MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

TGFb and Hippo Pathways Cooperate to EnhanceSarcomagenesis and Metastasis through theHyaluronan-Mediated Motility Receptor (HMMR) A C

Shuai Ye1, Ying Liu1, Ashley M. Fuller1, Rohan Katti1, Gabrielle E. Ciotti1, Susan Chor1, Md. Zahidul Alam1,Samir Devalaraja1, Kristin Lorent2, Kristy Weber3, Malay Haldar1, Michael A. Pack2, andT.S. Karin Eisinger-Mathason1

ABSTRACT◥

High-grade sarcomas are metastatic and pose a serious threat topatient survival. Undifferentiated pleomorphic sarcoma (UPS) is aparticularly dangerous and relatively common sarcoma subtypediagnosed in adults. UPS contains large quantities of extracellularmatrix (ECM) including hyaluronic acid (HA), which is linked tometastatic potential. Consistent with these observations, expressionof the HA receptor, hyaluronan-mediated motility receptor(HMMR/RHAMM), is tightly controlled in normal tissues andupregulated in UPS. Moreover, HMMR expression correlates withpoor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in thesepatients. YAP1, the transcriptional regulator and central effector ofHippo pathway, is aberrantly stabilized in UPS and was recentlyshown to control RHAMM expression in breast cancer cells.Interestingly, both YAP1 and RHAMM are linked to TGFb sig-naling. Therefore, we investigated crosstalk between YAP1 and

TGFb resulting in enhanced RHAMM-mediated cell migration andinvasion. We observed that HMMR expression is under the controlof both YAP1 and TGFb and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, wefound that RHAMM expression promotes tumor cell proliferationand migration/invasion. To test these observations in a robust andquantifiable in vivo system,we developed a zebrafish xenograft assayof metastasis, which is complimentary to our murine studies.Importantly, pharmacologic inhibition of the TGFb–YAP1–RHAMM axis prevents vascular migration of tumor cells to distantsites.

Implications: These studies reveal key metastatic signalingmechanisms and highlight potential approaches to prevent meta-static dissemination inUPS.YAP1 andTGFb cooperatively enhanceproliferation and migration/invasion of UPS and fibrosarcomas.

IntroductionSoft-tissue sarcomas (STS) are mesenchymal tumors arising from

muscle, fat, cartilage, and connective tissue etc. Because of theirkaryotype complexity, subtype heterogeneity, and the lack of commondriver mutations, adult sarcomas are not well understood. The paucityof molecular characterization has resulted in few therapeutic advancesbeyond standard resection/radiation over the last 30 years (1). Ourwork focuses on undifferentiated pleomorphic sarcoma (UPS) andfibrosarcomas, aggressive adult tumors found in skeletal muscle andconnective tissues. These are commonly diagnosed subtypes, relativeto other sarcomas, and high-grade occurrences are particularly resis-

tant to therapeutic approaches (2). We found that the central Hippoeffector, Yes associated protein 1 (YAP1), is stabilized in human UPStumors and promotes pro-proliferation and anti-differentiation tran-scriptional programs (3, 4). YAP1 is unusually stable in UPS andpotentially other sarcomas due to epigenetic silencing of its inhibitor,angiomotin (AMOT; ref. 5), and Hippo kinase copy number loss (3).These perturbations stabilize YAP1 at the protein level and enhance itsnuclear localization and subsequent transcriptional activity (6). In theabsence of a specific inhibitor for YAP1, we identified epigeneticmodulators including suberoylanilide hyroxamic acid (SAHA; Vor-inostat), and the BET bromodomain inhibitor JQ1, which can reduceYAP1 activity in combination. Importantly, SAHA/JQ1 inhibited UPSgrowth in murine models of UPS. Although SAHA/JQ1 treatment haswidespread effects, we use these tools to interrogate and then validateYAP1-mediated signaling and phenotypes.

Having established the role of YAP1 in UPS and fibrosarcomas, weare currently investigating the role of YAP1-associated pathways,including TGFb signaling, in sarcomagenesis. TGFb is a morphogenthat binds to specific serine-threonine kinase receptors, which phos-phorylate SMAD proteins when engaged (7). Downstream of receptoractivation, phosphorylated SMADs interact with transcriptional reg-ulators, including YAP1, to promote proliferation and metastasis (8).Whereas TGFb and YAP1 activity are linked in developmental con-texts (9, 10) and in various epithelial tumors (i.e., breast cancer; ref. 11),their relationship in mesenchymal tumors is entirely unknown. Fur-thermore, both TGFb (12) and YAP1 (13) are known to promotemetastasis in various epithelial contexts, an area that also remainsunclear in sarcoma. Our recent work has focused entirely on the role ofYAP1 in proliferation and tumorigenesis. Here we investigate the

1Abramson Family Cancer Research Institute, Department of Pathology &Laboratory Medicine, Penn Sarcoma Program, University of Pennsylvania Perel-man School of Medicine, Philadelphia, Pennsylvania. 2Division of Gastroenter-ology, Department of Medicine, Perelman School of Medicine, University ofPennsylvania Philadelphia, Pennsylvania. 3Department of Orthopedic Surgery,Penn Sarcoma Program, University of Pennsylvania Perelman School of Med-icine Philadelphia, Pennsylvania.

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

Corresponding Author: T.S. Karin Eisinger-Mathason, Abramson Family CancerResearch Institute, University of Pennsylvania, 414 BRB II/III, 421 Curie Boule-vard, Philadelphia, PA 19104-6160. Phone: 215-898-9086; Fax: 215-746-5511;E-mail: karineis@pennmedicine.upenn.edu

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doi: 10.1158/1541-7786.MCR-19-0877

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ability of these pathways to coordinate metastasis, as well as cellularproliferation.

Our goal was to identity critical overlapping downstream tran-scriptional targets of TGFb and YAP1 in sarcomas that promoteprimary tumorigenesis, metastatic dissemination, or both for com-binatorial therapy development. Previously, we identified NFkB asthe primary driver of proliferation and tumorigenesis in UPS cells.In this study, we report that TGFb signaling promotes NFkBactivity as well, lending further support to our assertion that Hippopathway deregulation and TGFb activation cooperatively enhancesarcomagenesis.

We also sought evidence that these pathways converge to regulatemetastasis. The hyaluronan-mediated motility receptor (HMMR)gene (14), which encodes the hyaluronic acid (HA) surface receptorRHAMM, is a known transcriptional target of TGFb signaling (15)and has recently been linked to YAP1 (16, 17). RHAMM is alsoassociated with fibrosarcoma progression (18). RHAMM physicallyinteracts with HA, a component of the extracellular matrix (ECM)and a major part of the tumor microenvironment in many cancers.HA binding engages RHAMM signaling that has both cytoplasmiceffects on MAPK activity and proliferation as well as nuclear effectson the transcription of key cell motility effectors (19, 20).

Although there is extensive literature linking RHAMM activity toTGFb signaling, the connection between RHAMM and YAP1 issignificantly more limited, and not well understood. Therefore, weexplored the relationship between YAP1 and RHAMM, as well asTGFb, in sarcomas. We found that YAP1 controls HMMR/RHAMMexpression in both murine and human sarcoma cell lines. Consistentwith these observations, pharmacologic inhibition of YAP1 and TGFbrepressed HMMR/RHAMM expression resulting in reduced tumorgrowth in our in vivo allograft assays. We also investigated the roleof TGFb and YAP1-mediated HMMR/RHAMM expression insarcoma cell motility and invasion, and found significant effectson these processes. Importantly, it is extremely difficult to distin-guish between effects on tumor size and metastasis in vivo, when agiven target affects both processes. Reduction in primary tumor sizecan correlate with less metastasis. This observation is particularlyaccurate in sarcomas, wherein low intratumoral oxygen (hypoxia)and tumor size are accurate predictors of clinical outcome (21–27).Therefore, we employed a novel zebrafish xenograft system thatallows us to specifically interrogate the effects of TGFb and YAP1inhibition on metastasis in vivo while bypassing the potentiallyconfounding influence of proliferation-associated factors (28–30).Using this system, we observed that pharmacologic inhibition ofboth pathways suppressed the metastatic cascade. Collectively, ourfindings suggest that TGFb signaling, which is upregulated in UPSand fibrosarcomas, cooperates with YAP1 to control HMMR/RHAMM, thus promoting tumorigenesis and metastasis. Interro-gation of this mechanism and its physiologic impact may helpelucidate important new biomarkers and therapeutic targets for thetreatment of human sarcoma.

Materials and MethodsGEMM

All experiments were performed in accordance withNIH guidelinesand were approved by the University of Pennsylvania InstitutionalAnimal Care and Use Committee. We generated KrasG12Dþ; Trp53fl/fl;tumors by injection of a calcium phosphate precipitate of adenovirusexpressing Cre recombinase (University of Iowa) into the rightgastrocnemius muscle of 3- to 6-month-old mice.

Allograft mouse modelAll experiments were performed in accordance with NIH guide-

lines and were approved by the University of Pennsylvania Insti-tutional Animal Care and Use Committee. For RHAMM-mediatedknockdown KP230 allografts, 1 � 106 cells were injected intothe flanks of 6-week-old nu/nu mice (Charles River Laboratories)with control tumor cells (scrambled shRNA) and experimentaltumor cells (RHAMM shRNA). Tumor size was measured everyother day and animals were euthanized after 19 days posttumorcells' injection. Tumor volume was calculated by using formula(ab2)p/6, where a is the longest measurement and b is the shortest.We also performed subcutaneous tumor experiments using a cellline derived from 30-methylcholanthrene (MCA)-induced murinefibrosarcoma in the C57BL/6 strain, obtained from the Schrieberlaboratory at Washington University of St. Louis (St. Louis, MO;ref. 31). Tumor cells were propagated in vitro for two passagesprior to implantation and injected cells were greater than 90%viable. We implanted 1 � 106 MCA tumor cells into shaved flanksof recipient C57BL/6 mice and harvested tumors at 14 days post-implantation for analysis. Single-cell suspensions were generatedby collagenase and DNAse digestion, incubated for 5 minutes atroom temperature with anti-mouse CD16/32 Fc Block, and subse-quently stained with fluorescently labeled antibodies for identifi-cation of macrophage and tumor cell populations by FACS. Cellswere sorted on a BD FACS Jazz instrument and RNA was isolatedfrom sorted cell pellets using Sigma GenElute Mammalian TotalRNA Miniprep Kit.

Cell linesHuman HT-1080 (fibrosarcoma), HCT-116 (colorectal cancer)

and HEK-293T cell lines were purchased from ATCC. STS-109 cellswere derived from human UPS tumors by Rebecca Gladdy, MD(University of Toronto, Toronto, Ontario, Canada) and RE cellswere derived from human UPS tumors by Kurt Weiss, MD (Uni-versity of Pittsburgh Medical College, Pittsburgh, PA). TC32 cells(Ewing sarcoma) were obtained from Patrick Grohar MD, PhD(Children's Hospital of Philadelphia, Philadelphia, PA). KP230,KP250, and KIA cell lines were derived from UPS mouse tumorsas described in Eisinger-Mathason and colleagues (4). Short tandemrepeat analysis was performed at the time of derivation andconfirmed in April 2015. Cells were purchased, thawed, and thenexpanded in the laboratory. Multiple aliquots were frozen downwithin 10 days of initial resuscitation. For experimental use, aliquotswere resuscitated and cultured for up to 20 passages (4–6 weeks)before being discarded. STS-48, STS109 and RE cells were culturedin DMEM with 20% (vol/vol) FBS and the other cells were culturedin DMEM with 10% (vol/vol) FBS and 1% penicillin/streptomycin.All cell lines were confirmed to be negative for Mycoplasmacontamination.

Drug treatments and reagentCells were treated with SB525334, SAHA (Sigma-Aldrich), trame-

tinib (Advanced Chemblock), and JQ1 (gift from Jun Qi, PhD,Harvard University, Boston, MA). Drugs were refreshed for cellstreated longer than 48 hours.

Lentiviral transductionShort hairpin RNA (shRNA)-mediated knockdown of Yap1 TRCN:

0000095864, 0000095866, 0000095867, 0000095868; YAP1 TRCN:0000107266, 0000107267; Hmmr TRCN: 0000071588, 0000071589,0000071590, 0000071591, 0000071592; HMMR TRCN: 0000061553,

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0000061554, 0000061555, 0000061556, 0000061557; Rela (p65 NFkB)TRCN: 0000055343, 0000055344, 0000055347; and scramble shRNAwere obtained from Dharmacon. shRNA plasmids were packaged byusing the third-generation lentivector system (VSV-G, p-MDLG, andpRSV-REV) and expressed in HEK-293T cells. Supernatant wascollected at 24 and 48 hours after transfection and subsequentlyconcentrated by using 10-kDa Amicon Ultra-15 centrifugal filter units(Millipore). Lenti-pcdh-EF1 was used as the empty vector control inour laboratory. Mouse and humanHMMRGene cDNA clone plasmidwere obtained from Sino Biological Inc.

IHCFifteen human UPS paraffin-embedded tissues obtained from

the Surgical Pathology group at University of Pennsylvaniawere stained for p-Smad3. Subcutaneous tumor paraffin-embedded tissues were stained for CD168 and Ki67. IHC wasperformed on 5-mm–thick tissue sections according to standardprotocols. Slides were digitally scanned by Pathology Core Labo-ratory at the Research Institute at the Children's Hospital ofPhiladelphia (Philadelphia, PA). Aperio ImageScope (Leica Biosys-tems) software was used for slide quantification. Modified macro(Nuclear v9 parameter) was used to distinguish intensity of staining.Fifteen tumor sections and 9 adjacent muscle sections were quan-titated. Five areas of tumor and 5 areas of adjacent muscle[identified by hematoxylin and eosin (H&E) staining] were capturedper section. Data from the Aperio IHC Nuclear algorithm were usedto calculate the “average intensity � % positivity” metric. Thismetric is independent of the number of nuclei within a given slideand considers the contribution of both positive and negative nucleito staining intensity. For each slide, the “accumulated intensity”output from Aperio (the cumulative intensity of all positivelystained nuclei) was divided by the total number of cells. Thiscalculation yielded an “average intensity” value (the average inten-sity of both positively and negatively stained nuclei), which wasthen multiplied by the “percent positive” output. Values weredivided by 1,000 for graphical representation purposes. The fol-lowing antibodies' concentrations were used: rabbit anti-p-Smad3(phospho S423/S425; 52903; 1:50; Abcam), rabbit anti-Ki-67(15580; 1:100; Abcam), and rabbit anti-CD168 antibody (124729,1:100; Abcam).

ImmunoblotsProtein lysate was prepared in SDS/Tris (pH7.6) lysis buffer,

separated by electrophoresis in 8%–10% SDS/PAGE gels, transferredto nitrocellulose membrane, and probed with the following antibodies:rabbit anti-YAP1 (4912; 1:1,000), rabbit anti-p-Yap (Ser397; 13619;1:1,000), rabbit anti-p-Smad2 (S465/467; 3108; 1:1,000), rabbit anti-Smad2 (5339; 1:1,000), rabbit anti-p-Smad3 (S423/425; 9520; 1:1,000),rabbit anti-p65 (8242; 1:500), rabbit anti-GAPDH (2118; 1:1,000),rabbit anti-p-44/42 Erk1/4 (Thr202/Tyr204; 4377; 1:1,000), rabbitanti-44/42 Erk1/4 (4695; 1:1,000; Cell Signaling Technology), rabbitanti-CD168 antibody (124729, 1:100; Abcam), rabbit FOXM1 (sc-502,1:500; Santa Cruz Biotechnology).

Oncomine, TCGA survival, and cBioPortal analysisWe used the publicly available database (32 and 33) through

Oncomine Research Premium edition software (version 4.5, LifeTechnologies) to query RHAMM expression and survival. RHAMMgene expression in overall survival from malignant fibrous histio-cytoma (MFH)/UPS in TCGA Adult Soft Tissue Sarcoma Data-set (34). Kaplan–Meier analyses were performed for OS of patients.

Through cBioPortal database, we queried HMMR gene amplifica-tion in 89 (60%) of 149 total sequenced patients.

Cell proliferation assayCells were plated in triplicate for each time point and incubated

overnight in tissue culture dishes. The following day, DMSO orSB525334, diluted in growth media, was added to the cells. Cells weretrypsinized, resuspended in PBS, and counted using a hemocytometeron the days indicated.

Wound-healing assayTo determine the effect of RHAMM on sarcoma cell migration,

wound-healing assays were performed with RHAMM-silenced celllines, overexpressed RHAMMcell lines, andTGFb receptor 1 inhibitor(SB525334; Tocris) pretreated cells for 48 hours. Wounds were madethrough the monolayer using a 200 mL pipette tip. Wounds weremeasured over a time course to calculate the migration rate accordingto the following formula: [(initial wound length)�(final woundlength)]/(initial wound length) � 100. The experiments were per-formed more than three times.

Matrigel invasion assayIn vitro cell invasion was performed using the BD Biocoat

Matrigel Invasion Chamber (BD Biosciences) with endogenousRHAMM-silenced cell lines HT-1080 and KP230 according to themanufacturer's protocol. Briefly, the Matrigel-coated chamberswere rehydrated in 1 mL DMEM/F12 for 2 hours in a humidifiedtissue culture incubator with a 37�C, 5% CO2 atmosphere prior tothe experiments. A total of 8 � 104 HT-1080 cells and 1.6 � 104 KP230 cells were seeded on to Matrigel-coated filters. After 24 hours ofincubation, the filters were stained using 0.2% crystal violet and thenumber of traversed cells was counted on an inverted microscope.For each membrane, five random fields were selected, and theinvasion rates were determined by the percent invasion formula:percentage invasion ¼ (mean number of cells invading through theMatrigel insert membrane)/(mean number of cell migratingthrough the control insert membrane) � 100.

qRT-PCRTotal RNA was isolated from tissues and cells using the TRIzol

reagent (Life Technologies) and RNeasy Mini Kit (Qiagen). Reveretranscription ofmRNAwas performed using theHigh-Capacity RNA-to-cDNAKit (Life Technologies). qRT-PCR was performed by using aViiA7 apparatus. All probes were obtained from TaqMan “Bestcoverage” (Life Technologies). HPRT was used as an endogenouscontrol.

Methods for zebrafish microinjectionAll procedures on zebrafish (Danio Rerio) were approved by the

Institutional Animal Care and Use Committee of the University ofPennsylvania (Philadelphia, PA). Fertilized zebrafish eggs of thetransgenic strain expressing enhanced GFP under the fli promoter(fli:EGFP) were incubated at 28�C in E3 solution and raised usingstandard methods. Embryos were transferred to E3 solution con-taining 0.2 mmol/L1-phenyl-2-thio-urea (PTU, Sigma) 6 hourspostfertilization to prevent pigmentation. Proteases were added toPTU-E3 solution on day 1 to dechorionate the fish embryos. At48 hours postfertilization, zebrafish embryos were anesthetizedwith 0.03% tricaine (Sigma) and then transferred to an injectionplate made with 1.5% agarose gel for microinjection. Approximately200 to 400 KIA/mCherry cells (�5 nL) suspended in complete

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DMEM medium further supplemented with 0.5 mmol/L EDTAwere injected into the yolk sac of each embryo using a XenoWorksDigital Microinjector (Sutter Instrument). Prepulled nonfilamen-tous borosilicate micropipettes were used for the microinjection(Tip ID 50 mm, base OD 1 mm, length 5.5 cm, FivephotonBiochemicals). After injection, the fish embryos were immediatelytransferred to PTU-E3 solution. Injected embryos were kept at 33�Cand were examined every day to monitor tumor migration usinga dissecting fluorescent microscope (Olympus MVX10). Confocalimages on embryos showing distant metastasis were captured usinga spinning disk microscope (Olympus Ix81 with an Andor iXon3EMCCD camera) and analyzed using software FIJI.

Statistical analysisStatistical analysis was performed using Prism (GraphPad Soft-

ware). Data are shown as mean� SEM or SD. Data were reported asbiological replicates with technical replicates indicated in figurelegends. Student t tests (unpaired two-tailed) were performed todetermine whether a difference between two values is statisticallysignificant different, with a P < 0.05 considered significant. ANOVAwas used for assays with multiple variables to compare.

ResultsTGFb signaling promotes sarcoma cell migration and isassociated with disease progression

Our recent work demonstrated that deregulation of the Hippopathway, which results in stabilization of YAP1, promotes UPSinitiation and growth (3, 5). In some contexts, YAP1-mediatedtranscription is dependent on its interaction with phosphorylatedSMAD3 (p-SMAD3). Therefore, we investigated the role of TGFb,which signals through activated SMAD3, in UPS. We observed thatlike YAP1 nuclear p-SMAD3 staining is significantly elevated inhuman UPS relative to skeletal muscle, suggesting a link betweenTGFb and YAP1-mediated UPS development (Fig. 1A). We alsointerrogated TGFb activation in the genetically engineered mousemodel of UPS, LSL-KrasG12D/þ; Trp53fl/fl (KP). In this model,adenovirus expressing Cre recombinase is injected into the gas-trocnemius muscle, activating oncogenic Kras expression anddeleting p53 in muscle progenitor cells (35, 36). This model anda similar one, KrasG12D/þ; Ink4a/arffl/fl (KIA), generate sarcomasthat are histologically, transcriptionally, and morphologically iden-tical to UPS and are thus the standard GEMMs in UPS stud-ies (35, 36). Although Kras mutation is rare in human sarcomas,activation of MAPK downstream of KRAS is common in UPS and isa reliable prognostic indicator (37). To fully characterize MAPKstatus in our experimental systems, we evaluated p-ERK1/2 levelsand sensitivity to MEKi in HT-1080 human fibrosarcoma cells,which express mutant oncogenic N-Ras (ATCC), and murine cellsderived from KP tumors. Interestingly, we observed that HT-1080cells maintain higher basal levels of MAPK activity than KP cells(Supplementary Fig. S1A). Consistently, we found that KP cells areless sensitive to MEKi (trametinib) and require higher doses oftrametinib to completely inhibit proliferation (SupplementaryFig. S1B and S1C). Finally, we found that treating each cell typewith a dose of trametinib that halts proliferation (HT-1080; 50nmol/L and KP; 150 nmol/L) results in roughly the same cellularresponses. Specifically, using Western blot analysis, we determinedthat p-ERK1/2, RHAMM, and other proliferation targets werereduced in response to trametinib in both cell types (SupplementaryFig. S1D and S1E).

Importantly, Yap1 is stabilized in KP GEMM tumors (5), anddeletion of YAP1 in this system (KPY) increased tumor latency andreduced tumor weight and volume relative to KP (5). Our assessmentof nuclear p-SMAD3 levels in KP tumors phenocopies human UPS.Consistent with our observation in human UPS, p-SMAD3 staining issignificantly upregulated in KP tumors relative to murine skeletalmuscle (Fig. 1B). Using cBioPortal analysis of The Cancer GenomeAtlas (TCGA) Soft Tissue Sarcoma Adult Dataset annotated in2017 (34), we also observed that more than 50% of human UPS andmyxofibrosarcomas, which are genetically indistinguishable fromUPS (34), bear alterations in the TGFb pathway (Fig. 1C). Weobserved similar alterations in liposarcomas using cBioPortal analysisof the MSKCC Broad dataset (Supplementary Fig. S2A). The majorityof tumors with enhanced expression of TGFb pathway membersincluding TGFbR1, TGFb1, and SMAD3, also bear mutations incommon sarcoma tumor suppressors like TP53 and ATRX, andexpress high levels of FOXM1, a YAP1 transcriptional target. Together,these data suggest that TGFb activity plays a role in UPS initiation orprogression. In contrast, mutations in the TGFb pathway are uncom-mon in colorectal cancer (Supplementary Fig. S2B) and colorectalcancer cell proliferation is independent of TGFb (38–41). These datasupport the conclusion that TGFb signaling promotes sarcoma cellproliferation but may not be involved in proliferation of certaincarcinoma cells. To validate these observations, we treated HT-1080and KP cells with increasing doses of the TGFb inhibitor SB525334(Fig. 1D and E). Treatment with 1 mmol/L SB525334 for 5 days hadno effect on HT-1080 proliferation, although higher doses weresignificantly more impactful. Interestingly, KP cell proliferation wasmore sensitive to TGFb inhibition. However, pretreatment with 1mmol/L SB525334 treatment had significant effects on migration inboth cell types, suggesting that cell migration is more uniformlysensitive to TGFb signaling among sarcoma subtypes (Fig. 1F andG). To demonstrate the specificity of our proliferation results tosarcoma, we stained a tissuemicroarray of normal colon and colorectalcancer samples with p-SMAD3 and treated HCT-116 colorectalcancer cells with SB525334.We observed that colorectal cancer tissuesstain positive for p-SMAD3, relative to normal tissue, (SupplementaryFig. S2C and S2D). However, TGFb inhibition has extremely minoreffects on colorectal cancer proliferation (Supplementary Fig. S2E,top). These findings are consistent with earlier reports indicating thatTGFb signaling specifically promotes migration and invasion incolorectal cancer (42). Finally, we evaluated the effect of SB525334on an additional sarcoma subtype, Ewing sarcoma. SB525334(5 mmol/L) dramatically reduced proliferation in the Ewing cell lineTC32 (Supplementary Fig. S2E, bottom). We conclude that TGFbsignaling plays important and perhaps specific roles in sarcoma growthand dissemination, whereas its effects are limited to tumor progressionin colorectal cancer.

TGFb and YAP1 cooperatively regulate HMMR/RHAMMexpression

In previous studies, we reported that YAP1mediates proliferation inUPS cells and tissues by upregulating NFkB activity. Our findingsrevealed that regulation of NFkB is a critical function of YAP1 inmuscle-derived sarcomas. Therefore, we hypothesized that TGFbsignaling is also required for NFkB activity in UPS. To test this idea,we treated KP230 cells with 1 mmol/L SB525334 and observeddecreased phosphorylation of p65/NFkB, which is indicative of tran-scriptional inactivation (Fig. 2A). We also saw loss of Foxm1 expres-sion in response to TGFb inhibition, which is consistent with loss ofYAP1 activity. FOXM1 is a pro-growth factor that promotes UPS

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proliferation. Although we have done significant work on YAP1 andFOXM1 in sarcoma cell proliferation and tumor formation, we haveyet to determine whether YAP1 also enhances metastasis in thiscontext or the cellular functions preceding metastatic dissemination,including cell migration and invasion. YAP1 is known to promotethese processes in epithelial cancers. However, its impact onmetastasisof mesenchymal tumors is unknown. In breast cancer, YAP1 regulatesHMMR/RHAMM, a surface receptor that responds to HA and isassociated with metastasis. Here we observed that TGFb inhibitiondecreases RHAMM expression in UPS cells (Fig. 2A). The specific

effects of TGFb inhibition on sarcoma cell proliferation, migration,and invasion suggest high levels of TGFb cytokine production in theUPS tumor microenvironment. To validate Tgfb expression in vivo,and to determine the source of Tgfb production, we fractionated KPas well as 30-methylcholanthrene (MCA)-induced fibrosarcomasand isolated tumor-associated macrophages (MAC), which areknown to produce significant levels of cytokines including Tgfb.We compared Tgfb expression in isolated KP and MCA fibrosar-coma tumor cells to infiltrating macrophages. Tgfb mRNA expres-sion was approximately 6 fold higher in infiltrating MACs than KP

Figure 1.

TGFb stimulates sarcoma cell proliferation andmigration.A,H&E and IHC of p-SMAD3 from human UPS tumor and skeletal muscle sections. Scale bar, 50 mm (tumorn¼ 15, muscle n¼ 9; � , P <0.001; SD).B,H&E and IHC for p-Smad3 fromKP230 tumor and skeletal muscle sections. Scale bar¼ 50 mm(� , P <0.001; SD).C, cBioPortalanalysis of TGFb pathway genetic alterations in patients with myxofibrosarcoma and UPS from the TCGA Soft tissue Sarcoma in Adults dataset. Proliferation assayof HT-1080 (D) and KP230 cells (E) treated with SB525334 1 mmol/L, 5 mmol/L, and 10 mmol/L (� , P < 0.05; SD). Scratch migration assay of HT-1080 (F) and KP230cells (G) pretreated with SB535334 (1 mmol/L) for 48 hours (� , P < 0.0001, n ¼ 3; SD).

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tumor cells (Fig. 2B, left) and approximately 30-fold higher inMACs from MCA-induced sarcomas relative to tumor cells(Fig. 2B, right). Together, these data suggest that macrophagesproduce Tgfb in the sarcoma microenvironment.

Collectively, our data suggest that TGFb signaling, which is acti-vated by tumor-infiltrating macrophage secretion of TGFb and YAP1cooperatively regulate HMMR/RHAMM expression. Therefore, wetested the contribution of YAP1 toHMMR/RHAMMexpression usingmultiple independent YAP1-specific shRNAs. Inhibition of YAP1suppressed HMMR levels in HT-1080 (Fig. 2C) STS-109 humanUPS (Fig. 2D) and KP230 cells (Fig. 2E). We observed similarresults at the protein levels by Western blot analysis of YAP1shRNA–expressing UPS and fibrosarcoma cells (Fig. 2F). Weconclude that both YAP1 and TGFb control RHAMM expressionin UPS and fibrosarcoma cells.

HMMR expression is linked to clinical outcomeOur laboratory studies revealed a clear link between HMMR/

RHAMM expression and the Hippo–TGFb axis. Next, we investi-gated expression of HMMR in human sarcomas and the relation-ship between HMMR and patient outcome. We found that HMMRis uniformly upregulated in UPS and myxofibrosarcoma tumors(Fig. 3A). Myxofibrosarcoma is genetically indistinguishable fromUPS (34). We also observed upregulation of HMMR mRNA in theMSKCCBroad (liposarcoma) and TCGA Soft Tissue Sarcomas inAdults datasets (UPS) via cBioPortal analyses (SupplementaryFig. S2A). Importantly, high HMMR expression correlated withpoor clinical outcome in patients with myxofibrosarcoma (Fig. 3B).Next, we investigated whether HMMR is coexpressed with otherYAP1 targets in UPS/myxofibrosarcoma and liposarcoma. Genes

that coexpressed significantly with HMMR in the TCGA Soft TissueSarcomas in Adults' datasets included, FOXM1, and BUB1B as wellas others that we identified as YAP1 targets in previous studies(ref. 3; Fig. 3C). On the basis of cBioPortal analyses of humansarcoma datasets, these targets are upregulated in human UPS/myxofibrosarcoma (Supplementary Fig. S3A). They are also inhib-ited in response to SAHA/JQ1 in KP cells (Supplementary Fig. S3B).Here we also show individual coexpression plots for HMMR versusFOXM1 or BUB1B in UPS/myxofibrosarcoma and liposarcoma(Fig. 3D; Supplementary Fig. S4A–S4C). Finally, we determinedthat RHAMM and the HA-binding protein HABP are highlyexpressed in human tissues by IHC (Supplementary Fig. S4D).These findings highlight the importance of HMMR/RHAMMexpression in human sarcoma, as well as the relationship betweenHMMR and YAP-mediated transcription in these tumors.

RHAMM expression is sensitive to epigenetic modulation ofYAP1 transcriptional activity

In previous studies, we reported that YAP1 activity is nearlyabolished in sarcoma cells treated with a combination of epigeneticmodulators including the histone deacetylase (HDAC) inhibitorSAHA (vorinostat), and the BET bromodomain inhibitor, JQ1 (5).Individually, SAHA is more effective at reducing proliferation andYAP1 expression relative to JQ1. However, we observed that thecombination of these drugs is by far the most effective pharmacologicmethod for inhibiting YAP1 expression and activity. Importantly,SAHA/JQ1 treatment does not trigger cell death, but rather inducesdifferentiation in sarcoma cells (5, 43). Although SAHA/JQ1 treatmenthas pleotropic effects, we use these tools to interrogate and thenvalidate YAP1-mediated signaling and related phenotypes. The

Figure 2.

TGFb and YAP1 cooperatively regulate RHAMM expression in soft-tissue sarcoma. A,Western blot analysis of KP230 cells treated with SB525334 1 mmol/L 48 hours.B, qRT-PCR ofmRNAderived from autochthonous KP andMCA-induced tumor cells (TCs) aswell as associatedmacrophages (MACs; � , P <0.0006; SD).C, qRT-PCRof HT-1080 STS109 (human UPS;D) and KP230 cells (E) transducedwith lentivirus expressing two shRNAs targeting Yap1 (�, P <0.01; SD). F,Western blot analysis ofHT-1080, STS109, KP230, and RE (human UPS) cells expressing control or YAP1 shRNAs.

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observation that YAP1 regulates HMMR/RHAMM expression ledus to hypothesize that SAHA/JQ1 treatment would inhibit thistarget. We treated KP230 cells and performed microarray analysisas well as qRT-PCR validation and found that both Yap1 andHmmrlevels were significantly inhibited by drug treatment (Fig. 4A–C).We also performed Western blot analysis of SAHA/JQ1–treatedcells for 0 to 72 hours and found that RHAMM expression is lost inHT-1080 (Fig. 4D), KP230 (Fig. 4E), and KIA (Fig. 4F) sarcomacells. Consistent with our prior studies, we observed less p65/NFkBphosphorylation and a decrease in overall YAP1 levels in treatedcells. Together, our findings indicate that RHAMM expression isdependent on YAP1 and TGFb signaling and could be pharmaco-logically repressed using inhibitors that modulate these pathways.The role of YAP1 in controlling NFkB activity led us to ask whetherHMMR/RHAMM is a target of this pathway or is regulated in anNFkB–independent manner. Therefore, we inhibited NFkB inKP230 cells using Rela-specific shRNA and observed decreases inHmmr (Fig. 4G) and Rhamm expression (Fig. 4H). We concludethat RHAMM expression is regulated by the YAP1–NFkB axis inaddition to TGFb signaling.

RHAMM modulates UPS growthOur current understanding of the YAP1–NFkB axis is restricted

to its effects on proliferation. Therefore, we began our functionalanalysis by modulating HMMR/RHAMM expression and evaluat-ing subsequent effects on proliferation. We used multiple indepen-

dent shRNAs targeting HMMR in HT-1080 and KP230 cells(Fig. 5A–C). Loss of HMMR/RHAMM significantly reduced cellproliferation in HT-1080 (Fig. 5D) and KP230 cells (Supplemen-tary Fig. S5A). We also performed the reverse experiment byectopically expressing RHAMM in HT-1080 cells (Fig. 5F) andobserved significantly enhanced cell proliferation (Fig. 5G).Although the effects were more modest, we observed similar effectsin KP cells (Supplementary Fig. S5B). We attribute the modestyof the effects to the significantly weaker expression of pcdh-Rhammin KP cells (Supplementary Fig. S5C and S5D) than pcdh-RHAMMin HT-1080 cells (Fig. 5E and F). We investigated the effectsof Hmmr/Rhamm loss in vivo in an allograft KP model by subcu-taneously implanting KP cells expressing control or Hmmr/Rhamm–specific shRNAs and found that Rhamm expression issignificantly lost based on IHC staining of tumor sections (Fig. 5Hand I). Inhibition of Hmmr/Rhamm reduced both Ki67 stainingand tumor volume/weight (Fig. 5J–L). We also confirmed thatRhamm protein expression was decreased in explanted tumor tissueby Western blot analysis (Fig. 5M). Finally, we tested the hypoth-esis that altered Rhamm expression resulted in a feedback loop,which modulated SMAD3 phosphorylation and TGFb activity. Lossof RHAMM in HT-1080 and KP230 cells had no reproducibleeffects on p-SMAD3 (Supplementary Fig. S5E). Collectively, thesefindings indicate that Rhamm expression promotes UPS prolifer-ation and tumorigenesis in vivo, but does not impact upstreamTGFb signaling.

Figure 3.

RHAMM expression correlates with poor outcome and YAP1-mediated sarcomagenesis. A, Oncomine gene expression analysis of RHAMM mRNA levels in normaltissues including skeletal muscle and adipose tissue as well as multiple sarcoma subtypes from the Detwiller and colleagues and Barretina and colleagues datasets.Eachbar represents an individual patient sample.B,Kaplan–Meier curve of overall survival of patientswithMFS (n¼ 17; TCGA sarcomadataset).C, cBioPortal analysisof genes coexpressed with HMMR in the Adult Soft Tissue Sarcoma TCGA dataset (MFS and UPS only). D, Coexpression graphs of YAP1-associated targets CDKN3and FOXM1.

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RHAMM promotes UPS migration and invasionAlthough our investigation into YAP1 transcriptional targets

has focused primarily on proliferation modulators, YAP1 is alsoknown to have effects on migration/invasion in vitro and metastasisin vivo (44–47). RHAMM is also known to mediate cellular changesin response to ECM and plays a role in tumor cell dissemination.Therefore, we explored the effects of RHAMM inhibition onsarcoma cell migration and invasion using scratch migration andMatrigel-coated transwell invasion assays. In HT-1080 cells,HMMR/RHAMM loss inhibited both migration and invasion, butmore effectively reduced migration (Fig. 6A, top and B) relative toinvasion. Invasive cells are labeled with crystal violet (Fig. 6A,bottom and C). In KP230 cells, Rhamm loss inhibits both migrationand invasion to a similar extent (Fig. 6D–F). KIA cell migration wasalso sensitive to Rhamm loss (Supplementary Fig. S6A and S6B). Todetermine the specificity of Rhamm as a Yap1 target in this system,we performed a rescue migration assay using Yap1 targeting shRNAand pcdh-Rhamm. Ectopic expression of Rhamm increased migra-tion in a scratch assay, whereas loss of Yap1 reduced migration. Incombination, we observed that ectopic expression of Rhamm inYap1-depleted cells returned migration to control levels (Fig. 6G).Therefore, we conclude that Rhamm expression promotes migra-tion and invasion in response to Yap1 upregulation, collectivelyenhancing sarcomagenesis.

TGFb and YAP1 inhibition prevent metastasis in vivoMany proteins, including YAP1 and RHAMM, promote prolif-

eration as well as aspects of metastatic progression. Until recently, ithas been difficult to investigate molecules with these dual functionsin vivo. Reduction in distant metastases cannot be specifically andconclusively attributed to changes in migratory or invasive char-acteristics when primary tumor size is also decreased in response to

target depletion. Therefore, we have incorporated a zebrafish xeno-graft approach into our studies. In recent years, zebrafish embryoxenograft models have gained attention for their utility in rapidassessment of cell migration in vivo, high-throughput drug screen-ing, and lack of adaptive immunity, which supports engraftment ofcell lines and PDX systems (28–30). As a result of the short timeframe of these experiments (3–48 hours) and the imaging-basedoutput, we were able to adapt this system to specifically evaluate theeffects of the TGFb–YAP1 axis on sarcoma metastasis. We injectedfluorescently tagged (mCherryþ) KIA cells, which are highly met-astatic, into the yolk sac of a 2-day old Fli-EGFP embryo. Thesezebrafish express EGFP in the endothelial population. Three hourspostinjection, we observed tumor cell dissemination toward the tailand head regions of the embryo, indicating that sarcoma cells willmigrate in zebrafish (Fig. 7A). Using spinning disk microscopy togenerate high-resolution images of live zebrafish embryos, weobserved substantial migration and colonization at both the headand tail of the embryo 24 hours post tumor cell injection (Fig. 7B).We also noticed that the caudal artery, which contains the tumorcells, is significantly disrupted as they cluster and proliferate.Interestingly, at 3 hours postinjection, many tumor cells can bevisualized individually and they generally remain inside the caudalartery, although the extravasation process has begun. However, by24 hours postinjection, some cells appear clustered or fused togetherand many have fully extravasated (Fig. 7C). We and others haveperformed similar studies using MDA-MB-231 breast cancer cellsand other tumor cell types and have observed predominantly single-cell migration in MDA-MB-231 (48), whereas other tumor celltypes cluster prior to extravasation (49). This clustering may be animportant contributor to extravasation and metastatic outgrowth insarcoma and will be investigated in future studies. For more detailedanalyses of sarcoma cell dissemination, we performed 3D surface

Figure 4.

TGFb and YAP1 cooperatively regulate RHAMM via NFkB signaling pathway. A, Broad institute Morpheus software heatmap based on gene expression frommicroarray analysis of KP230 cells treatedwith DMSOor SAHA (2mmol/L)/JQ1 (0.5mmol/L) for 48 hours. qRT-PCR ofHT-1080 (B) andKP230 cells (C) treated as inA(� , P < 0.01; n¼ 3).D,Western blot analysis of HT-1080 KP230 (E) and KIA (F) treated as inA.G, qRT-PCR of KP cells p65/Rela shRNA (� , P < 0.01; n¼ 3).H,Westernblot of KP cells expressing Rela shRNA.

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interaction analysis on merged fluorescent images as shownin Fig. 7C (Supplementary Fig. S7A). Yellow peaks indicate trans-endothelial migration, which is mostly complete by 24 hours. 3Dmodeling of the tail region of injected embryos confirms this finding(Supplementary Fig. S7B). To address the role of Hmmr/Rhamm intumor dissemination in vivo, we used shRNA to inhibit Rhammexpression in GFP-expressing KIA cells and injected them into flk-mCherry zebrafish embryos. In these fish, the vasculature is labeledwith mCherry. We measured metastatic capacity of control andRhamm shRNA–expressing cells and found a dramatic decrease indissemination when Rhamm is lost (Fig. 7D and E). Metastaticcapacity is defined as number of fish showing migrated cells/number of fish injected. We also used a pharmacologic approachto inhibit TGFb and YAP1 signaling and found that both treatmentssuppressed metastasis by 75% (Fig. 7F). Using this powerfulxenograft tool, we were able to specifically assess the role of these

pathways in tumor cell dissemination and observed that the TGFb–YAP1–Rhamm axis promotes metastasis and can be therapeuticallytargeted in vivo.

DiscussionThe goal of our recent work has been to delineate mechanisms by

which Hippo pathway deregulation contributes to sarcomagenesis.Our observation that constitutive NFkB signaling is the mainmolecular driver of muscle-derived UPS (5) led us to exploreadditional pathways that promote NFkB activity independent ofor in cooperation with YAP1, the transcriptional effector of theHippo pathway. Here we report that TGFb signaling increasestranscription of YAP1-associated pro-proliferation targets includ-ing FOXM1, enhances NFkB activity, and promotes metastasis.TGFb signaling has been shown to promote YAP1 activity through

Figure 5.

RHAMM promotes sarcomagenesis in vivo. qRT-PCR of HT-1080 (A) and KP cells (B) expressing multiple independent shRNAs targeting HMMR/Hmmr. C,Westernblot analysis of HT-1080 and KP230 cells expressing multiple independent shRNAs targeting HMMR/Hmmr (� , P < 0.01). D, Proliferation assay of HT-1080 cellsexpressing control or multiple independent RHAMM shRNAs (�, P < 0.01). E, Proliferation assay of HT-1080 cells ectopically expressing pcdh-RHAMM (� , P < 0.01).qRT-PCR (F) and Western blot (G) of HT-1080 cells ectopically expressing pcdh-RHAMM (�, P < 0.01). IHC Images (H) and quantification (I) of Rhamm and Ki67staining (J) from 5 unique subcutaneously implanted KP230 cells expressing control or Hmmr/Rhamm targeting shRNAs (� , P < 0.01; scale bar, 50 mm). Tumorvolume; SEM (K) and tumor weights; SEM (L) from subcutaneous allografts of KP230 cells treated with lentivirus expressing shRNAs (� , P < 0.01). M,Western blotanalysis of 3 allograft tumors harvested 17 days after subcutaneous injection.

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phosphorylated SMAD3 (50, 51). In the nucleus, p-SMAD3 phys-ically interacts with the YAP1/TEAD1-4 complex and initiatestranscription. This mechanism, which has been characterized inepithelial cancer cells, now appears likely to extend to sarcomas(Fig. 8).

Very little is known about the role and importance of TGFbsignaling in muscle-derived sarcomas like UPS. Other groups havelinked this pathway to osteosarcoma, fibrosarcoma, and chondro-sarcoma (52–56), but only one limited study has suggested thatTGFb is relevant in UPS (ref. 57; formerly known as MFH). Ourwork has revealed a clear link between TGFb activity and UPS. Weobserved that p-SMAD3 expression is significantly upregulated inhuman and murine UPS relative to control skeletal muscle tissue.Importantly, we also show that pharmacologic inhibition of TGFb

significantly reduces UPS cell proliferation, migration, and inva-sion. Interestingly, migration/invasion are more sensitive to TGFbinhibition, suggesting that the critical function of the TGFb path-way in UPS and fibrosarcoma is in control of tumor cell dissem-ination and metastatic processes.

Therefore, we sought to identify potential transcriptional tar-gets of TGFb, associated with migration/invasion, which areproducts of coregulation by YAP1 in sarcoma. HMMR/RHAMMis one of the most well established TGFb targets in various cancercontexts (15, 58, 59). RHAMM signaling specifically promotesmetastasis and associated cellular processes, particularly in fibro-sarcoma (14, 20, 60, 61). Recently, Wang and colleagues reportedthat RHAMM is regulated by YAP1 in breast cancer motility (16).Together, these findings suggested that RHAMM might be a

Figure 6.

RHAMM promotes migration and invasion of sarcoma cells. A, Top, scratch migration (bottom) and and invasion assay (bottom) of HT-1080 cellsexpressing control or HMMR/RHAMM shRNAs. Quantification of migration (B) and invasion assays (C) from A (� , P < 0.01; n ¼ 3; SD). D, Top,scratch migration (bottom) and crystal violet invasion assay (bottom) of KP230 cells expressing control or Hmmr/Rhamm shRNAs. Quantificationof migration (E) and invasion assays (F) from D (� , P < 0.01; n ¼ 3; SD). G, Rescue scratch migration assay of HT-1080 cells expressing shYAP1, pcdh-HMMR, or pcdh-HMMRþshYap1 (� , P < 0.01; n ¼ 3; SD).

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Figure 7.

TGFb inhibitor andSAHA/JQ1 treatment preventmetastasis in a zebrafish xenograftmodel.A,KIA/mCherry sarcoma tumor cellswere injected into the yolk sac ofFli-EGFP transgenic zebrafish embryos at 48 hours postfertilization. Tumor cell migration and metastasis were detected under fluorescent microscopy at 3 hourspostinjection. White arrow indicates primary injection site. Blue arrowhead indicates distant metastasis toward tail. B, Representative images of zebrafish embryosdisplaying overt extravasation at 24 hours postinjection. Top, representative images showing metastasis in zebrafish head, trunk, and tail respectively. “h”, heart;white arrow, primary injection site; star, tumor migration into the pericardiac space; blue arrowheads, metastasis. Bottom, magnified image on tail metastasis; whitearrowheads, extravasated tumor foci. Cartoons indicate areas being imaged. C, Representative high-resolution 3D confocal images of zebrafish tail regionsshowing initial and overt extravasation at 3 hours and 24 hours postinjection, respectively (scale bar, 100 mm). Yellow signals, transendothelial migration oftumor cells; dashed boxes, extravasation detailed in zoom-in-views on the right; white arrowheads, extravasated tumor foci. Cartoons indicate areas beingimaged. Annotations: A, anterior; P, posterior; D, dorsal; V, ventral. D, KIA/Cop-GFP sarcoma tumor cells expressing Scr or Rhamm shRNA #1 were injected intothe yolk sac of Flk-mCherry transgenic zebrafish embryos at 48 hours postfertilization. Tumor cell migration and metastasis were detected under fluorescentmicroscopy at 3 hours postinjection. White arrow, metastatic cells. Cartoons indicate areas being imaged. E, Quantification of normalized metastatic capacity.Metastatic capacity is defined as number of fish showing migrated cells/number of fish injected (head, trunk, tail metastasis all combined), normalized to 100%for Cop-GFP–expressing KIA cells as in D or SB-525334- and SAHA/JQ1-treated tumor cells (n ≥ 2 injections; each injection included at least 20 zebrafishembryos; F). Statistical analysis was performed using one-way ANOVA followed by Dunnett multiple comparison test (� , P < 0.05; SD).

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critical target downstream of cooperative TGFb and YAP1-medi-ated transcription in sarcoma. Moreover, RHAMM is highlyexpressed in human UPS and fibrosarcomas, relative to normalmesenchymal tissues, and its expression is linked to poor clinicaloutcome. Our findings suggest that dual inhibition of YAP1 andTGFb may significantly reduce primary sarcomagenesis andmetastasis. Furthermore, our results suggest the HMMR/RHAMMexpression mediates the effects of these pathways on proliferationand dissemination.

To test the effects of pharmacologic inhibition of the YAP1–TGFb–RHAMM axis on sarcoma cell dissemination, we initiated a zebrafishxenograft model that allows specific investigation of cell motilityin vivo (29, 62, 63). This system has significant advantages overstandard in vitro migration/invasion assays. Injected tumor cells aresubjected to the fluidic properties native to circulating cells includingshear stress and interaction with multiple additional cell types. Thissystem facilitates powerful imaging approaches due to the transparentnature of 2-day old embryos, which also lack fully developed immunesystems. Pretreatment of murine UPS cells with a TGFb inhibitor orSAHA/JQ1 significantly reduced tumor cell dissemination from thesite of injection to the tail region of the fish.

Collectively, our findings suggest that TGFb signaling cooperateswith YAP1 to modulate critical genes likeHMMR to promote primarysarcomagenesis andmetastatic progression in UPS and fibrosarcomas.

Furthermore, we have established the potential utility of pharmaco-logic inhibition of the TGFb/YAP1 axis in this context while inves-tigating RHAMM as an important biomarker of metastasis andtherapeutic intervention in this context.

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

Authors’ ContributionsConception and design: S. Ye, M.A. Pack, T.S.K. Eisinger-MathasonDevelopment of methodology: S. Ye, Y. Liu, M.Z. Alam, M.A. PackAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Y. Liu, A.M. Fuller, R. Katti, G.E. Ciotti, S. Chor, M.Z. Alam,S. Devalaraja, K. Lorent, K. Weber, M. Haldar, M.A. PackAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ye, Y. Liu, A.M. Fuller, S. DevalarajaWriting, review, and/or revision of the manuscript: S. Ye, A.M. Fuller, R. Katti,M.Z. Alam, K. Weber, T.S.K. Eisinger-MathasonAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S. Ye, S. ChorStudy supervision: T.S.K. Eisinger-Mathason

AcknowledgmentsThe authors wish to acknowledge Dr. Robert Schreiber, PhD (Washington

University of St. Louis, St. Louis, MO), Dr. Rebecca Gladdy (University of

Figure 8.

Model summarizing the role of TGFband Hippo pathways in through theHMMR/RHAMM–modulated sarcoma-genesis and metastasis.

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Toronto, Toronto, Ontario, Canada), and Dr. Kurt Weiss MD (University ofPittsburgh Medical College, Pittsburgh, PA) for providing us with sarcoma celllines. We would also like to acknowledge Dr. John Tobias PhD (University ofPennsylvania, Philadelphia, PA) for his assistance with bioinformatics analysis andDr. Patrick Grohar MD, PhD (Children's Hospital of Philadelphia, Philadelphia,PA) for his input and advice. This work was funded by the NCI (R01CA229688),“Steps to Cure Sarcoma”, the University of Pennsylvania Abramson CancerCenter, the Penn Sarcoma Program, and the SLAY sarcoma fund.

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 23, 2019; revised December 13, 2019; accepted January 21, 2020;published first January 27, 2020.

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Hippo and TGFb Cooperatively Drive Sarcomagenesis