yap/taz initiates gastric tumorigenesis via upregulation of ......horseradish peroxidase (jackson...

Tumor Biology and Immunology

YAP/TAZ Initiates Gastric Tumorigenesis viaUpregulation of MYCWonyoung Choi1, Jeongsik Kim2, Jaeoh Park2, Da-Hye Lee2, Daehee Hwang2,Jeong-Hwan Kim3, Hassan Ashktorab4, Duane Smoot5, Seon-Young Kim3,Chan Choi6, Gou Young Koh1,7, and Dae-Sik Lim2

Abstract

YAP and TAZ play oncogenic roles in various organs, but therole of YAP/TAZ in gastric cancer remains unclear. Here, weshow that YAP/TAZ activation initiates gastric tumorigenesisin vivo and verify its significance in human gastric cancer. Inmice, YAP/TAZ activation in the pyloric stem cell led to step-wise tumorigenesis. RNA sequencing identified MYC as a deci-sive target of YAP, which controls MYC at transcriptional andposttranscriptional levels. These mechanisms tightly regulatedMYC in homeostatic conditions, but YAP activation altered thisbalance by impeding miRNA processing, causing a shifttowards MYC upregulation. Pharmacologic inhibition of MYCsuppressed YAP-dependent phenotypes in vitro and in vivo,verifying its functional role as a key mediator. Human gastric

cancer samples also displayed a significant correlation betweenYAP and MYC. We reanalyzed human transcriptome data toverify enrichment of YAP signatures in a subpopulation ofgastric cancers and found that our model closely reflected themolecular pattern of patients with high YAP activity. Overall,these results provide genetic evidence of YAP/TAZ as oncogenicinitiators and drivers for gastric tumors with MYC as thekey downstream mediator. These findings are also evident inhuman gastric cancer, emphasizing the significance of YAP/TAZsignaling in gastric carcinogenesis.

Significance: YAP/TAZ activation initiates gastric carcinogen-esis with MYC as the key downstreammediator. Cancer Res; 78(12);3306–20. �2018 AACR.

IntroductionGastric cancer is the fifth most common type of cancer world-

wide and ranks as the third most frequent cause of cancer-relateddeaths (1). Despite numerous studies focusing on the geneticalterations associated with gastric cancer, our knowledge is lim-ited about the signaling pathways that drive malignant transfor-mation (2–6). The Hippo signaling pathway (also known asHippo-YAP/TAZ signaling) is a growth control and tumor sup-pressor pathway associated with carcinogenesis, regeneration,and metabolism (7–11). In this pathway, the core kinases

MST1/2 and LATS1/2 negatively regulate the effector moleculesYAP and TAZ. The scaffolding proteins SAV1 and MOB1A/B bindto MST1/2 and LATS1/2, respectively, to modulate their kinaseactivities. When the Hippo pathway is activated, LATS1/2 phos-phorylates YAP/TAZ, inhibiting their activity. Conversely, whenthe pathway is deactivated, YAP/TAZ accumulates in the nucleus,driving gene expression by forming complexes with transcriptionfactors (most notably with TEAD). This effect leads to cell pro-liferation and inhibition of apoptosis.

Several studies have suggested a potential link betweenYAP/TAZ signaling and gastric cancer (12–15). YAP expressionis upregulated in gastric tumor samples compared with normaltissue, and the total amount of YAP expression as well as itsnuclear localization are significantly correlated with poorer clin-ical outcomes (12–14).Moreover, a peptidemimicking the role ofVGLL4 (a candidate for inhibiting the YAP–TEAD interaction)inhibits tumor growth in both xenograft and carcinogen-inducedmurine gastric tumormodels (15).However, the clear significanceof Hippo-YAP/TAZ signaling in gastric cancer has never beenestablished inhumangenomic/transcriptomic studieswith a largenumber of samples (2–6). Most important, the lack of a genet-ically engineered mouse model has made it difficult to clarifywhether the relationship between the upregulation of YAP/TAZand carcinogenesis is direct and causal. The fundamental questionof whether YAP/TAZ activation is the cause or consequence ofgastric cancer in vivo thus remains to be answered.

Anatomically, themurine stomach can be roughly subclassifiedinto three parts. The proximal part is the forestomach, which iscovered with squamous epithelium. The distal part is the glan-dular stomach, which can be subdivided into corpus and pylorus(antrum). Surfacemucous epithelial cells in the gastricmucosa arecolumnar, and epithelial cells in the glands are cuboidal, but their

1Graduate School of Medical Science and Engineering, Korea Advanced Instituteof Science and Technology (KAIST), Daejeon, Republic of Korea. 2NationalCreative Research Initiatives Center for Cell Division andDifferentiation, Depart-ment of Biological Sciences, KAIST, Daejeon, Republic of Korea. 3MedicalGenomics Research Center, Korea Research Institute of Bioscience and Bio-technology, Daejeon, Republic of Korea. 4Department of Medicine and CancerResearch Center, Howard University College of Medicine, Washington DC.5Department of Internal Medicine, Meharry Medical College, Nashville, Tennes-see. 6Department of Pathology, Chonnam National University Hwasun Hospital,Chonnam National University Medical School, Hwasun, Jeonnam, Republic ofKorea. 7Center for Vascular Research, Institute for Basic Science, Daejeon,Republic of Korea.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Dae-Sik Lim, Korea Advanced Institute of Science andTechnology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea(South). Phone: 82-42-350-2635; Fax: 82-42-350-2610; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-3487

�2018 American Association for Cancer Research.

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morphologic details and cellular makeup differ (16, 17). Variousmarkers are available for stem cell populations in the corpus andpylorus, and some of them overlap. Stem cells in the corpusexpress Sox2, Troy, Mist1, and eR1, whereas stem cells in thepyloric antrum express Sox2, Lgr5, CCK2R, eR1, Axin2, and Mist1(18–25).Mousemodel studies have shown that oncogenic eventsin gastric stem cells can frequently lead to carcinogenesis,although mutations targeted to chief cells also can cause thesecells to dedifferentiate and acquire the potential to grow andregenerate gastric glands, sometimes forming metaplasia (17, 20,21, 22, 26–29).

In this study, we asked whether pyloric stem cells expressingLgr5 may serve as the cells of origin for cancers of the distalstomach that exhibit YAP/TAZ activation (22). For this purpose,we used conditional knockouts of Lats1 and Lats2 to activateYAP/TAZ and showed that this perturbation was sufficient totrigger dysplastic changes and eventually tumorigenesis in thepyloric epithelium. We performed transcriptome analysis to scru-tinize the underlyingmolecularmechanisms and uncoveredMYCas a critical target of YAP in the tumorigenic pathway. We cor-roborated the significance of MYC by showing that its pharma-cologic inhibition could rescue YAP-induced oncogenic pheno-types. Using human gastric cancer tissue microarray samples, weconfirmed a positive correlation of YAP and MYC. Finally, byanalyzing public microarray data, we show that YAP signaturegenes are highly enriched in a subset of gastric tumors, supportingits importance in carcinogenesis of human gastric cancer.

Materials and MethodsMice

All mouse experiments were performed under approval bythe Institutional Animal Care and Use Committee of KoreaAdvanced Institute of Science and Technology. Lats1fl/fl andLats2fl/fl mice were described previously (30–32). Lgr5-EGFP-IRES-creERT2mice were kindly provided by Dr. Young-Yun Kong(Seoul National University) and described previously (33).R26-tdTomato reporter mice were purchased from Jacksonlaboratory, and also described previously (34). Inducible Cre-mediated gene deletion was initiated by single dose intraperito-neal injections into 8- to 10-week-oldmice of 75mg tamoxifen/kgbody weight. Tamoxifen (Cayman Chemical) was dissolved incorn oil (Sigma) at a concentration of 20 mg/mL.

Histology, IHC, and immunofluorescence stainingMouse stomachwas harvested and cut open through the greater

curvature, fixed with 10% neutral-buffered formaldehyde at 4�Covernight, and embedded in paraffin. Sections (3 mm) were cutand stainedwith hematoxylin and eosin (H&E). For IHC staining,slides were deparaffinized, quenched for endogenous peroxidaseswith 3% hydrogen peroxide, and subjected to antigen retrieval incitrate buffer (10 mmol/L sodium citrate, 0.05% Tween 20, pH6.0) using a pressure cooker. Blocking was performed with 3%BSA and 0.3% Triton X-100 in PBS for 1 hour at room temper-ature. The primary antibodies used were anti-MYC (Millipore,06-340, 1:100), and anti-Hes1 [gift fromDr. Ryoichiro Kageyama(Kyoto University, Kyoto, Japan), rabbit polyclonal, 1:500]. Pri-mary antibody detection was performed using goat anti-rabbithorseradish peroxidase (Jackson ImmunoResearch, catalog no.111-035-003, 1:500) and the Dako EnVision System (catalog no.K5007). Immunofluorescence staining was performed with

anti-YAP (Cell Signaling Technology, 4912s, 1:200), anti-TAZ(Sigma, HPA007415, 1:200), anti-Ki67 (Abcam, ab16667,1:100), anti-RFP (Rockland Immunochemicals, RK600-401-379, 1:200), anti-MUC2 (Abcam, ab90007, 1:150), and anti-b-catenin (BD Biosciences, 610154, 1:100). Goat anti-rabbitAlexa Fluor 594 (Thermo Fisher Scientific, A-11037, 1:1,000),donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific,A-21206, 1:1,000), or donkey anti-mouse Alexa Fluor 488(Thermo Fisher Scientific, A-21202, 1:1,000) was used to detectfluorescent signals. Images were obtained with a Leica DMLBmicroscope (Leica) or confocal microscope (Zeiss, LSM 780, andLSM 800). Alcian blue staining was performed using Alcian Blue(pH 2.5) Stain Kit (Vector Laboratories, H-3501). Staining inten-sities were calculated using ImageJ software. Mouse histologyslides forH&E stainingwere confirmedby apathologist (C.Choi).

Growth in low attachment assayCells were trypsinized and 1,000 cells per well in 100 mL of

DMEM with 10% FBS were seeded in 96-well ultralow attach-ment plates (Corning, 3474). For drug treatment, JQ1 (CaymanChemical, final concentration 500 nmol/L), 10058-F4 (Sigma,final concentration 50 mmol/L) or vehicle (DMSO, MP Biome-dicals) was added to the media. Colonies were counted 5–7 dayslater. Quantification of cell viability was measured with a WST-1assay (LPS solution, CYT3000) according to the manufacturer'sprotocol.

JQ1 treatment in vivoVehicle for injection was prepared with 10% cyclodextrin

(Sigma, H-107) in distilled water. JQ1 was first dissolved inDMSO at a concentration of 50 mg/mL, and then diluted 1:10with vehicle to a final concentration of 5 mg/mL. Mice wereintraperitoneally injected daily with JQ1 at 50mg/kg bodyweightfor 5 weeks.

Microarray data analysisMicroarray data from the Asian Cancer Research Group cohort

were downloaded from NCBI Gene Expression Omnibus(GSE62254). Gene set enrichment analysis (GSEA) was per-formed using C6 Oncogenic Signatures from the Molecular Sig-natures Database (MSigDB) version 5.1 (35). The microarraysamples were reclassified based on YAP signature gene expressionusing the Signature Evaluation Tool (36). Survival analysis wasperformed using the log rank test and Kaplan–Meier method viaGraphPad Prism.

Tissue microarrayGastric adenocarcinoma tissuemicroarrayswere obtained from

Chonnam National University Hwasun Hospital National Bio-bank of Korea. The patients fromwhom the samples were derivedunderwent surgical tumor resections between 2009 and 2012 atChonnam National University Hwasun Hospital. All sampleswere obtained with informed consent and approved by theInstitutional Review Board of Chonnam National UniversityHwasun Hospital (CNUHH-2017-086).

Statistical analysisAll statistical analyses, including two-tailed t tests and log-rank

tests for survival analyses, were performed using GraphPad Prismsoftware (version 7). Analyses were performed with two-tailedt tests, unless otherwise indicated.

YAP/TAZ Initiates Gastric Tumorigenesis

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Data availabilityThe data discussed in this publication have been deposited in

NCBI's Gene Expression Omnibus and are accessible throughGEO Series accession number GSE104823 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE104823).

ResultsKnockout of Lats1 and Lats2 in mouse pyloric epithelial stemcells induces gastric tumorigenesis

To activate YAP/TAZ, we used conditional knockout mice forLats1 and Lats2 because LATS kinases are direct negative upstreamregulators of YAP/TAZ.Wemated Lats1 and Lats2-floxedmicewithLgr5-EGFP-IRES-creERT2 to generate Lats1fl/fl;Lats2fl/fl;Lgr5-CreERmice, designated as Lats1/2iDLgr5 mice, for activating YAP/TAZspecifically in Lgr5þ epithelial stem cells in a tamoxifen-induciblemanner (Supplementary Fig. S1A). These mice were born inMendelian ratios and showed no noticeable defects unlessinjected with tamoxifen for Cre induction. Tamoxifen was intra-peritoneally injected into Lats1/2iDLgr5 mice at 8–10 weeks ofage to induce gene deletion, consequently activating YAP/TAZ.Lats1fl/fl;Lats2fl/fl littermates of Lats1/2iDLgr5mice (referred tohere aswild type; WT) were used as control and injected with identicaltamoxifendoses at the same frequencies. By tracingCre expressionwith reportermice (Lgr5-CreER;tdTomato) and performing recom-bination PCR on the pyloric epithelial tissue, we confirmedefficient Cre-induced gene deletion upon tamoxifen injection inLats1/2iDLgr5 mice (Supplementary Fig. S1B and S1C).

Lgr5marks adult stem cells in various tissues, so we harvestedall organs known to contain Lgr5þ stem cells (i.e., stomach,small intestine, colon, skin, liver, pancreas, and ovary; refs. 22,33, 37–40). To our surprise, we observed Lats1 and Lats2knockout phenotypes only in the pyloric stomach and skin(hair follicular hyperplasia). Unexpectedly, the small intestineand colon, the best known locations for Lgr5þ stem cell popu-lations, appeared to be histologically normal (SupplementaryFig. S1D). The reason for this result is unclear, but to investigatethe role of YAP/TAZ in gastric cancer, we focused only on thestomach in this study.

To analyze tumorigenesis in the stomach, we sacrificed mice atsequential time points after tamoxifen induction (SupplementaryFig. S1A). At gross examination, there was no significant massprotrusion on the gastric epithelial surface, but there were diffuseflat elevations in the pyloric region (Supplementary Fig. S1E). Inhistology sections, we found evidence of microscopic changes inthe pyloric epithelium in Lats1/2iDLgr5 mice beginning at 8 weekspostinduction (Fig. 1A). These changes include slight tubuledistortion andmild nuclear size variation, indicating hyperplasia.Dysplastic changes appeared 12 weeks postinduction with somenuclei showing a slight loss of polarity and mild cytologic atypia(low-grade intraepithelial neoplasia). At 16 weeks, these lesionsbecame more severe, characterized by crowding of tubules linedby atypical cells (high-grade intraepithelial neoplasia). At 20 and24 weeks, cells in the lesions displayed hyperchromatic nucleiand loss of polarity with glands budding and invading into thelamina propria (intramucosal invasive carcinoma/intramucosalcarcinoma; Fig. 1A and B). The intense Ki67 staining of theselesions suggested that they were highly proliferative (Supplemen-tary Fig. S2A). Pyloric stomach of WT mice exhibited weak YAPexpression in the antral glands, while TAZ expression was restrict-ed in the isthmus region and lost at the base of glandswhere Lgr5þ

cells reside (Supplementary Fig. S2B). However, Lats1/2iDLgr5miceexhibited intense nuclear immunostaining of YAP/TAZ through-out the period of analysis, reflecting elevated activity of thesesignaling effectors (Fig. 2A; Supplementary Fig. S2C). Lats1/2iDLgr5

mice also showed strong increases in staining for markers ofintestinal metaplasia (MUC2 and Alcian blue), indicating thatmetaplasia preceded dysplastic changes (SupplementaryFig. S3A). These lesions appeared at very high penetrance(Supplementary Table S1A), confirming the robustness of ourYAP/TAZ–induced gastric tumor model.

To confirm that these tumors originated from gastric stem cellslacking Lats1 and Lats2, we bred Lats1/2iDLgr5 mice with R26-tdTomato reporter mice to generate Lats1fl/fl;Lats2fl/fl;Lgr5-CreER;R26-tdTomato (Lats1/2iDLgr5;tdTomato) mice. Gastric lesionsformed after Cre induction in these mice were positive for tdTo-mato, indicating that they were derived from Lats1/2-deleted stemcells (Fig. 2B). As pyloric Lgr5þ cells are reportedly almost qui-escent, we asked whether Lgr5þ cell proliferation changes earlyafter Lats knockout (22, 23). When we compared Lgr5-CreER;tdTomato mice with Lats1/2iDLgr5;tdTomato mice 1 week post-tamoxifen induction, we noticed that while there was no differ-ence in the number of Lgr5þcells, loss of Lats markedly increasesLgr5þ cell proliferation as tracked by tdTomato expression(Supplementary Fig. S3B). Also, whereas most Lgr5þ cells inLgr5-CreER;tdTomato mice were quiescent, Lats-deficient Lgr5þ

cells showed significantly more Ki67 staining, implying loss ofLats triggersmore rapid cell cycling in these cells by enhancingYAPand TAZ (Supplementary Fig. S3B and S3C). Together, theseresults indicated that YAP/TAZ activation by Lats1/2 deletion inpyloric stem cells triggered stepwise progression from low-gradeintraepithelial neoplasia to intramucosal carcinoma in the nor-mal gastric epithelium, validating this signaling cascade as acritical driver of gastric tumorigenesis in vivo.

Activation of YAP/TAZ promotes the oncogenic transformationof gastric epithelial cells

To verify the oncogenic role of YAP/TAZ in vitro, we askedwhether activation of YAP/TAZ could promote cellular trans-formation and confer the hallmark features of cancer on agastric epithelial cell line (HFE-145). YAP-5SA and TAZ-4SAharbor serine-to-alanine mutations in LATS phosphorylationsites that result in phenotypes similar to those caused byhyperactive mutant forms of YAP and TAZ (41, 42). To reca-pitulate our in vivo results, we overexpressed YAP-5SA andTAZ-4SA in HFE-145 cells. As an index for cellular transforma-tion, we asked whether these active mutants promote colonyformation in an anchorage-independent condition. In a softagar assay, both YAP-5SA and TAZ-4SA overexpression led to3.25-fold and 1.74-fold increases in colony size, while onlyYAP-5SA overexpression led to a 5.42-fold increase in thenumber of colonies (Fig. 2C; Supplementary Fig. S3D). Thedifferential effect of YAP and TAZ on colony formationsuggested a more active role of YAP in tumorigenesis.

We next asked whether active YAP/TAZ promotes invasionthrough the extracellular matrix, another hallmark of malignantcells. In a transwell invasion assay, YAP-5SA and TAZ-4SA over-expression enhanced cellular invasion through the extracellularmatrix by 3.58- and 1.89-fold, respectively (Fig. 2D; Supplemen-tary Fig. S3E). Collectively, these in vitro data, combined with thein vivo findings, indicated that YAP/TAZ activation promotesmalignant transformation of gastric epithelial cells.

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Transcriptome analysis of the YAP/TAZ-induced murinegastric tumor reveals MYC as a promising downstreamtarget

Because YAP and TAZ are transcriptional coactivators, we nextattempted to identify the downstream targets responsible for

relaying tumorigenic signals. For this purpose, we performedRNA sequencingon gastric lesions formed8weeks after tamoxifeninduction in our model mice. We first harvested the epitheliallining of the stomach and verified the activation of YAP/TAZ byquantifying expression levels of the known downstream target

Figure 1.

Knockout of Lats1 and Lats2 inpyloric stem cells induces gastrictumorigenesis. A, Serial H&E staining(top) and enlarged images (bottom)of the pyloric stomach from WT miceat 12 weeks and Lats1/2iDLgr5mice at 8,12, 16, 20, and 24 weeks postinductionby tamoxifen. Arrowheads indicatehyperplasia (8 weeks), low-gradeintraepithelial neoplasia (12 weeks),high-grade intraepithelial neoplasia(16 weeks), and intramucosalcarcinoma (20 and 24 weeks) at eachsequential time point. Scale bars, 100mm. B, Enlarged H&E staining imageof Lats1/2iDLgr5 pyloric stomach at20 weeks post-tamoxifen induction.Arrows indicate glandular buddingand invasion of lamina propria.Arrowheads indicate hyperchromaticcells with loss of polarity and encircledareas indicate mitotic figures.Scale bar, 50 mm.






genes Ctgf, Cyr61, and Ankrd1 (Supplementary Fig. S4A). Byanalyzing and comparing transcriptome data from the gastrictissues of Lats1/2iDLgr5 and WT mice, we identified almost 2700differentially expressed genes (i.e., 1158 upregulated genes and1543 downregulated genes in the Lats1/2iDLgr5 mice comparedwithWTmice). A subsequent gene ontology analysis of biologicalprocess terms revealed significant enrichment of genes related tocell-cycle progression (Fig. 3A).

We also analyzed the differentially expressed genes usingGSEA to identify the signaling pathways downstream of theoncogenic cascade. First, we found significant enrichment ofYAP signature gene sets in the Lats1/2iDLgr5 group, validating oursequencing results (Fig. 3B). Consistent with our gene ontologyanalysis, we found that E2F targets, G2M checkpoint-relatedgenes, and MYC target genes, all molecular signatures related tocell-cycle progression, topped the GSEA list (Fig. 3C and D;Supplementary Fig. S4B). Epithelial-to-mesenchymal transition(EMT)-related gene sets were also highly upregulated in thetumors, suggesting a link between this process and YAP/TAZsignaling (Supplementary Fig. S4B and S4C).

Among the enriched gene sets, we focused onMYC target genes.MYC is widely known for its role as an oncogene, promoting cell-cycle progression and regulating the expression of many impor-tant genes critical for cell growth andproliferation (43). It also hasbeen suggested as adownstream target of YAP (44, 45). In an effortto extend our own observations and the results in these previousreports, we asked whether MYC may act as a critical downstreamtarget relaying YAP/TAZ signals in gastric tumorigenesis.

MYC is upregulated by YAP through posttranscriptionalregulation

First, we confirmed whether or not MYC was active in ourmodel mice. Using IHC with mice at 8 weeks post-tamoxifeninduction, we found that pylorus of Lats1/2iDLgr5miceweremostlycovered with intense nuclear MYC expression, while MYC expres-sion in WT mice was limited to the basal layer (Fig. 4A). BecauseYAP reportedly regulates Wnt/b-catenin andNotch signaling, andbecause MYC acts downstream of these pathways, it is possiblethat the upregulation of MYC we observed is indirect (46, 47). Inour RNA sequencing results comparingWT and Lats1/2iDLgr5mice,

Figure 2.

YAP/TAZ activation induces gastric tumorigenesis. A, Immunofluorescent staining and quantification of YAP and TAZ intensity in WT and Lats1/2iDLgr5 micepyloric stomach, 8 weeks post-tamoxifen induction. � , P < 0.05 versus WT. n ¼ 3 mice for each group. B, Immunofluorescent staining of tdTomato (RFP) inLats1/2iDLgr5-tdTomatomice stomach, 8 weeks post-tamoxifen induction. C, Colony size measurements and colony counts from soft agar assays with HFE-145 cellsexpressing vector, YAP-5SA (active mutant), or TAZ-4SA (active mutant). � , P < 0.05 versus empty vector. n ¼ 3 for each group. D, Transwell invasion assayswith HFE-145 cells expressing vector, YAP-5SA, or TAZ-4SA. Quantification of cells that have penetrated through the extracellular matrix in the transwellinvasion assay. � , P < 0.05 versus empty vector. n ¼ 3 for each group. All scale bars, 100 mm. The data (A, C, and D) are presented as means � SEM.

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however, we did not observe any significant enrichment ofWnt/b-catenin or Notch target gene sets. (SupplementaryFig. S5A).Moreover, we could not detect any significant differencein the staining of b-catenin and Hes1 in the pyloric epithelium ofWT and Lats1/2iDLgr5 mice (Supplementary Fig. S5B).

To further confirm that YAP/TAZ activation upregulates MYC,we examined MYC expression in the YAP-5SA– and TAZ-4SA–overexpressed HFE-145 cells. As expected, YAP-5SA and TAZ-4SAoverexpression increased MYC protein levels (Fig. 4B, left),but to our surprise, MYC mRNA levels remained unaltered(Fig. 4B, right).

To determine whether this regulation occurs at the posttran-scriptional level, we overexpressed YAP-5SA/S94A, which harborsanother serine-to-alanine mutant preventing YAP binding to theTEAD transcription factor (48). YAP5SA/S94A overexpressionalso increased MYC protein expression but not MYC mRNAlevels (Fig. 4C). We additionally checked on whether proteinstability is altered with YAP5SA overexpression, performing the

cycloheximide chase assay; however, the half-life of MYC did notchange upon YAP activation (Fig. 4D), indicating that changes inprotein stability did not mediate the MYC upregulation.

YAP reportedly regulates MYC through repression of miRNAprocessing (44), so we asked whether the same mechanismoperates in the gastric epithelial cells. We sequenced miRNAsfrom HFE-145 cells overexpressing YAP-5SA or a vector con-trol. Among the miRNAs significantly downregulated uponYAP activation, we found two (miR-34a-5p and miR-21-5p)that are known to repress MYC and three (miR-374b-5p,miR-34a-5p, and miR-449b-5p) that are predicted to targetthe MYC 30-UTR (Fig. 4E; Supplementary Fig. S5C; refs. 49,50). Using a miRNA reporter assay, we confirmed all fourmiRNAs from our sequencing results can bind the MYC 30-UTR(Fig. 4F). Moreover, we verified that two of these miRNAs(miR-374b-5p and miR-449b-5p) functionally repress MYCprotein expression in the HFE-145 and AGS cell lines(Supplementary Fig. S5D).

Figure 3.

Transcriptome analysis of YAP/TAZ-induced gastric tumors. A, Gene ontology analysis of genes significantly up- and downregulated in Lats1/2iDLgr5 mice pyloricstomach compared with WT on RNA sequencing. Two mice for each group at 8 weeks post-tamoxifen induction were used for RNA sequencing. B, GSEA ofdifferentially expressed genes using a GSEA preranked method, showing upregulation of YAP signature genes in Lats1/2iDLgr5mice pyloric stomach compared withWT and corresponding heatmap of top enriched genes. C, GSEA result showing enrichment of E2F targets and G2M checkpoint gene sets from differentiallyexpressed genes of Lats1/2iDLgr5 mice pyloric stomach compared with WT. D, GSEA result showing enrichment of MYC target genes in Lats1/2iDLgr5 mice pyloricstomach compared with WT and corresponding heatmap of top enriched genes.






Figure 4.

MYC is upregulated by YAP at the posttranscriptional level. A, H&E, IHC staining, and quantification of MYC intensity in WT and Lats1/2iDLgr5 mice stomach, 8 weekspost-tamoxifen induction. Arrowheads, MYC stained in the nuclei. Scale bars, 100 mm. �, P < 0.05 versus WT. n ¼ 3 mice for each group. B, Western blot (left) andqRT-PCR (right) for HFE-145 cells with vector, YAP5SA, or TAZ4SA overexpression, short exposure (S.E.), and long exposure (L.E.). n¼ 4 for each group in qRT-PCR.C, Western blot (left) and qRT-PCR (right) results for HFE-145 cells overexpressing vector, YAP5SA, or YAP5SA/S94A. n ¼ 4 for each group in qRT-PCR. D,Cycloheximide chase analysis for HFE-145 cells expressing vector or YAP5SA. Left, Western blots for the indicated antibodies at 0, 30, 60, 90, and 120 minutes aftercycloheximide (50 mg/mL) addition. Right, Quantification of blot intensities using ImageJ software. E, Heatmap of differentially expressed mature miRNAs inHFE-145 cells overexpressing vector or YAP5SA. miRNAs with a relative expression change of < 0.5 or > 2-fold, and P < 0.05 compared with the control (vector)were considered significantly different between the two groups. miRNAs known to repress MYC (miR-34a-5p and miR-21-5p) and predicted to bind MYC 30UTR(miR-374b-5p, miR-34a-5p, and miR-449b-5p) are indicated. n ¼ 2 for each group. F, miRNA reporter assays examining the binding of miR-21-5p, miR-34a-5p,miR-374b-5p, miR-449b-5p, or all miRNAs (cotransfection of all four miRNAs) to the MYC 30-UTR. � , P < 0.05 versus control. n ¼ 3 for each group. G,Western blotresults for HFE-145 cells expressing vector, or YAP5SA with or without DDX-17 (encoding p72). The data (A, B, C, and F) are presented as means � SEM.

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We went on to validate detailed mechanisms that have beensuggested to regulate MYC through miRNA. According to theliterature (44), nuclear YAP sequesters p72 (a regulatory compo-nent of the miRNA processing machinery) to prevent its bindingto DROSHA/DGCR8, leading to a decrease in mature miRNAprocessing. This causes downregulation of the miRNAs thatrepressMYC, thereby resulting in increasedMYC expression uponYAP activation. Thus, we asked whether overexpression of p72against a background of high YAP activity could restore miRNAprocessing, consequently repressing MYC. Indeed, we found thatoverexpression of DDX17, the gene that encodes p72, in theYAP-5SA-expressing cells significantly downregulated MYC pro-tein expression, even under YAP active conditions (Fig. 4G). Onthebasis of thesedata andonpreviousfindings,we confirmed thatMYC is upregulated by YAP at the posttranscriptional level via itsregulation through miRNA processing.

MYC is a direct transcriptional target of YAPWe then asked whether YAP/TAZ also transcriptionally regu-

lates MYC. YAP5SA overexpression did not increase MYC mRNAlevels (Fig. 4B and C), so we evaluatedMYC levels upon YAP/TAZdepletion.We tested this effect in HFE-145 cells and in two gastriccancer cell lines (AGS, and MKN74) that display high YAP/TAZandMYCprotein expression. Knockdownof YAP andTAZ in thesecell lines via the expression of short hairpin RNAs induced asignificant reduction in both MYC mRNA and protein levels(Fig. 5A and B; Supplementary Fig. S6A).

To confirm MYC as a direct transcriptional target of YAP,we performed a chromatin immunoprecipitation assay in theYAP-5SA–overexpressing HFE-145 cells. In the resulting data, welooked for YAP binding at the previously reported MYC enhancerregions and in promoter regions with at least three TEAD-bindingconsensus motifs (CATTCC; ref. 45). As shown in Fig. 5C, MYCenhancers 4 and 6 showed enrichment of YAP binding (Fig. 5C;Supplementary Fig. S6B). Altogether, these results are consistentwith a role for YAP and TAZ in the transcriptional regulationof MYC.

After confirming that YAP/TAZ regulates MYC at the transcrip-tional and posttranscriptional levels, we incorporated these twomechanisms into a new model of MYC regulation (Fig. 5D). Inthis model, under baseline conditions, YAP binds toMYC enhan-cers to transcriptionally control MYC expression. At the sametime, miRNAs processed by the DROSHA–DGCR8–p72 complexrepress MYC. These two regulatory mechanisms oppose oneanother to maintain tight, homeostatic control of MYC levels.When YAP is activated, however, there is no additional transcrip-tional regulation; instead, increased nuclear YAP sequesters p72and downregulates miRNA processing. The outcome is decreasedMYC repression, causing a shift toward elevated MYC expression(Fig. 5D).

Inhibition of MYC hinders YAP/TAZ oncogenic activityTo confirm MYC's functional importance as a key signaling

molecule downstream of YAP/TAZ, we asked whether inhibitionof MYC limits tumorigenic potential. We took pharmacologicapproaches and first used JQ1, a BET bromodomain inhibitor,which has been widely employed to inhibit MYC both in vitro andin vivo (51–53). As expected, JQ1 treatment in HFE-145 cellseffectively inhibited MYC even in the presence of YAP-5SA over-expression (Fig. 6A and B). Because YAP activation promotescellular transformation (Fig. 2C), we next asked whether this

transformation can be rescued by addition of JQ1. We performeda low-attachment growth assay, which facilitates test drug effi-ciency during cellular transformation compared with the soft agarassay (54). Consistent with our other results, YAP-5SA overex-pression increased colony formation in low attachment condi-tions. In line with our hypothesis, the addition of JQ1 effectivelyhindered the cellular transformation phenotype enhanced byYAP-5SA (Fig. 6C and D).

To corroborate our findings, we tested the effect of a morespecific MYC inhibitor under the same conditions. 10058-F4inhibits the dimerization of MYC-MAX, thereby blocking tran-scription of MYC downstream targets (55), as shown in Fig. 6E.Similar to JQ1, 10058-F4 inhibition of MYC also suppressedthe anchorage-independent growth intensified by YAP-5SA over-expression (Fig. 6C and D).

Extending these results, we asked whether MYC inhibitionblocks the tumorigenic phenotype in vivo. 10058-F4 was unsuit-able for in vivo application because of its rapid metabolism, so wetested this effect with JQ1 (56). Starting 3 weeks after tamoxifeninduction, we treated Lats1/2iDLgr5mice with either JQ1 or vehicledaily for 5 weeks and compared their gastric epithelium with thatof vehicle-treated WT mice (Fig. 6F). As expected, the vehicle-treated Lats1/2iDLgr5 animals showed gastric epithelial hyperplasiaat 8 weeks post-tamoxifen induction. Mice treated with JQ1,however, were comparable with vehicle-treated WT mice, withno significant evidence of hyperplasia. The JQ1-treated mice alsoshowed reduced MYC expression, supporting the efficacy of JQ1in MYC blockade (Fig. 6G; Supplementary Table S1B). Together,these data strongly suggested that pharmacologic inhibition ofMYC blocked the oncogenic activity of YAP/TAZ. They alsocorroborate the role of MYC as a key signaling mediator down-stream of YAP/TAZ in gastric tumorigenesis.

YAP and MYC expression are correlated in human gastriccancers

Having shown that MYC mediates the oncogenic signalsassociated with YAP/TAZ activation, we next looked for acorrelation between YAP and MYC expression in human gastriccancers. After staining a tissue microarray composed of 1,090samples from patients with gastric cancer for the YAP and MYCproteins, we measured the signal intensity for each in a semi-quantitative manner (Fig. 7A). We then converted these inten-sity values and the area of distribution for each sample intonumeric scales to create a scoring system ranging from 0 to 12(see Supplementary Methods for a more detailed description).Because these scores reflect the activity of each molecule, wealigned the scores of YAP and MYC and found a significantcorrelation between them (Fig. 7B). When we split the scoresinto two groups at their median value (6, with scores �6designated as high and scores

Figure 5.

MYC is a direct transcriptional target of YAP. A and B, Western blots and qRT-PCR analyses of HFE-145 and AGS (gastric cancer cell line) upon simultaneousknockdown of YAP and TAZ, using shGFP as a control. � , P < 0.05 versus shGFP. n ¼ 3 for each group in qRT-PCR. C, Chromatin immunoprecipitationresults for HFE-145 cells overexpressing vector or YAP5SA, performed using a YAP-specific antibody. The CTGF promoter and 30-UTR were used as positiveand negative controls, respectively. � , P < 0.05 versus vector. n ¼ 3 for each group. D, Integrated model of the transcriptional and post-transcriptionalregulation of MYC by YAP. Left, baseline regulation status; right, upregulation of MYC when YAP is active. qRT-PCR and chromatin immunoprecipitationdata are presented as means � SEM.

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Figure 6.

Pharmacologic blockade of MYC rescues YAP-induced tumorigenesis. A, Western blot analysis for YAP, TAZ, and MYC in HFE-145 cells treated with vehicle(DMSO) or JQ1 (500 nmol/L, 24 hours).B,Western blot analysis of HFE-145 cells expressing vector or YAP5SA and treated with vehicle (DMSO) or JQ1 (500 nmol/L,24 hours). C, Microscopic images of growth in low attachment assays, with HFE-145 cells expressing either vector or YAP5SA and treated with either vehicle(DMSO), JQ1 (500 nmol/L), or 10058-F4 (50 mmol/L). D, Colony count (top) and cell viability assays with WST-1 (bottom) in growth in low attachmentassays. � , P < 0.05 versus Vector-DMSO; #, P < 0.05 versus YAP5SA-DMSO. n ¼ 5 for each group. E, qRT-PCR analyses of MYC target genes (CAD, CDK4, andCCND2) in HFE-145 cells treated with DMSO vehicle or 10058-F4 (50 mmol/L, 72 hours). � , P < 0.05 versus vehicle (DMSO). n ¼ 3 for each group. F, Schematicdiagram for JQ1 injection in vivo. G, H&E and IHC staining of MYC in WT (treated with vehicle) and Lats1/2iDLgr5 mice (treated with either vehicle or JQ1).Scale bars, 100 mm. The data in D and E are presented as means � SEM.






Figure 7.

YAP signaling is activated in a subset of human gastric cancers.A,Representative IHC staining images from tissuemicroarrays for YAP andMYC.B, Scatter plot of IHCscores for YAP and MYC from tissue microarray (r ¼ 0.4449; P < 0.0001). C, Quantification of MYC staining in patients with high or low YAP expression, andPearson x2 test to analyze the correlation between YAP and MYC expression based on IHC scores (x2 ¼ 124.211; P < 0.001; OR ¼ 5.554; 95% CI ¼ 4.033–7.647).D, Schematic diagram and GSEA results showing enrichment of YAP target gene sets in MSS/EMT subgroup of ACRG gastric tumors (GSE62254). Falsediscovery rate (FDR) q-values < 0.25 are considered significant in GSEA. E, Schematic diagram for reclassifying ACRG gastric tumors based on YAP signature geneexpression using Signature Evaluation Tool. YAP signature gene upregulated group consisted of 122 cases, and downregulated group consisted of 178 cases.F,Kaplan–Meier plots of disease-free andoverall survival in patientswhose tumors have higheror lower levels of YAP signaturegenes, subclassified fromACRGgastrictumors (GSE62254). Statistical analyses were performed using log-rank test. G, Composition of YAP signature gene upregulated and downregulated subgroupsin the original classification of ACRG gastric tumors. H, GSEA results of ACRG gastric tumors subclassified on the basis of YAP signature gene expression (YAPsignature upregulated vs. downregulated), showing enrichment of MYC targets, G2M checkpoint gene sets and E2F target genes in subgroups expressinghigh YAP signatures. I, GSEA results for Wnt signaling gene set in the reclassified ACRG dataset. Left, MSS/EMT compared with the other three groups.Right, YAP signature upregulated compared with the downregulated subgroup.

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YAP/TAZ signaling has never been addressed in human geno-mics/transcriptomics studies with large numbers of samples,leaving unclear the significance of this signaling cascade inhuman gastric cancer (2–6). We thus reanalyzed transcriptomedata from a public dataset to evaluate the global expressionpatterns of YAP/TAZ target genes in human gastric cancer (3).The microarray dataset reported by the Asian Cancer ResearchGroup (GSE62254) includes gene expression profiles from300 patients with gastric cancer along with associated clinicalinformation. In the original study, the authors classified gastriccancers into four distinct groups based on gene expressionpatterns: (i) microsatellite instability (MSI), (ii) microsatellitestability/epithelial-to-mesenchymal transition (MSS/EMT),(iii) MSS/TP53þ, and (iv) MSS/TP53-. Of these four groups,MSS/EMT showed the worst prognosis, which was validated inother patients with gastric cancer cohorts (Fig. 7D; ref. 3).

YAP activity has also been reported as an indicator of poorprognosis, andweobserved an enrichment of EMTgene sets in ourRNA sequencing data from Lats1/2iDLgr5 tumors (SupplementaryFig. S4B and S4C). Therefore, we hypothesized that the MSS/EMTsubgroup may show upregulation of YAP signature genes. Weperformed a GSEA based on the classifications provided in theoriginal study and found that the MSS/EMT subgroup did indeedshow significant enrichment of YAP signature genes (Fig. 7D;Supplementary Table S2). Assuming that the expression level ofwell-known YAP target genes can be used as a readout of YAPactivity (57), we askedwhether YAP activity can act as a prognosticindicator in this dataset. After splitting the dataset based on YAPsignature gene expression levels using bioinformatics software(Fig. 7E; ref. 36), we found that patients with high YAP activityshowed significantly shorter periods of disease-free and overallsurvival (Fig. 7F). When this subclassification was applied to theoriginal classification of ACRG, 98% of the MSS/EMT group wasconsisted of YAP upregulated cases, while the composition wasaround 30 % in the other three groups (Fig. 7G). On the basis ofthese analyses, we concluded that YAP is significantly activated ina subset of patients with gastric cancer and that its activity isclosely related to their clinical prognosis.

To assess the similarity of the molecular expression patternsbetween our RNA sequencing data and the published humanmicroarray data, we performed GSEA in the dichotomizedGSE62254 dataset based on YAP signature gene expression. Wefound highly significant levels of overlap among the gene setsenriched in the YAP-activated tumor subgroup and in the RNAsequencing data from ourmousemodel (Figs. 3C andD and 7H).And in line with the sequencing results, Wnt signaling was notenriched in either MSS/EMT or YAP-activated subgroup when thedataset was reclassified (Fig. 7I). This also supports that upregula-tion ofMYC is a direct consequence of YAP activation, rather thanan indirect effect through Wnt signaling pathway. Altogether,these results corroborate that our YAP/TAZ-induced gastric tumormodel mimics the molecular expression pattern of human gastriccancer with YAP activation.

DiscussionIn this study, we developed a novel gastric tumor model by

activating YAP/TAZ in gastric epithelial stem cells. Althoughgastric cancer has been reported to arise from mature differenti-ated cells and from stem cells, we add a layer of evidence thatpyloric stem cells can serve as the origin of tumorigenesis. We

demonstrate that YAP/TAZ activation by Lats1/2 deletion inpyloric stem cells induces a step-wise tumorigenic progressionfrom low-grade intraepithelial neoplasia to intramucosal carci-noma. This finding suggests that Lats1/2-mediated inhibition ofYap/Taz is required to restrict the proliferation of Lgr5-postivegastric epithelial stem cells. It is necessary to keep the mice for alonger period to confirm whether these mice develop advancedstages of cancer and eventually metastasize into distant organs.However, most of Lats-deleted mice becamemoribund at around6 months post-tamoxifen injection, which precluded long-termfollow-up. It seems their development of skin lesions (i.e., hairfollicular hyperplasia and crust formation all over the body) maybe one of the causes of morbidity. Because Lgr5 is the mostprominent intestinal stem cell marker in crypt basal cells (33),it is unclear why we observed no related phenotypes in the smallor large intestines and saw no histologic changes in any of theother organs (i.e., liver, pancreas, and ovary) reported to harborLgr5þ cells. Further studies are ongoing to disclose themechanismunderlying the differences in these phenotypes.

It is interesting that Latsknockout can causedistinct phenotypesin different organs. Previously, our group reported that liver-specific Lats depletion induces an expansion of immature biliaryepithelial cells and fibroblasts but not tumor formation (58).Importantly, rather than causing hyperproliferation, we found theactivation of YAP in hepatocytes induces a transdifferentiationinto cells of the biliary epithelial lineage as a consequence of TGFbupregulation. This change also triggers senescencemechanisms inYAP-activated hepatocytes, as reflected in the RNA sequencingresults. In contrast to what we observed in the gastric epithelium,we observed p53 pathway upregulation and E2F target down-regulation. This implies the LATS-YAP signaling cascaderegulates diverse physiologic functions via various mechanismsin a context-dependent manner.

We injected tamoxifen to induce Cre expression at thedesired time point. Intriguingly, one study reports that injec-tions of 5 mg tamoxifen per 20 g of mouse body weight cantrigger injury to the gastric epithelium and cause metaplasia(59). In our conditions, however, we detected no pathologicchanges with tamoxifen injections alone, even at early (24- to120-hour) time points. This distinction may be attributable tothe smaller doses of tamoxifen we injected (75 mg/kg mousebody weight ¼ 1.5 mg/20 g).

Using RNA sequencing, we identified MYC as the key down-streammolecular target of YAP/TAZ in gastric tumorigenesis.MYCis a well-known oncogene involved in cell-cycle progression (43),so our transcriptome analysis points to the activation of cell-cycle–related genes, corroborating the significance of MYC as thekey molecular target. In functional studies, we confirmed thatMYC inhibition significantly rescues the cancer-related pheno-types of YAP. Furthermore, we found a positive correlationbetween YAP and MYC in human gastric cancers, also supportingYAP's regulation of MYC as a key molecular mediator of gastrictumorigenesis. Of note, Yorkie (the Drosophila homolog of YAP)anddMyc directly regulate one another inflies (60, 61). Twootherstudies showed that YAP/TAZ regulatesMYC inmammalian cells,but the mechanisms by which YAP/TAZ did so were discrepant(44, 45). Mori and colleagues found that YAP regulates MYC atthe posttranscriptional level through miRNA repression. NuclearYAP directly interacts with p72 (encoded byDDX17), a regulatorycomponent of the miRNA processing machinery, and sequestersit from DROSHA and DGCR8, thereby inhibiting miRNA






processing (44). As a consequence, miRNAs that inhibit MYC aredownregulated upon YAP activation, leading to increased MYCprotein expression. However, Zanconato and colleagues (45)suggested a different mechanism, showing that MYC is a directtranscriptional target of YAP/TAZ. In their study, they found thatYAP/TAZ binds MYC enhancers and that knockdown of YAP/TAZreduces MYC mRNA levels.

In our own experiments, we confirmed the existence of bothtranscriptional and posttranscriptional regulatory mechanismsand integrated them into a model explaining the tight regula-tion of MYC expression by YAP/TAZ. Under physiologicconditions, MYC is maintained in an equilibrium betweentranscription and repression. When oncogenic YAP is activated,however, it induces a shift toward increased MYC expressionthrough downregulating miRNA repression of MYC. Neverthe-less, MYC is a transcriptional target of YAP that is involved inregulating cell growth, and that role has been conserved fromflies to humans.

It is important to note that CTGF, AREG, andBIRC5 are all well-known targets of YAP that are reportedly related to gastric cancer(62–64). It is therefore plausible that these genes may contributedirectly to YAP-induced gastric tumorigenesis. We are convinced,however, that it is MYC that has the most influence relaying YAPsignals because MYC inhibition rescues the observed YAP-depen-dent oncogenic phenotypes.

The gastric tumor model we present here seems to reflect wellthe molecular pattern of a subgroup of human gastric cancers. Asdescribed above, none of the human genomics/transcriptomicsstudies have revealed the significance of Hippo-YAP/TAZ signal-ing in gastric cancer. Here, through analyzing a public microarraydataset containing a large number of patient samples, we showthat a subset of human gastric cancers (the MSS/EMT subtype)shows strong YAP target gene enrichment. When we reclassifiedthe dataset based on YAP signature gene expression, we observedan inverse correlationbetween clinical outcomes andYAPactivity.Furthermore, the enriched gene sets in the YAP-activated humangastric cancer subgroup were similar to the upregulated genes inour murine gastric tumors, reflecting the clinical relevance of thismouse model.

Previous studies reporting poor clinical outcome with YAPactivity show inconsistent results. Song and colleagues reportedthat nuclear YAP1 expression did not correlate with overallsurvival when all patients with gastric cancer were analyzedtogether, but was an independent prognostic factor only for theintestinal type (13). Other studies by Hu and colleagues, andKang and colleagues showed that YAP expression was correlat-ed with poor clinical outcome regardless of the histologicsubtype (12, 14). Analyses from the original article of ourmicroarray data showed that MSS/EMT subgroup, which ismainly consisted of diffuse-type cancer, displayed the worstclinical outcome (3). The underlying reasons to the differencesshown in these reports remain elusive at this point. One of thepossibilities is that classifying gastric cancer by Lauren's sub-types could have high inter-observer variabilities (65, 66). Asthere is no "gold standard" for defining the Lauren's classifi-cation, discordance between pathologists vary from 20% to30% and there also exists a considerable portion of mixed-typegastric cancer. Another possibility is the difference in measuringYAP activity between the studies. Previous studies used IHCstaining, whereas our data used YAP signature gene expressionas the indicator for YAP activity.

YAP/TAZ is a well-known potent oncogene in many differentcancers. Nevertheless,mutations in YAP/TAZor the core upstreamHippo pathway regulators are rarely reported (11). This lack ofgenomic evidence leads to the hypothesis that various extracel-lular cues may be responsible for the upregulation of YAP/TAZ incancers. Gastric cancer is associated with diverse carcinogenicstimuli including Helicobacter pylori infection, smoking, and diet(e.g., ingestednitrates/nitrites andhigh salt). These triggers inducerepetitive injury and regeneration cycles in which the gastricepithelial cells and their microenvironment are affected bymechanical tension and extracellular ligands. Because YAP/TAZseems to act as a nexus for these various signals, it may be possibleto link such changes as potential cues for YAP/TAZ activation(10, 67). This idea is supported by the upregulation of YAPexpression in Helicobacter-infected murine gastric epitheliumand with the activation of YAP signaling upon cholinergicneural stimulation of the gastric tissue (15, 68). In addition,RUNX3, which is not a member of the canonical Hippo pathway,reportedly regulates the role the YAP–TEAD complex plays ingastric tumorigenesis (69). Thus, YAP/TAZ activation mayresult from various carcinogenic stimuli rather than fromgenomic mutations, which might explain why YAP/TAZ has beenoverlooked in previous studiesmainly focused on genomicmuta-tions (2–6).

Together, our model provides solid genetic evidence thatYAP/TAZ activation can initiate gastric tumoriogenesis in vivo.We also demonstrate that this signaling cascade is enriched in asubset of human gastric cancers, strengthening its clinical impor-tance. Therefore, we propose further investigation of YAP/TAZ infuture translational studies. Because the YAP/TAZ–active gastriccancer subgroup of patients showed worse prognoses, YAP/TAZactivity may be a useful marker for predicting clinical outcomes.Our results also have led us to investigate the therapeutic impactof YAP/TAZorMYCblockade in patients showing upregulation ofthis signaling cascade.

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

Authors' ContributionsConception and design: W. Choi, G.Y. Koh, D.-S. LimDevelopment of methodology: W. Choi, J. Kim, D.-S. LimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Choi, J. Kim, J. Park, D.T. Smoot, S.-Y. Kim,C. Choi, D.-S. LimAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Choi, C. Choi, D.-S. LimWriting, review, and/or revision of the manuscript: W. Choi, D. Hwang,G.Y. Koh, D.-S. LimAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Hwang, J.-H. Kim, H. Ashktorab, C. Choi,D.-S. LimStudy supervision: D.-H. Lee, G.Y. Koh, D.-S. Lim

AcknowledgmentsWe thank Dr. Young-Yun Kong (Seoul National University) for providing

Lgr5-EGFP-IRES-creERT2 mice for this study. DDX17 plasmid was a generousgift from Dr. Didier Auboeuf (INSERM, France). JQ1 for in vivo treatment waskindly provided by Dr. Hee-Dong Park (LG Chem, Korea). The biospecimensand data used for this study were provided by the Biobank of ChonnamNational University Hwasun Hospital, a member of the Korea Biobank Net-work. This study was supported by a grant from the National Creative ResearchInitiatives Center Program (NRF-2010-0018277 to D-S. Lim), Korean Founda-tion for Cancer Research (KFCR-2016-004 toW. Choi), Individual Basic Science

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& Engineering Research Program (NRF-2016R1D1A1B03935764 to D.-H. Lee),and Institute for Basic Science funded by the Ministry of Science and ICT,Republic of Korea (IBS-R025-D1 to G.Y. Koh).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 9, 2017; revised February 7, 2018; accepted April 12,2018; published first April 18, 2018.

References1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al.

Cancer incidence and mortality worldwide: sources, methods and majorpatterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86.

2. Cancer Genome Atlas Research Network. Comprehensive molecular char-acterization of gastric adenocarcinoma. Nature 2014;513:202–9.

3. Cristescu R, Lee J, NebozhynM, KimKM, Ting JC,Wong SS, et al. Molecularanalysis of gastric cancer identifies subtypes associatedwith distinct clinicaloutcomes. Nat Med 2015;21:449–56.

4. Kakiuchi M, Nishizawa T, Ueda H, Gotoh K, Tanaka A, Hayashi A, et al.Recurrent gain-of-function mutations of RHOA in diffuse-type gastriccarcinoma. Nat Genet 2014;46:583–7.

5. Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, et al. Exome sequencingidentifies frequent mutation of ARID1A in molecular subtypes of gastriccancer. Nat Genet 2011;43:1219–23.

6. Wang K, Yuen ST, Xu J, Lee SP, Yan HH, Shi ST, et al. Whole-genomesequencing and comprehensive molecular profiling identify new drivermutations in gastric cancer. Nat Genet 2014;46:573–82.

7. Camargo FD,Gokhale S, Johnnidis JB, FuD, BellGW, JaenischR, et al. YAP1increases organ size and expands undifferentiated progenitor cells. CurrBiol 2007;17:2054–60.

8. Lee JH, Kim TS, Yang TH, Koo BK, Oh SP, Lee KP, et al. A crucial role ofWW45 in developing epithelial tissues in the mouse. EMBO J 2008;27:1231–42.

9. Lee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, et al. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, andliver tumorigenesis. Proc Natl Acad Sci U S A 2010;107:8248–53.

10. Yu FX, Zhao B, Guan KL. Hippo pathway in organ size control, tissuehomeostasis, and cancer. Cell 2015;163:811–28.

11. Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the roots of cancer.Cancer Cell 2016;29:783–803.

12. Kang W, Tong JH, Chan AW, Lee TL, Lung RW, Leung PP, et al.Yes-associated protein 1 exhibits oncogenic property in gastric cancer andits nuclear accumulation associates with poor prognosis. Clin Cancer Res2011;17:2130–9.

13. Song M, Cheong JH, Kim H, Noh SH, Kim H. Nuclear expression of Yes-associated protein 1 correlateswith poor prognosis in intestinal type gastriccancer. Anticancer Res 2012;32:3827–34.

14. Hu X, Xin Y, Xiao Y, Zhao J. Overexpression of YAP1 is correlated withprogression, metastasis and poor prognosis in patients with gastric carci-noma. Pathol Oncol Res 2014;20:805–11.

15. Jiao S,WangH, Shi Z, DongA, ZhangW, Song X, et al. A peptidemimickingVGLL4 function acts as a YAP antagonist therapy against gastric cancer.Cancer Cell 2014;25:166–80.

16. Schepers A, Clevers H. Wnt signaling, stem cells, and cancer of thegastrointestinal tract. Cold Spring Harb Perspect Biol 2012;4:a007989.

17. Hayakawa Y, Fox JG, Wang TC. The origins of gastric cancer from gastricstem cells: lessons from mouse models. Cell Mol Gastroenterol Hepatol2017;3:331–8.

18. Arnold K, Sarkar A, Yram MA, Polo JM, Bronson R, Sengupta S, et al.Sox2(þ) adult stem and progenitor cells are important for tissueregeneration and survival of mice. Cell Stem Cell 2011;9:317–29.

19. Stange DE, Koo BK, Huch M, Sibbel G, Basak O, Lyubimova A, et al.Differentiated Troyþ chief cells act as reserve stem cells to generate alllineages of the stomach epithelium. Cell 2013;155:357–68.

20. Hayakawa Y, Ariyama H, Stancikova J, Sakitani K, Asfaha S, Renz BW, et al.Mist1 expressing gastric stem cells maintain the normal and neoplasticgastric epithelium and are supported by a perivascular stem cell niche.Cancer Cell 2015;28:800–14.

21. Matsuo J, Kimura S, Yamamura A, Koh CP, Hossain MZ, Heng DL, et al.Identification of stem cells in the epithelium of the stomach corpus andantrum of mice. Gastroenterology 2017;152:218–31.

22. Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH,et al. Lgr5(þve) stem cells drive self-renewal in the stomach andbuild long-lived gastric units in vitro. Cell Stem Cell 2010;6:25–36.

23. HayakawaY, JinG,WangH,ChenX,WestphalenCB,Asfaha S, et al. CCK2Ridentifies and regulates gastric antral stem cell states and carcinogenesis.Gut 2015;64:544–53.

24. Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wieden-mann B, et al. Stromal R-spondin orchestrates gastric epithelial stem cellsand gland homeostasis. Nature 2017;548:451–5.

25. Sakitani K, Hayakawa Y, DengH, AriyamaH, Kinoshita H, KonishiM, et al.CXCR4-expressing Mist1þ progenitors in the gastric antrum contribute togastric cancer development. Oncotarget 2017;8:111012–25.

26. Choi E, Hendley AM, Bailey JM, Leach SD, Goldenring JR. Expression ofactivated Ras in gastric chief cells of mice leads to the full spectrum ofmetaplastic lineage transitions. Gastroenterology 2016;150:918–30.

27. Shimada S, Mimata A, Sekine M, Mogushi K, Akiyama Y, Fukamachi H,et al. Synergistic tumour suppressor activity of E-cadherin and p53 in aconditional mouse model for metastatic diffuse-type gastric cancer. Gut2012;61:344–53.

28. Li XB, YangG,ZhuL, TangYL, ZhangC, JuZ, et al.Gastric Lgr5(þ) stemcellsare the cellular origin of invasive intestinal-type gastric cancer inmice. CellRes 2016;26:838–49.

29. Syu LJ, Zhao X, Zhang Y, Grachtchouk M, Demitrack E, Ermilov A, et al.Invasive mouse gastric adenocarcinomas arising from Lgr5þ stem cells aredependent on crosstalk between theHedgehog/GLI2 andmTORpathways.Oncotarget 2016;7:10255–70.

30. KimM,KimM,Lee S, Kuninaka S, SayaH, LeeH, et al. cAMP/PKA signallingreinforces the LATS-YAP pathway to fully suppress YAP in response to actincytoskeletal changes. EMBO J 2013;32:1543–55.

31. Heallen T, ZhangM,Wang J, Bonilla-ClaudioM, Klysik E, Johnson RL, et al.Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte prolif-eration and heart size. Science 2011;332:458–61.

32. Heallen T, Morikawa Y, Leach J, Tao G, Willerson JT, Johnson RL, et al.Hippo signaling impedes adult heart regeneration. Development 2013;140:4683–90.

33. Barker N, van Es JH, Kuipers J, Kujala P, van den BornM, CozijnsenM, et al.Identification of stem cells in small intestine and colon by marker geneLgr5. Nature 2007;449:1003–7.

34. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, et al.A robust and high-throughput Cre reporting and characterization systemfor the whole mouse brain. Nat Neurosci 2010;13:133–40.

35. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA,et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci U S A2005;102:15545–50.

36. Jen CH, Yang TP, Tung CY, Su SH, Lin CH, Hsu MT, et al. SignatureEvaluation Tool (SET): a Java-based tool to evaluate and visualize thesample discrimination abilities of gene expression signatures. BMC Bio-informatics 2008;9:58.

37. Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, et al. Lgr5marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 2008;40:1291–9.

38. Huch M, Bonfanti P, Boj SF, Sato T, Loomans CJ, van de Wetering M, et al.Unlimited in vitro expansion of adult bi-potent pancreas progenitorsthrough the Lgr5/R-spondin axis. EMBO J 2013;32:2708–21.

39. HuchM,Dorrell C, Boj SF, van Es JH, Li VS, vandeWeteringM, et al. In vitroexpansion of single Lgr5þ liver stem cells induced by Wnt-driven regen-eration. Nature 2013;494:247–50.

40. Ng A, Tan S, Singh G, Rizk P, Swathi Y, Tan TZ, et al. Lgr5 marks stem/progenitor cells in ovary and tubal epithelia. Nat Cell Biol 2014;16:745–57.






41. Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J, et al. Inactivation of YAPoncoprotein by the Hippo pathway is involved in cell contact inhibitionand tissue growth control. Genes Dev 2007;21:2747–61.

42. Lei QY, Zhang H, Zhao B, Zha ZY, Bai F, Pei XH, et al. TAZ promotes cellproliferation and epithelial-mesenchymal transition and is inhibited bythe hippo pathway. Mol Cell Biol 2008;28:2426–36.

43. Dang CV. MYC on the path to cancer. Cell 2012;149:22–35.44. Mori M, Triboulet R, Mohseni M, Schlegelmilch K, Shrestha K, Camargo

FD, et al. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer. Cell 2014;156:893–906.

45. Zanconato F, ForcatoM, BattilanaG, Azzolin L,Quaranta E, Bodega B, et al.Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancersdrives oncogenic growth. Nat Cell Biol 2015;17:1218–27.

46. Rosenbluh J, Nijhawan D, Cox AG, Li X, Neal JT, Schafer EJ, et al. beta-Catenin-driven cancers require a YAP1 transcriptional complex for survivaland tumorigenesis. Cell 2012;151:1457–73.

47. Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B,Gurung B, et al. Hippo pathway activity influences liver cell fate. Cell2014;157:1324–38.

48. Zhao B, Ye X, Yu J, Li L, LiW, Li S, et al. TEADmediates YAP-dependent geneinduction and growth control. Genes Dev 2008;22:1962–71.

49. Christoffersen NR, Shalgi R, Frankel LB, Leucci E, Lees M, Klausen M, et al.p53-independent upregulation of miR-34a during oncogene-inducedsenescence represses MYC. Cell Death Differ 2010;17:236–45.

50. Singh SK, KagalwalaMN, Parker-Thornburg J, AdamsH,Majumder S. RESTmaintains self-renewal and pluripotency of embryonic stem cells. Nature2008;453:223–7.

51. Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, et al. BETbromodomain inhibition as a therapeutic strategy to target c-Myc. Cell2011;146:904–17.

52. Bandopadhayay P, Bergthold G, Nguyen B, Schubert S, Gholamin S, TangY, et al. BET bromodomain inhibition of MYC-amplified medulloblasto-ma. Clin Cancer Res 2014;20:912–25.

53. Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, et al.Selective inhibition of BET bromodomains. Nature 2010;468:1067–73.

54. RotemA, Janzer A, Izar B, Ji Z, Doench JG, Garraway LA, et al. Alternative tothe soft-agar assay that permits high-throughput drug and genetic screensfor cellular transformation. Proc Natl Acad Sci U S A 2015;112:5708–13.

55. Yin X, Giap C, Lazo JS, Prochownik EV. Lowmolecular weight inhibitors ofMyc-Max interaction and function. Oncogene 2003;22:6151–9.

56. Guo J, Parise RA, Joseph E, Egorin MJ, Lazo JS, Prochownik EV, et al.Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the

Myc-Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2-thioxothia-zolidin-4-one, in mice. Cancer Chemother Pharmacol 2009;63:615–25.

57. Cordenonsi M, Zanconato F, Azzolin L, Forcato M, Rosato A, Frasson C,et al. The Hippo transducer TAZ confers cancer stem cell-related traits onbreast cancer cells. Cell 2011;147:759–72.

58. Lee DH, Park JO, Kim TS, Kim SK, Kim TH, Kim MC, et al. LATS-YAP/TAZcontrols lineage specification by regulating TGFbeta signaling and Hnf4al-pha expression during liver development. Nat Commun 2016;7:11961.

59. HuhWJ, Khurana SS, Geahlen JH, Kohli K, Waller RA, Mills JC. Tamoxifeninduces rapid, reversible atrophy, and metaplasia in mouse stomach.Gastroenterology 2012;142:21–4.

60. Neto-Silva RM, de Beco S, Johnston LA. Evidence for a growth-stabilizingregulatory feedback mechanism between Myc and Yorkie, the Drosophilahomolog of Yap. Dev Cell 2010;19:507–20.

61. Ziosi M, Baena-Lopez LA, Grifoni D, Froldi F, Pession A, Garoia F, et al.dMyc functions downstream of Yorkie to promote the supercompetitivebehavior of hippo pathway mutant cells. PLoS Genet 2010;6:e1001140.

62. Jiang CG, Lv L, Liu FR, Wang ZN, Liu FN, Li YS, et al. Downregulation ofconnective tissue growth factor inhibits the growth and invasion of gastriccancer cells and attenuates peritoneal dissemination. Mol Cancer2011;10:122.

63. Wang B, Yong H, Zhu H, Ni D, Tang S, Zhang S, et al. Abnormalamphiregulin expression correlates with gastric cancer prognosis.Oncotarget 2016;7:76684–92.

64. Tu SP, Jiang XH, Lin MC, Cui JT, Yang Y, Lum CT, et al. Suppression ofsurvivin expression inhibits in vivo tumorigenicity and angiogenesis ingastric cancer. Cancer Res 2003;63:7724–32.

65. Palli D, Bianchi S, Cipriani F, Duca P, Amorosi A, Avellini C, et al.Reproducibility of histologic classification of gastric cancer. Br J Cancer1991;63:765–8.

66. Shibata A, Longacre TA, Puligandla B, Parsonnet J, Habel LA. Histologicalclassification of gastric adenocarcinoma for epidemiological research:concordance between pathologists. Cancer Epidemiol Biomarkers Prev2001;10:75–8.

67. Hansen CG, Moroishi T, Guan KL. YAP and TAZ: a nexus for Hipposignaling and beyond. Trends Cell Biol 2015;25:499–513.

68. Hayakawa Y, Sakitani K, Konishi M, Asfaha S, Niikura R, Tomita H, et al.Nerve growth factor promotes gastric tumorigenesis through aberrantcholinergic signaling. Cancer Cell 2017;31:21–34.

69. Qiao Y, Lin SJ, Chen Y, VoonDC, Zhu F, Chuang LS, et al. RUNX3 is a novelnegative regulator of oncogenic TEAD-YAP complex in gastric cancer.Oncogene 2016;35:2664–74.


Choi et al.




2018;78:3306-3320. Published OnlineFirst April 18, 2018.Cancer Res Wonyoung Choi, Jeongsik Kim, Jaeoh Park, et al. YAP/TAZ Initiates Gastric Tumorigenesis via Upregulation of MYC

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