in each the primary cell lines was determined by immunoblotting (Fig. 3A). The carcinosarcoma cell lines with high endogenous RACGAP1 expression (CS008, CS019) showed significantly increased invasiveness compared with carcinosarcoma cell lines with low RACGAP1 expression (CS013, CS017), as determined using a Matrigel-coated Bowden chamber assay (Figs. 3B and C).

Figure 3.

RACGAP1 expression correlates with invasive ability in primary carcinosarcoma cell lines. A, RACGAP1 protein expression in four unique patient-derived primary cell lines is shown by immunoblotting. B, representative images of crystal violet–stained primary carcinosarcoma cells that have invaded into 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 ± SEM of two independent experiments. **, P < 0.01; ***, P < 0.001; one-way ANOVA with Tukey multiple comparisons test. ns, not significant.

Figure 3.

RACGAP1 expression correlates with invasive ability in primary carcinosarcoma cell lines. A, RACGAP1 protein expression in four unique patient-derived primary cell lines is shown by immunoblotting. B, representative images of crystal violet–stained primary carcinosarcoma cells that have invaded into 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 ± SEM of two independent experiments. **, P < 0.01; ***, P < 0.001; one-way ANOVA with Tukey multiple comparisons test. ns, not significant.

Close modal

RACGAP1 regulates STAT3 phosphorylation and survivin expression in uterine carcinosarcoma

On the basis of a prior report that RACGAP1 facilitates the Tyr705 phosphorylation of STAT3 and promotes its translocation to the nucleus (15), we evaluated the relationship of RACGAP1, phospho-STAT3 (Y705), and total STAT3 in uterine carcinosarcoma (Fig. 4A). The expression of survivin (encoded by the BIRC5 gene, baculoviral inhibitor of apoptosis repeat containing 5), a transcriptional target of STAT3, was also evaluated by immunoblotting and qRT-PCR. In carcinosarcoma tumor tissues, RACGAP1 expression was significantly correlated with levels of phospho-STAT3 (Fig. 4B) and with survivin expression (Fig. 4C). Furthermore, we found that RACGAP1 knockdown decreased phosphorylation of STAT3 and reduced survivin expression in CS99 cells (Fig. 4D). On the basis of these data, STAT3 and survivin are bona fide downstream targets of RACGAP1 in uterine carcinosarcoma.

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 = 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 = 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 = 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 = −0.76; *, 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, r = −0.75; *, 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.

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 = 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 = 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 = 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 = −0.76; *, 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, r = −0.75; *, 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.

Close modal

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

YM155 is a first-in-class small molecule that selectively suppresses survivin at the mRNA and protein level (16, 17). Results of cytotoxicity assays showed that YM155 potently inhibited the proliferation of primary and established carcinosarcoma cell lines, with IC50 concentrations ranging from 1.9 nmol/L (for CS99) to 24.3 nmol/L (for CS017; Supplementary Table S5). Target inhibition was confirmed by immunoblotting that showed reduced survivin protein expression at these YM155 concentrations (Supplementary Fig. S2). The IC50 concentrations inversely correlated with RACGAP1 and survivin mRNA expression levels (Fig. 4E and F). On the basis of these data, high RACGAP1 expression predicts sensitivity to anti-survivin therapy. The effect of YM155 on the invasiveness of CS99 cells was determined using the Matrigel-coated Boyden chamber assay (Fig. 4G). In a dose-dependent fashion, YM155 significantly reduced the invasive capacity of CS99 cells (Fig. 4H).

RACGAP1 overexpression promotes invasiveness of endometrial cells

Next, the effect of RACGAP1 overexpression was evaluated in the endometrioid adenocarcinoma cell line Hec1b. Expression of full-length RACGAP1 following transfection was confirmed by immunoblotting (Fig. 5A). The invasive capacity of Hec1b/RACGAP1–transfected cells was significantly increased compared with Hec1b/empty vector–transfected cells (Fig. 5B). These data suggest that increased RACGAP1 expression is sufficient to promote invasive ability in this cell line model of endometrioid endometrial cancer, which is the more common and less aggressive histologic type of uterine corpus cancer.

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 = 57). RACGAP1 and BIRC5 (survivin) mRNA expression were positively correlated in this independent cohort of 57 patients with carcinosarcoma; Pearson correlation coefficient, r = 0.31; *, P < 0.05. D, RACGAP1 expression and higher stage of disease in the TCGA cohort of uterine carcinosarcoma (n = 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).

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 = 57). RACGAP1 and BIRC5 (survivin) mRNA expression were positively correlated in this independent cohort of 57 patients with carcinosarcoma; Pearson correlation coefficient, r = 0.31; *, P < 0.05. D, RACGAP1 expression and higher stage of disease in the TCGA cohort of uterine carcinosarcoma (n = 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).

Close modal

Correlation of RACGAP1 and survivin expression in uterine carcinosarcoma

To validate our findings in an independent cohort, we obtained deidentified clinical data and gene expression data from the TCGA uterine carcinosarcoma cohort (N = 57 patients). First, we determined the correlation of RACGAP1 and survivin (BIRC5) in these patients. As in our institutional cohort, RACGAP1 and survivin mRNA levels were significantly positively correlated in the TCGA cohort (Fig. 5C). The difference in the absolute correlation coefficient of RACGAP1 and survivin in our institutional cohort versus the multi-institutional TCGA cohort could be related to the different method of measuring mRNA levels. In the TCGA cohort, mRNA expression levels of RACGAP1 and survivin were determined by RNA-seq on an Illumina HiSeq platform. In the correlation analysis for our institutional cohort, mRNA levels were quantified by qRT-PCR, using optimized primers and linear amplification conditions, as well as internal normalization.

Increased risk of extrauterine disease in patients with high RACGAP1-expressing carcinosarcoma

As our data showed RACGAP1 regulates the migratory and invasive behavior of carcinosarcoma cells, we hypothesized that patients with high RACGAP1-expressing tumors would be more likely to present 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 carcinosarcoma patients from the TCGA was done to evaluate the potential relationship of RACGAP1 and stage. As shown in Fig. 5D, patients with higher stage of disease (indicating cancer spread beyond the uterus) had significantly higher RACGAP1 mRNA expression compared with patients without spread of disease. These findings in an independent cohort of patients with carcinosarcoma support the role of RACGAP1 as a metastatic driver in this disease, consistent with the findings in our cell line models of carcinosarcoma.

To our knowledge, this study is the first to apply RNA-seq technology to compare clinical tumor samples of uterine carcinosarcoma, a highly aggressive malignancy, with normal endometrial tissue. Using this approach, we have identified RACGAP1 as a critical upregulated gene and driver of the metastatic phenotype in uterine carcinosarcoma. In functional assays, we show that RACGAP1 depletion abolishes the migratory and invasive capacity of uterine carcinosarcoma cells, while RACGAP1 overexpression confers a metastatic phenotype to endometrial adenocarcinoma cells. In addition, we have found that high RACGAP1 expression is significantly correlated with extrauterine spread of disease in patients, substantiating its clinical relevance in uterine carcinosarcoma.

Our data show that RACGAP1 is a key regulator of STAT3 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 carcinosarcoma cells, we found that RACGAP1 depletion reduces STAT3 phosphorylation and survivin expression. Analysis of two independent cohorts of patients with uterine carcinosarcoma showed that RACGAP1 and survivin expression are significantly positively correlated in tumor samples.

On the basis of these novel findings, we evaluated the therapeutic potential of a first-in-class survivin inhibitor YM155 using primary carcinosarcoma cell lines developed from human carcinosarcoma tumors. YM155 is a first-in-class inhibitor of survivin that shows promising therapeutic activity in preclinical and clinical trials. For many targeted therapies, overexpression of the target (in this case, survivin) correlates with increased sensitivity to the therapeutic agent. Recently, survivin overexpression was identified in anaplastic thyroid cancer; the authors performed a high-throughput screen of >3,000 drugs and identified the survivin inhibitor YM155 as one of the most active agents in that aggressive disease (18). In our studies, we found that YM155 showed potent antiproliferative activity in primary and established uterine carcinosarcoma lines and moreover, abolished the invasive capacity of carcinosarcoma cells. Furthermore, the expression levels of RACGAP1 and survivin predicted sensitivity to YM155 in primary carcinosarcoma cell lines. Phase I trials have shown that YM155 is well-tolerated with a very favorable safety profile (19, 20). Phase II trials in several disease sites have demonstrated single-agent activity, for example, in patients with advanced, refractory non–small cell lung cancer and castration-resistant prostate cancer (21, 22). The antitumor activity of YM155 in patients with uterine carcinosarcoma awaits future investigation.

RACGAP1, also known as MgcRacGAP, was initially identified as a GTPase-activating protein (GAP) expressed in testis and male germ cells (23). Unlike other GAPs, RACGAP1 is an essential component of the centralspindlin complex (with the kinesin KlF23), where its GAP activity is required for cytokinesis and inactivation of Rac1 at the cleavage furrow (24–28). RACGAP1 also regulates tethering of the mitotic spindle to the plasma membrane during cytokinesis (29). Apart from its role in cytokinesis, RACGAP1 was shown to regulate STAT3 activation in leukemia cells (30), and may also play a role in regulating endothelial permeability (31). Recently, RACGAP1 expression has been linked with aggressive clinical behavior in several cancers, 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 targeting the metastatic phenotype in diverse tumor types.

Very little has been previously published regarding underlying drivers of the development and progression of this disease, and there is a dire lack of effective therapies for this highly lethal disease. This knowledge gap is one of the motivators for the current study, which has led to our discovery of a key metastatic driver and identification of a promising, novel targeted therapy. In summary, RACGAP1 promotes the metastatic phenotype in uterine carcinosarcoma via a STAT3/survivin signaling pathway. The survivin inhibitor YM155 potently suppresses carcinosarcoma cell proliferation and abrogates the invasive capacity of carcinosarcoma cells. We suggest that inhibition of the RACGAP1–STAT3 survivin pathway should be investigated as a novel therapeutic strategy for this lethal malignancy, as well as for other high RACGAP1–expressing cancers.

No potential conflicts of interest were disclosed.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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, G.S. Huang

Acquisition 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. Huang

Analysis 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. Huang

Writing, 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. Huang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Lin, G.S. Huang

Study supervision: G.S. Huang

The results shown are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.

This work was supported by the Albert Einstein Cancer Center Support Grant of the NIH under award number P30CA013330 and Albert Einstein Cancer Center Pilot Award (to G.S. Huang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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