molecular cell biology - cancer research · molecular cell biology tgfb promotes genomic...

Molecular Cell Biology

TGFb Promotes Genomic Instability after Loss ofRUNX3Vaidehi Krishnan1, Yu Lin Chong1, Tuan Zea Tan1, Madhura Kulkarni1,2,Muhammad Bakhait Bin Rahmat1, Lavina Sierra Tay1, Haresh Sankar1,Doorgesh S. Jokhun3,4, Amudha Ganesan1, Linda Shyue Huey Chuang1,Dominic C. Voon1, GV Shivashankar3,4, Jean-Paul Thiery5,6, and Yoshiaki Ito1

Abstract

Studies of genomic instability have historically focused onintrinsic mechanisms rather than extrinsic mechanisms based inthe tumor microenvironment (TME). TGFb is the most abun-dantly secreted cytokine in the TME, where it imparts variousaggressive characteristics including invasive migration, drug resis-tance, and epithelial-to-mesenchymal transition (EMT). Here weshow that TGFb also promotes genomic instability in the form ofDNA double strand breaks (DSB) in cancer cells that lack thetumor suppressor gene RUNX3. Loss of RUNX3 resulted intranscriptional downregulation of the redox regulator heme oxy-genase-1 (HO-1 or HMOX1). Consequently, elevated oxidativeDNA damage disrupted genomic integrity and triggered cellularsenescence,whichwas accompaniedby tumor-promoting inflam-

matory cytokine expression and acquisition of the senescence-associated secretory phenotype (SASP). Recapitulating the abovefindings, tumors harboring a TGFb gene expression signature andRUNX3 loss exhibited higher levels of genomic instability. Insummary, RUNX3 creates an effective barrier against furtherTGFb-dependent tumor progression by preventing genomic insta-bility. Thesedata suggest a novel cooperationbetween cancer cell–extrinsic TGFb signaling and cancer cell–intrinsic RUNX3 inacti-vation as aggravating factors for genomic instability.

Significance: RUNX3 inactivation in cancer removes anantioxidant barrier against DNA double strand breaks inducedby TGFb expressed in the tumor microenvironment. Cancer Res;78(1); 88–102. �2017 AACR.

IntroductionGenomic instability promotes the acquisition of the cancer

phenotype by allowing mutational accumulation (1). In mosthereditary cancers, genomic instability arises by the inactivationof DNA damage repair genes like BRCA1, BRCA2, TP53, ormismatch repair genes. In sporadic cancers, genomic instabilityarises due to cancer cell–specific defects like DNA replicationstress, aberrant AID/APOBEC activity, or micronuclei-mediatedchromothripsis (2). However, it has remained unexplored wheth-er genomic instability can be fuelled in a cell-extrinsic manner bycytokines from the tumor microenvironment.

In this regard, the TGFb is the most abundantly secretedcytokine by both tumors and their surrounding stromal cells(3). The secreted TGFb, in turn, might elicit paradoxical effects

on cancers by activating antiproliferative signaling or by impartingpro-oncogenic properties (3). The contradictory facets of TGFbsignaling and the molecular switches that enable this transitionhave been a subject of intense study. For example, in pancreaticcancers, TGFb induces Sox4 that converts TGFb from a tumor-promoting into a tumor-inhibiting factor by inducing apoptosis(4). In contrast, in lung cancers, TGFb functions as a tumor-promoting factor that induces angiogenesis, metastasis, andpoorer patient survival, through the induction of EMT (5, 6).EMT is a central developmental process whereby cells lose theirepithelial identity and gain mesenchymal features (7). TGFbregulates EMT by SMAD-dependent and SMAD-independentpathways such as the PI3K–AKT, ERK MAPK, p38 MAPK,and JUN N-terminal kinase (JNK). These intricate signalingpathways actively crosstalk and impart potent oncogenic pheno-types such as increased migration, invasion, drug resistance, andstemness (8).

Despite extensive studies on the pro-oncogenic effects of TGFb,it remains unknown whether TGFb disrupts cancer cell genomicintegrity. In an earlier work, TGFbwas shown to control the DNAdamage response (DDR) pathway by regulating ATM (9). Alongsimilar lines, TGFb inhibition type I receptor led to reduced Chk2,Rad17, and p53 phosphorylation and Smad2 and Smad7 local-ized with DSB repair proteins (10). Recently, TGFb was shown totrigger aneuploidy and genomic instability in cells undergoingEMT by inducing mitotic abnormalities (11). Here, we identifiedthat stromal TGFbmay be involved in triggering another distinctform of genomic instability, specifically by the generation ofoxidative DNA damage in cells deficient for the tumor suppressorgene, RUNX3.

1Cancer Science Institute of Singapore, National University of Singapore,Singapore. 2Lee Kong Chian School of Medicine, NTU, Singapore. 3Mechano-biology Institute, National University of Singapore, Singapore. 4Department ofBiological Sciences, National University of Singapore, Singapore. 5Yong Loo LinSchool of Medicine, National University of Singapore, Singapore. 6UMR 1186INSERM Institute, Gustave Roussy, Villejuif, France.

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

Corresponding Authors: Yoshiaki Ito, Cancer Science Institute of Singapore,National University of Singapore, MD-6, 14-Medical Drive, Singapore 117599,Singapore. Phone: 65-6516-2242; Fax: 65-6873-9664; E-mail: [email protected];Jean-Paul Thiery, [email protected]; and Vaidehi Krishnan, [email protected]

doi: 10.1158/0008-5472.CAN-17-1178

�2017 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(1) January 1, 201888

on May 26, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 26, 2017; DOI: 10.1158/0008-5472.CAN-17-1178

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-1178&domain=pdf&date_stamp=2017-12-19

http://cancerres.aacrjournals.org/

The RUNX family of proteins is comprised of three hetero-dimeric transcription factors (TF), RUNX1, RUNX2, and RUNX3.Of these, RUNX1 and RUNX3 are inactivated in cancers bymutation or epigenetic deregulation, emphasizing their status asbonafide tumor suppressors (12). In the developmental context,RUNX proteins have emerged as critical modulators of TGFbsignaling through their physical interaction with R-SMADs andwith the bonemorphogenetic protein (BMP)-specific SMADs andSMAD4. RUNX cooperates with SMADs to induce synergistic genetranscription of the cell-cycle inhibitor, p21, and the apoptosisinducer, BIM in a TGFb-dependentmanner (13, 14). In pancreaticcancer, the SMAD4 gene dosage was recently reported to controlRUNX3 transcription; in turn, the expression levels of RUNX3control the switch between local proliferation and metastasis(15). RUNX3, in conjunction with TGFb, therefore plays key rolesin dictating cancer progression.

Here, we uncovered that TGFb promotes genomic instability inRUNX3-deficient cells by inducing oxidative stress–associatedDNA damage by the downregulation of the redox regulator,HMOX1. By providing defense against TGFb-dependent promo-tion of cancer progression, our results unveil a unique facet ofRUNX3-dependent tumor suppression.

Materials and MethodsCell lines and cell culture

A549, NCI-H1299, NCI-H292, PC-14, and NCI-H23 cells wereobtained from ATCC. AZ-521 was from the Korean ResearchInstitute of Bioscience and Biotechnology (KRIBB, Korea).HGC-27 was from Riken Cell Bank. Early passage cells wereutilized for all experiments. Cells were cultured in RPMI medium(RPMI1640, Nacalai Tesque) supplemented with 10% (v/v) FBS(Biowest).

RNA-Sequencing and raw data processingSamples were harvested and total RNA was extracted according

to the manufacturer's instructions (Qiagen RNeasy Mini Kit,Qiagen). Samples were processed further for RNA-Seq analysis,as described under Supplementary Methods.

Gene knockdown and plasmid transfectionA549 cellswere transfectedwith pooled siRNA (Dharmacon) as

described in "Experimental Procedures" (Supplementary Infor-mation). Jet Prime transfection reagent (Polypus) was used forsiRNA transfection into NCI-H1299, PC-14, NCI-H23, AZ-521,and HGC-27, according to the manufacturer's instructions.

ImmunofluorescenceCells were fixed with 4% paraformaldehyde for 15 minutes at

room temperature, permeabilized with 0.5% Triton X-100 in PBSfor 15minutes and blocked using 2%BSA, 5% FBS in 0.1% TritonX-100 for 30 minutes. Antibodies diluted in 2% BSA in 0.1%Triton X-100 were incubated overnight at 4�C. Alexa Fluor–conjugated secondary antibodies (1:1,000) were added for 1hour, at room temperature. Coverslips were mounted with Pro-long Gold Anti-fade (Invitrogen) with DAPI. Images were cap-tured using the Olympus FluoView FV1000 Confocal microscopeusing the 60�oil objective. Cells containing greater than5 gH2AXfoci or 53BP1 foci were considered as positive for DNA damageaccumulation. gH2AXantibodywas fromEMDMillipore (catalogno. 05-636) and the 53BP1 antibody was from Abcam (catalogno. ab-36823).

Senescence-associated b-galactosidase assaySenescence-associated b-galactosidase (SA-b-gal) assay was

done according to the manufacturer's instructions (Abcam).Briefly, cells were washed twice with PBS, fixed with fixationbuffer for 15 minutes at room temperature, and stained withb-galactosidase detection solution overnight at 37�C. Cells wereimaged and blue cells were scored using the ImageJ (NIH,Bethesda, MD) software.

Reactive oxygen species measurement by flow cytometryFor detection of total reactive oxygen species (ROS) levels, cells

were washed twice with PBS and incubated with 10 mmol/Lcarboxy-H2DCFDA (6-carboxy-20,70-dichlorodihydrofluoresceindiacetate) for 20minutes or 5 mmol/L CellROXDeep Red Reagent(Molecular Probes) for 30 minutes. The FACS LSRII flow cyt-ometer (Becton Dickinson) was used for data acquisition. Dataanalysis was performed using the FlowJO single-cell analysissoftware.

ResultsTGFb-mediated EMT in non–small cell lung cancer cells

The exposure of the non–small cell lung cancer (NSCLC)cell line, A549, to recombinant TGFb (5 ng/mL) inducesthe acquisition of a mesenchymal morphology in 48 hours(Fig. 1A). TGFb repressed the expression of epithelial markers,CDH1 and OCCLN, Adherens junction, and tight junction pro-teins, respectively, and increasedmesenchymal gene expression ofCDH2, FN1, and SERPINE1 (Fig. 1B and C). Western blotsdisplayed fibronectin, vimentin, and N-cadherin upregulation(Fig. 1D, top) and E-cadherin downregulation (Fig. 1D, bottom).The EMT-TFs SNAI1 and SNAI2 were upregulated whereas ZEB1,ZEB2, TWIST1, and TWIST2 remained unaltered (Fig. 1E).SMAD4 depletion abrogated TGFb-mediated EMT, indicating therequirement of canonical SMAD signaling in this context(Supplementary Fig. S1A and S1B).

TGFb induces DNA damage in RUNX3-depleted cellsTo elucidate the function of RUNX3 during TGFb signaling,

RUNX3 was silenced using siRNA (RUNX3-KD). Nontargetingoligonucleotides were used as the negative control (CONT-KD;Fig. 1F). Western blot analyses confirmed almost complete deple-tion of RUNX3 (Fig. 1G). Control and RUNX3-KD cells exhibitedsimilar SMAD2/3 phosphorylation, indicating comparableupstream TGFb signaling (Fig. 1G). RUNX3 levels were down-regulated by approximately 40% by TGFb (Fig. 1G, Supplemen-tary Fig. S1C). Interestingly, upon RUNX3 depletion, cells appear-ed sparser in cell density with abnormal flattened morphologyafter TGFb exposure (Supplementary Fig. S1D andS1E). AlthoughRUNX3 depletion or TGFb treatment individually reduced cellgrowth and bromodeoxyuridine (BrdUrd) uptake (�80% ofcontrol), RUNX3 depletion coupled with TGFb treatment greatlylowered the 24-hour BrdUrd uptake, implying exit from the cellcycle (��, P < 0.001; Supplementary Fig. S1F and S1G).

We hypothesized that cells may withdraw from the cell cycle asa stress-dependent response mechanism triggered by DNA dam-age accumulation. The presence of gH2AX (phosphorylated-his-tone H2AX at ser139) foci, expressed at DSB sites, was monitored(16). gH2AX antibody was validated using H2AX siRNA (Sup-plementary Fig. S1H). gH2AX accumulated to a limited extentafter TGFb treatment (Fig. 2A). In contrast, approximately 40% of

DNA Damage during TGFb Signaling

www.aacrjournals.org Cancer Res; 78(1) January 1, 2018 89




RUNX3-KD cells displayed robust accumulation of gH2AX fociafter TGFb treatment. Such gH2AX foci colocalized with 53BP1andATM(Ser1981), bonafidemarkers forDSBs (Fig. 2B–D).DSBaccumulation was rescued through the overexpression of siRNA-resistant wild-type RUNX3 construct, demonstrating specificity ofthe knockdown experiments (Supplementary Fig. S2A). Karyo-typing confirmed DSB accumulation in approximately 20% ofRUNX3-KD cells exposed to TGFb (Supplementary Fig. S2B). Toexamine the dependence of SMAD signaling on DNA damageaccumulation, RUNX3 was codepleted with SMAD4. Following

TGFb treatment, gH2AX positivity was abolished in RUNX3/SMAD4 double knockdown (DKD) cells (Fig. 2E and F). Incontrast, the codepletion of RUNX3 with SNAI1 and SNAI2 didnot impact DNA damage accumulation (Supplementary Fig. S2Cand S2D). Thus, an upstream SMAD-dependent, but SNAI1/2-independent event, triggered DNA damage following TGFb expo-sure. The above results were independently validated using anoth-er TGFb-responsive NSCLC cell line, NCI-H1299. Similar to A549cells, TGFb triggered DNA damage upon RUNX3 depletion inNCI-H1299 (Fig. 2G and H).

Figure 1.

Characterization of TGFb-dependentEMT in NSCLC cells.A,A549 cellswereseeded into 6-well dishes at the celldensity of 0.1 � 106 cells/well. Cellswere either exposed to vehicle controlor TGFb. Cells were imaged at theindicated time-points to track spindle-shaped morphologic changes inducedduring TGFb-mediated EMT. Scale bar,200 mm. B, Gene expression levels ofCDH1 and OCLN plotted relative toTGFb-untreated controls. GAPDH wasused for normalization of geneexpression. C, CDH2, FN1, andSERPINE1 relative gene expressionlevels are shown. D, Cells were leftuntreated or exposed to TGFb. Top,Western blots were probed withantibodies against fibronectin,vimentin, and N-cadherin. GAPDHwasused as the loading control. Bottom,Western blots were probed withantibodies against E-cadherin. E, Cellswere either exposed to vehicle controlor TGFb for 48 hours. Relative geneexpression levels of SNAI1, SNAI2,TWIST1, TWIST2, ZEB1, and ZEB2 areshown. F, Schematic representation ofknockdown experiment. Cells wereseeded into 6-cm dishes at the celldensity of 0.2 � 106 cells/well andtransfectedwith pooled siRNA againstRUNX3 or control siRNA. On day 4,cells were trypsinized and seeded atthe cell density of 0.3� 106 cells/6-cmdish and subjected to a second roundof siRNA. Twenty-four hours later,either vehicle control or TGFb wasadded for 48 hours.G,Experimentwasdone as described in F. Total cellextracts were Western blotted withthe indicated antibodies. KU-70served as the loading control.Graphs show mean � SD. Asterisksrepresent significant differences.� , P < 0.05; �� , P < 0.01;�� , P < 0.001; n.s., not significant.

Krishnan et al.

Cancer Res; 78(1) January 1, 2018 Cancer Research90




Figure 2.

RUNX3 depletion triggers the accumulation of endogenous DNA damage in the presence of TGFb. A, A549 cells were transfected with control or RUNX3 siRNA andexposed to either vehicle control or TGFb for 48 hours. Samples were fixed and stained with antibody against gH2AX. A zoomed image (200%) of theinset is shown on the right. Scale bar, 50 mm. B, Quantification of gH2AX foci by the "point-maxima method," as described in Materials and Methods. Graph showspercent positivity for gH2AX (left). Right, number of gH2AX foci per cell is shown. Data are representative of four independent experiments. C, Following RUNX3-KD,cells were exposed to TGFb for 48 hours. Top, coimmunofluorescence staining with antibodies against gH2AX and 53BP1. Bottom, coimmunofluorescencestaining with antibodies against gH2AX and pATM (Ser1981). Scale bar, 50 mm.D, Experiment was done as described inA. Imageswere captured using the ArrayscanHCS reader (Cellomics). gH2AX fluorescence intensity per cell was computed and plotted using the Cellomics Arayscan software. E, Cells were subjectedto either RUNX3-KD, SMAD4-KD, or RUNX3/SMAD4-DKD and either treated with vehicle control or TGFb for 48 hours. Coimmunofluorescence was performed withgH2AX and 53BP1 antibodies. Images depict immunofluorescence staining for gH2AX and 53BP1 in control cells not exposed to TGFb or in RUNX3-KD,SMAD4-KD, or RUNX3/SMAD4-DKD cells treated with TGFb. Scale bar, 50 mm. F, For experiment described in E, percent cells accumulating >5 gH2AX foci werequantified and plotted. G, NCI-H1299 cells were seeded into 6-cm dishes at the cell density of 0.2 � 106 cells/dish and transfected with control or RUNX3 siRNA.Following exposure to vehicle control or TGFb for 48 hours, immunofluorescence staining was done using antibody against gH2AX. Scale bar, 40 mm. H, Forexperiment described in G, relative expression levels of RUNX3 (left) is shown. Right, percent cells expressing greater than 5 gH2AX foci are plotted. Graphs showmean � SD. Asterisks represent significant differences. �, P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






Figure 3.

Increased intracellular ROS is responsible for heightened DNA damage accumulation in cells undergoing TGFb-mediated EMT. A, A549 cells were subjected toCONT-KD or RUNX3-KD and synchronized inmitosiswith nocodazole (100 ng/mL, overnight). Followingmitotic shake-off, cells were released into TGFb andBrdUrd(25 mmol/L). After 6 hours, nocodazole (100 ng/mL) was readded to prevent cells from exiting the next cell cycle. Samples were fixed after 24 hours andcoimmunofluorescence staining was done using anti-BrdUrd FITC and anti-gH2AX antibodies. Blue arrowheads, gH2AX/BrdUrd double-positive cells; yellowarrowheads, gH2AX–positive/BrdUrd-negative cells. Scale bar, 20 mm. B, RUNX3-KD cells treated with TGFb (48 hours) were fixed for immunofluorescence.Coimmunofluorescence staining was performed with gH2AX antibody and pRPA (Ser33) antibodies. (Continued on the following page.)

Krishnan et al.





We tested whether the inactivation of other key DNA repairregulators similarly elevated DNA damage after TGFb exposure.Seven candidate genes (ATM, BRCA1, PRKDC, FANCD2, LSD1,PARP1, and TP53) were silenced using siRNA and cells wereexposed to TGFb (Supplementary Fig. S2E). Apart from RUNX3depletion, silencing the above repair genes did not induce DNAdamage after TGFb (Supplementary Fig. S2F). RUNX3, by pre-venting the accumulation of DSBs, therefore performs a uniquegenome maintenance function following TGFb exposure.

RUNX3 depletion induced heightened ROS accumulation afterTGFb exposure

The biological basis underlying the accumulation of DNAdamagewas investigated. Tomonitor the cell-cycle phase atwhichDSBs accumulated, cells were synchronized at mitosis andreleased into TGFb and BrdUrd-containing media. Coimmuno-fluorescence revealed that approximately 60% of the cells har-boring DNA damage were at G1 (yellow arrowheads; Fig. 3A). Inthe remaining 40%, gH2AX signals were observed in S-phase cells(blue arrowheads; Fig. 3A) and colocalized with pRPA (Ser33)signals, a protein recruited to the sites of single-stranded DNA(ssDNA) associated with replication stress (Fig. 3B).

Endogenous DSBs arise by factors like telomere attrition,impaired chromatin structure, or ROS accumulation (17).Heightened ROS can lead to ssDNA breaks, which at closeproximity or upon collision with the transcription or the DNAreplication apparatusmay convert into DSBs (18). Consistently,the TGFb pathway is known to induce ROS accumulationthrough two independent pathways—the upregulation of thepro-oxidant, NADPH oxidase, NOX4, and the downregulationof the activity of NRF2, a central antioxidant regulator (19–21).While NOX4 upregulation is elevated by mitochondrial com-plex III and SMAD signaling, NRF2 inhibition is associated withthe elevated expression of its repressive partner ATF3, by TGFb(22). The increased ROS, in turn, promotes redox signaling byregulating TGFb signaling in a feed-forward manner by con-trolling the activities of the JNK and p38 MAPK pathways (23).

We confirmedNOX4 upregulation by TGFb in a SMAD-depen-dent manner (Supplementary Fig. S3A and S3B). Transcriptionalprofiling revealed the downregulation of some NRF2-dependentgenes like GCLM, GCLC, and GPX2 following TGFb treatment(Supplementary Fig. S3C). While NRF2 expression remainedunchanged, ATF3 was transcriptionally upregulated by TGFb(Supplementary Fig. S3D). Pathways known to contribute toincreased ROS production during TGFb signaling thus remainconserved in our cellular context.

ROS levels were quantified in TGFb-treated cells using carboxy-H2DCFDA, a nonfluorescent compound that is converted to agreen fluorescent form via ROS-mediated oxidation. Following

TGFb exposure, ROS levels increased by approximately 3.8-fold,as indicated by mean fluorescence intensity (MFI) values (��, P< 0.001). However, intracellular ROS levels were elevated byapproximately 6-fold in RUNX3-KD cells upon TGFb exposure(��, P < 0.001; Fig. 3C and D). Independently, another reporterreagent CellROX Deep Red Reagent, confirmed that althoughTGFb exposure and RUNX3-KD independently increased ROSlevels to approximately 1.4-fold, the combination of RUNX3depletion with TGFb exposure increased ROS levels to greaterthan 2-fold (��, P < 0.01; Fig. 3E and F). Thus, RUNX3 loss resultsin heightened oxidative stress specifically after TGFb exposure.

We asked whether ROS sequestration using antioxidantsrestores ROS levels to reduce DNA damage. N-acetylcysteine isa pan-antioxidant; deferiprone is an iron chelator while manga-nese (III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP) is asynthetic metalloporphyrin. Although deferiprone did not rescuegH2AX accumulation (data not shown), both NAC as well asMnTBAP supplementation curtailed the accumulation of gH2AXin RUNX3-KD cells (Fig. 3G and H). The differential effects ofthese antioxidants on the rescue of DNA damage probably reflectthe type of ROS species responsible for DNA damage uponRUNX3 depletion. We conclude that the DNA damage inducedby the exposure of RUNX3-deficient cells to TGFb was owing tooxidative stress.

HMOX1 downregulation in RUNX3-inactivated cellsWe reasoned that RUNX3 might regulate the expression of

genes involved in combating ROS production after TGFb treat-ment. Hence, transcriptional profiling was conducted usingRNA-Sequencing (RNA-Seq; Fig. 4A). Principal component anal-ysis of the dataset showed the clustering of biological replicates inclose proximity, indicating their concordance (SupplementaryFig. S4A). TGFb gene expression signature (126 genes) and EMTscoring methods (254 genes), described earlier were utilized tocompare expression signatures (24–26). As expected, TGFb-trea-ted samples showed higher enrichment for TGFb signature (0.14for control vs. 0.84 after TGFb, �, P < 0.05), validating theBioinformatic scoring (Fig. 4B). EMT scores were moderatelyhigher upon RUNX3 depletion even in the absence of exogenousTGFb (0.01 vs. 0.29 forCONT-KDandRUNX3-KD, respectively, �,P < 0.05; Fig. 4C). Furthermore, differential gene expressionanalysis of genes contributing to the EMT score unveiled a subsetof genes that underwent downregulation or upregulation afterRUNX3 depletion, mimicking TGFb exposure (SupplementaryFig. S4B and S4C). These analyses thus provided a comprehensivegenomic perspective on earlier reports of RUNX3 being a negativeregulator of EMT (27).

Fragments Per Kilobase of transcript per Million mapped reads(FPKM) values of key oxidant andpro-oxidants from the RNA-Seq

(Continued.) Blue arrowheads, gH2AX-positive/pRPA-negative; yellow arrowheads, gH2AX/pRPA double-positive cells. Zoomed image of the inset (300%) showscolocalization of gH2AX/pRPA signals. Scale bar, 50 mm. C, Intracellular ROS detection-A549 cells were transfected with either control or RUNX3 siRNA and treatedwith vehicle control or TGFb for 24 hours and incubated with FITC-labeled carboxy-H2DCFDA reporter. Histogram represents images of carboxy-H2DCFDAfluorescence obtained through flow cytometry. D, For experiment described in C, quantification of MFI per cell, normalized as fold relative to CONT-KDcells not treated with TGFb. E, A549 cells were transfected with either control or RUNX3 siRNA and treated with vehicle control or TGFb for 24 hours Cells wereincubated with APC-labeled Cell-ROX reporter reagent. Histogram represents images of Cell-ROX fluorescence obtained through flow cytometry. F, Forexperiment described in E, quantification of MFI per cell normalized as fold relative to CONT-KD cells not treated with TGFb. G, Experiment was carried out asdescribed in the schematic. N-acetylcysteine (5 mmol/L) or MnTBAP chloride (100 mmol/L) was added 24 hours prior to TGFb exposure for 48 hours.Immunofluorescence stainingwas performedwith gH2AX antibody. Scale bar, 50mm.H,Percent cells containing greater than 5 gH2AX foci per cell were counted andare represented in the graph (n ¼ 300). Two independent experiments were done as triplicates. Graphs show mean � SD. Asterisks represent significantdifferences. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






Figure 4.

HMOX1 downregulation is responsible for increased DNA damage in RUNX3-depleted cells undergoing TGFb-mediated EMT. A, A549 cells were seeded into 6-cmdishes at the cell density of 0.2 � 106 cells/dish and transfected with control siRNA or RUNX3 siRNA. Three days later, cells were trypsinized and seededat the cell density of 0.3� 106 cells/6-cmdish and subjected to a second roundof siRNA. Twenty-four hours later, cellswere exposed to either vehicle control or TGFbfor 48 hours. Cells were harvested and RNA extraction and sequencing were performed. RNA-Seq reads were mapped to human genome HG19 and FPKMvalueswere calculated per gene.B,Graphdepicts the average TGFb enrichment score calculated across biological replicates.C,Graphdepicts the averageEMT scorecomputed for biological replicates. D, Heatmap depicting expression of genes encoding for antioxidants. Gene expression derived from FPKM values wasplotted. Biological replicate for each sample is represented individually as 1 and 2. (Continued on the following page.)

Krishnan et al.





datasets were assessed. Pro-oxidant gene expression largelyremained unchanged upon RUNX3 depletion (data not shown).In contrast, among the 21 critical antioxidant defense genesexamined, RUNX3 depletion resulted in transcriptional down-regulation of the metabolic enzyme and a redox regulator, Hemeoxygenase-1 (HMOX1 orHO-1; Fig. 4D and E). HMOX1 is a rate-limiting enzyme of an important metabolic pathway that detoxi-fies heme (Fe-protoporphyrin IX), an important cofactor of sev-eral Heme-containing proteins like hemoglobin, cytochrome c,and catalase. Three metabolic byproducts are generated byHMOX1-carbon monoxide, heme and biliverdin, of which, bil-iverdin is reduced by the enzyme biliverdin reductase to generatethe antioxidant, bilirubin (28).

The downregulation of HMOX1 following RUNX3 depletionwas validated by qPCR in gastric (AZ521, HGC-27) and lungcancer cells (NCI-H23, PC-14, NCI-H1299), two tissues whereRUNX3 is an established tumor suppressor. RUNX3 knockdownattenuated HMOX1 expression by approximately 50%–80% inevery cell line examined (Fig. 4F). HMOX1 expression levels alsostrongly correlated with RUNX3 status in Spearman correlationanalysis of gene expression of 436 samples in TCGA (The CancerGenome Atlas Consortium) lung adenocarcinoma datasets (Sup-plementary Fig. S5A). In hierarchical clustering analysis, lungcancers with high RUNX3 levels had either high HMOX1 orintermediate HMOX1 expression, while lung cancers with lowRUNX3 levels had low HMOX1 expression (r ¼ þ0.3028; P ¼1.33e�10).Western blot analysis confirmed that basal and TGFb-inducedHMOX1 protein levels were downregulated after RUNX3depletion (Fig. 4G).

According to earlier reports, TGFb addition induces HMOX1in human retinal pigment epithelial cells, A549 cells, and bovinechoroid fibroblasts (29). In contrast, TGFb suppressed HMOX1expression through the elevated expression of the negativeregulators, MafK and Bach1, in NMuMG (30). In our analysis,HMOX1 was transcriptionally upregulated by TGFb across threedifferent cell lines, A549, NCI-H1299, and NCI-H292 (Supple-mentary Fig. S5B). The induction of MMP-9 by TGFbwas used asa marker for TGFb signaling. We then examined the effect ofRUNX3 status on HMOX1 induction by TGFb. Upon RUNX3depletion, HMOX1 transcript and protein levels were substan-tially diminished across all timepoints examined (maximalexpression at 8 hours following TGFb exposure; Fig. 4H and I;Supplementary Fig. S5C). Thus, RUNX3-dependent HMOX1accumulation is an early response following TGFb exposure.Finally, we evaluated whether RUNX3-mediated HMOX1 upre-gulation was modulated by ROS production. Cells were chal-lenged with acute oxidative stress (250 mmol/L hydrogen per-oxide) and HMOX1 levels were measured. Whereas HMOX1 was

robustly upregulated within 6 hours after H2O2 exposure, theknockdown of RUNX3 markedly attenuated HMOX1 induction(Supplementary Fig. S5D). The data presented above usingseveral cancer lines and genomic datasets thus establish RUNX3as an important regulator of HMOX1 transcription.

Ectopic HMOX1 expression prevents TGFb-elicited DNAdamage in RUNX3-depleted cells

The consequence of HMOX1 downregulation to DNA damageaccumulation was then investigated. GFP-tagged HMOX1 wasintroduced ectopically in RUNX3-depleted cells, followingwhich,cells were treated with TGFb. As a positive control, a siRNA-resistant version of wild-type GFP-tagged RUNX3 was overex-pressed. ROS levels were compared in the GFP-positive and GFP-negative populations using the CellROX assay. Cellular ROSamounts restored to basal levels upon RUNX3 overexpression,indicating the specificity of the siRNA experiments (��, P <0.01; Fig. 4J). Importantly, HMOX1 overexpression reduced ROSlevels to basal levels upon RUNX3 depletion (��, P < 0.01; Fig. 4J).Next, gH2AXpositivity was scored followingHMOX1overexpres-sion and TGFb exposure. As a positive control, a siRNA-resistantversion of wild-type GFP-tagged RUNX3 was overexpressed. TheDNA-binding deficient mutant of RUNX3 (R178Q) was alsotested for its ability to rescue DNA damage. Importantly,RUNX3-KD cells overexpressing HMOX1 or wild-type (WT)-RUNX3 did not accumulate DNA damage after TGFb exposure(��, P < 0.001; Fig. 4K and L; Supplementary Fig. S6A and S6B).Surprisingly, R178Q-RUNX3 mutant also rescued DNA damagein the presence of TGFb (Supplementary Fig. S6B). We concludeHMOX1 downregulation as themechanism underlying increasedDNA damage accumulation in RUNX3-deficient cells. Intriguing-ly, the genome maintenance function of RUNX3 does not seemto rely on its DNA-binding property (elaborated further in"Discussion").

Many intracellular signaling cascades and transcription factorshave been found to regulateHMOX1 transcription (31). Of these,HMOX1 transcription is mainly regulated by the master regulatorof antioxidant gene expression, NRF2 (32). HMOX1 enhancer isbound by the BACH1 repressor complex at the basal transcrip-tional state, but upon oxidative stress, BACH1 is replaced byNRF2, to stimulateHMOX1 transcription (33). Taking advantageof the BACH1-dependent repression of HMOX1, HMOX1 levelswere increased in RUNX3-KD cells by silencing BACH1 (Supple-mentary Fig. S6C and S6D). BACH1/RUNX3-DKD cells did notaccumulate DNA damage upon TGFb exposure (��, P < 0.001;Supplementary Fig. S6E and S6F), indicating that DNA damageaccumulation can be rescued by genetic manipulations thatelevate HMOX1 levels.

(Continued.) E, A549 cells were subjected to control or RUNX3 knockdown for 72 hours. HMOX1 levels were measured using qPCR. F, Cell lines shown in the graphwere transfected with RUNX3 for 72 hours. HMOX1 levels were measured using qPCR. G, Cells were transfected with either CONT siRNA or RUNX3 siRNAand treated with vehicle control or TGF-b for 36 hours. Samples wereWestern blotted with RUNX3 and HMOX1-specific antibodies. GAPDHwas used as the loadingcontrol. H, Experiment was performed as described in A. Cells were exposed to TGFb and harvested at the indicated timepoints, followed by qPCR analysisof HMOX1. I, CONT-KD or RUNX3-KD cells were exposed to TGFb and harvested at the indicated timepoints. Western blot analysis of RUNX3 and HMOX1 is shown.GAPDH was used as the loading control. J, Cells were transfected with RUNX3 siRNA. GFP-tagged siRNA-resistant RUNX3 or GFP-HMOX1 was expressedfor 24 hours, followingwhich, cellswere exposed to TGFb for another 24 hours. Cellswere incubatedwith Cell-ROX reagent and subjected to flow cytometry. TheMFIfor Cell-ROX fluorescence in the GFP-positive and GFP-negative populations are shown. K, Experiment was performed as described in A. GFP-HMOX1 wasexpressed 24hours before TGFb addition. Following48hours of TGFb exposure, cellswerefixedand stainedwith 53BP1 antibody. Scale bar, 50mm.L,For experimentdescribed in K, percent cells expressing greater than five 53BP1 foci/cell were quantified within the GFP-positive and GFP-negative populations (n ¼ 300).Two independent experiments were done as triplicates. For graphs shown in B and C, data represent mean� SEM. For all other graphs, data represent mean� SD.Asterisks represent significant differences. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






Figure 5.

RUNX3 depletion induces cellular senescence and inflammatory cytokine expression in cells undergoing TGFb-mediated EMT.A, Cells were transfected with controlor RUNX3 siRNA and treated with vehicle control or TGFb for 48 hours. Immunofluorescence staining was performed with gH2AX antibody and AlexaFluor-594–conjugated phalloidin. The phalloidin and gH2AX images were imaged individually and merged using Adobe Photoshop software. Merged imagesrepresent coimmunofluorescence for phalloidin and gH2AX staining. Scale bar, 50 mm. B, Experiment was performed as described inA. Scatter plot shows projectednuclear area across samples. C, Experiment was performed as described in A. Images were captured and subjected to quantitative image analysis to computenuclear area. The average gH2AX foci/cell within cells with the nuclear area 100–250 mm2 or greater than 250 mm2 is shown. (Continued on the following page.)


Krishnan et al.




TGFb provokes cellular senescence in RUNX3-depleted cellsafter TGFb exposure

The accumulation of DNA damage either results in apoptosisor persistently activates the DNA damage checkpoint to mediatecellular senescence (34). As increased apoptosis was notobserved in RUNX3-KD cells exposed to TGFb, we asked wheth-er cells underwent senescence. Senescence is a cell-fate associ-ated with flattened cell shape, higher nuclear area, cell-cycle exit,and positivity for the SA-b-gal. Indeed, RUNX3-depleted cellsupon TGFb exposure were more flattened in cell shape andexhibited atypical disorganized actin, as evident by stainingwith the F-actin probe, Phalloidin (Fig. 5A). RUNX3-KD cellsupon TGFb exposure also had a significantly larger nuclear area(��, P < 0.001; Fig. 5B). Image quantification revealed a closecorrelation between nuclear area and gH2AX focus accumula-tion (r ¼ 0.382; Fig. 5C). Finally, approximately 40%–50% ofRUNX3-KD cells exposed to TGFb exhibited positivity for thesenescence hallmark, SA-b-gal (Fig. 5D and E). Thus, whereasTGFb triggers EMT in cells with intact RUNX3, DNA damage andsenescence are triggered upon the loss of RUNX3.

To study whether reduced HMOX1 was responsible for senes-cence following RUNX3 depletion, rescue experiments were per-formed. GFP-HMOX1was overexpressed and cells were subjectedto flow cytometry to sort for GFP-positive cells. As a positivecontrol, siRNA-resistant wild-type GFP-RUNX3 was overex-pressed. GFP-positive cells were cultured and exposed to TGFbfor 48 hours. Cellular senescence was substantially rescued inRUNX3-depleted cells upon the ectopic expression of wild-typeRUNX3 or HMOX1 (Fig. 5F).

Taken together, the instatement of HMOX1 expression inRUNX3-depleted cells restored cellular ROS, rescued DNA dam-age, and premature senescence following TGFb exposure.

TGFb induces SASP in RUNX3-depleted cellsAlthough senescence was originally discovered as a tumor-

suppressive mechanism, it is now well established that the senes-cent response can be also protumorigenic. In a phenomenonreferred to as the SASPand regulatedby factors likeNFkB,C/EBPb,mTOR, MacroH2A.1, and GATA4, senescent cells secrete a largerepertoire of cytokines and metalloproteinases (35). The suste-nance of senescent cells within tumors thus creates a pro-onco-genic inflammatory microenvironment promoting tumor pro-gression (36).

Genes differentially expressed were subjected to NOISeq filtra-tion (methods) and GO analysis (Supplementary Table S1).Interestingly, the terms "cytokine-activity", "metalloproteinases"were the "molecular functions" enriched in the RUNX3-depleted

transcriptomes when compared with control cells exposed toTGFb (Fig. 5G). The cluster of genes representing this groupCXCL1, INHBA, BMP2, CCL2, CXCL3, CXCL2, IL32, IL8, AREG,GDF15, and IL1A represent cytokines and chemokines secreted bysenescent cells exhibiting the SASP phenotype (36). The metallo-proteinase gene cluster encoding MMP10, MMP9, MMP2, andMMP1 are also enzymes known to be enriched in the secretomesof senescent cells (Fig. 5H, heatmap). Consistently, miR-146a, amiRNA induced during DNA damage-induced senescence andSASP,was highly expressedonlywhenRUNX3-depleted cellswereexposed to TGFb (37) (Fig. 5I). The differential upregulation ofSASP genes in RUNX3-KD cells exposed to TGFb was confirmedthrough qPCR analysis (Fig. 5J).

Cellular senescence is ATM- and ATR-dependent but p53-independent

In cells harboring unrepaired DNA damage, the DDR kinasesATM and ATR relieve the inhibition of p53 and p16INK4a toinduce growth arrest and senescence. To assess the roles of ATM,ATR, and p53 in the induction of senescence, we inhibited ATMand ATR using two specific small-molecule inhibitors, KU-55933(10 mmol/L) and VE-821 (2 mmol/L), respectively (38, 39). Tostudy the function of p53 as a senescence inducer, p53 wascodepleted with RUNX3 using siRNA. The role of p16INK4a wasnot studied as this gene is deleted in A549 cells. To confirm theinactivation of the ATM/ATR pathways by the small-moleculeinhibitors, immunofluorescence staining was performed withpATM/ATR substrate antibody that detects ATM and ATR sub-strates phosphorylated at the consensus SQ/TQ sites. The con-current inhibition of ATM and ATR significantly reduced bothpSQ/TQ signals and gH2AX positivity (Fig. 6A). Importantly, theinactivation of ATM and ATR significantly mitigated cellularsenescence, establishing a role for these DDR kinases as transdu-cers of senescence signals (��, P < 0.001; Fig. 6B and C). Incontrast, p53 depletion had little effect on alleviating the extent ofsenescence (Fig. 6B and C). Taken together, the senescence acti-vated in RUNX3-deficient cells upon TGFb exposure was relianton upstream DNA damage signaling by ATM and ATR but wasindependent of p53 status.

TGFb provokes DNA damage and senescence upon RUNX1deficiency

We asked whether RUNX1 and RUNX2 perform redundantroles with RUNX3 in genome maintenance during TGFb sig-naling. qPCR analysis confirmed efficient siRNA-mediateddepletion of RUNX1, RUNX2, and RUNX3 (Supplementary Fig.S7A). Interestingly, TGFb exposure triggered the accumulation

(Continued.) One-way ANOVA nonparametric test was used for statistical analysis. �� , P < 0.001. D, Cells were treated as described in A. Samples were fixed andstained for SA-b-gal enzyme overnight at 37�C. Inset image was zoomed (200%) and is shown below. Scale bar, 50 mm. E, SA-b-gal–positive cells(blue) were scored and plotted. Quantification of SA-b-gal positivity across two independent experiments, each done as triplicates. F, Cells were transfected withRUNX3 siRNA. GFP-tagged siRNA-resistant RUNX3 or GFP-HMOX1 were expressed for 24 hours, following which, cells were sorted by flow cytometryinto GFP-positive andGFP-negative populations and plated. After TGFb exposure for 48 hours, the SA-b-gal assaywas performed.G,RNA-Seq data described in Fig.4A was subjected to pathway analysis. Gene ontology (GO) pathways enriched under the category "Molecular function" are shown. Control and RUNX3-KDcells treated with TGFb were subjected to differential gene expression analysis, as described in Materials and Methods. Genes regulated by RUNX3 in aTGFb-independent manner were excluded. The online tool, Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7, was used for GO analysis.Genes expressing more than 200% of control were considered upregulated, whereas those with expression less than 50% of control were considered asdownregulated. List of differentially expressed genes are shown in Supplementary Table S1. H, Heatmap depicting overexpression of SASP genes. Gene expressionderived from FPKM values was plotted. Biological replicates for each sample, is represented individually. I, miR-146a FPKM values derived from RNA-Seqanalyses are shown. J, Experiment was performed as described in A. Samples were harvested after 48 hours of TGFb treatment. qPCR analyses were done for theindicated genes. Graphs show mean � SD. Asterisks represent significant differences. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






of DNA damage following both RUNX1 and RUNX3 depletionbut not following RUNX2 depletion (Supplementary Fig. S7Band S7C). The combined knockdown of RUNX1 and RUNX3did not further increase the extent of DNA damage accumula-tion (RUNX1-KD vs. RUNX3-KD vs. RUNX1/RUNX3-DKD-20% 35%, and 38% gH2AX positivity, respectively). RUNX1and RUNX3 depletion significantly reduced HMOX1 levelsafter TGFb exposure and their combined knockdown did notfurther exacerbate the extent of HMOX1 downregulation (Sup-plementary Fig. S7D). Finally, RUNX1 depletion also triggeredsenescence following TGFb treatment although to a lesserextent than that following RUNX3 depletion (SupplementaryFig. S7E). Taken together, although both RUNX1 and RUNX3are required to prevent the accumulation of DNA damagefollowing TGFb exposure, RUNX3 depletion evoked strongerDNA damage phenotypes and HMOX1 downregulation thanRUNX1.

Exacerbation of genomic instability in RUNX3-inactivated lungcancers with "a TGFb high" expression gene signature

To assess the in vivo significance of RUNX3 loss specifically inthe context of TGFb signaling, we interrogated TCGA datasets oflung adenocarcinomas and evaluated the relationship betweengenomic instability, RUNX3 status, and TGFb signature. Inhuman lung adenocarcinomas andmousemodels, RUNX3 showstumor-suppressive function and its inactivation promotes lungcancer progression (40). Genomic instability was measured asmutation rate (number ofmutations accumulationpermega-baseof genome) and copy number alteration or CNA (presence ofgeneswith copy number�3or1). First, tumorswere stratified aseither "TGFb-high" or "TGFb-low" based on TGFb expressionsignature (Fig. 7A andB). Then, such tumorswere further stratifiedas "RUNX3-high" or "RUNX3-low" (median gene expressionvalues). No significant differences in genomic instability werefound when "TGFb-high" and "TGFb-low" tumors were com-pared or when "RUNX3-low" and "RUNX3-high" tumors werecompared.

In contrast, when "TGFb-high" tumorswere further stratifiedonthe basis of RUNX3 status, tumors with low RUNX3 levels hadsignificantly greater CNA (P ¼ 0.0001) and accumulation ofmutations (P ¼ 0.0221) as compared with tumors with highRUNX3 expression (Fig. 7A and B). Interestingly, the tumors thatfell into the quadrant of "TGFb high/RUNX3 low" exhibited atrend towards poorer survival as comparedwith those tumors thatfell into the quadrant of "TGFb low/RUNX3 high" (P ¼ 0.095,HR ¼ 1.845, n ¼ 181; Fig. 7C). A comparison of overall survivalwith RUNX3 and TGFb status are shown in Supplementary Fig.S8A and S8B. Thus, closely recapitulating the data shown in earliersections, RUNX3 inactivation in lung adenocarcinomas exacer-bates genomic instability and survival of tumors harboring aTGFbexpression signature.

HMOX1 depletion radiosensitizes cellsAlthough increased ROS levels promote tumorigenesis, it has

been proposed that escalating ROS to lethal levels has the poten-tial to block tumor progression by inducing apoptosis. Moreover,increased ROS levels can accentuate the cytotoxicity of somechemotherapeutics like cisplatin, anthracyclines, and irradiation(41). Indeed, HMOX1 inhibition through its competitive small-molecule inhibitor, Zinc protoporphyrin IX synergizes with cis-platin (42).

We explored the possibility of exploiting increased oxidativestress as a therapeutic strategy against TGFb-high/RUNX3-lowtumors. Cells were exposed to increasing doses of radiation andpercent survival was plotted. TGFb treatment of RUNX3-deplet-ed cells resulted in a 5-fold increase in radiosensitization(Fig. 7D and E). Finally, we depleted HMOX1 using siRNAand studied whether it recapitulates the loss of RUNX3 inenabling radiosensitization following TGFb. Similar to RUNX3loss, siRNA-mediated HMOX1 knockdown resulted in highlevels of oxidative stress, specifically upon exposure to TGFb(��, P < 0.001; Supplementary Fig. S8C–S8E). HMOX1 deple-tion also engendered cellular senescence and DNA damage,although gH2AX signals generated were more diffuse than thatfollowing RUNX3 depletion (discussed below; SupplementaryFig. S8F–S8H). The depletion of HMOX1 also rendered radio-sensitization similar to RUNX3 depletion. More importantly,the extent of radiosensitization mediated by HMOX1 depletionwas increased by TGFb (Fig. 7F and G). Put together, the abovedata emphasize oxidative stress induction through HMOX1inhibition as a therapeutic vulnerability for tumors exposedto pro-oncogenic TGFb.

DiscussionHere, we uncovered that the pro-oncogenic effects for TGFb

signaling are exacerbated upon the loss of RUNX3. We show thatthe reduced expression of HMOX1 in RUNX3-deficient cellscaused a catastrophic imbalance toward greater ROS productionby TGFb, causing oxidativeDNAdamage and genomic instability.Consequent to DNA damage, the DDR kinases ATM and ATRmediate senescence, which is accompanied by the expression ofproinflammatory cytokines. The latter phenomenon, known asSASP, is known to favor tumor progression (Fig. 7H). Important-ly, the inactivation ofmajorDNA repair genes in TGFb-stimulatedcells did not result in DNA damage, indicating a unique role forRUNX3 in the defense against TGFb-dependent promotion ofcancer progression.

Over the years, we and others have studied the tumor suppres-sing potential of RUNX3 in early-stage cancers (43). The inacti-vation of RUNX3 at the earliest stages of cancer progression seemsto be a mechanism evolved by cancer cells to simultaneouslysuppress the antiproliferative arm of TGFb signaling whileaccentuating its pro-oncogenic roles. In support, RUNX3 lossimpairs p21 and Bim transcription during TGFb signaling, thusimpairing apoptosis while promoting gastric hyperplasia (13,14). Second, RUNX3 loss supports pro-oncogenic TGF-b sig-naling by spontaneously upregulating a subset of EMT genes,thus mimicking a partial EMT phenotype (Supplementary Fig.S4B and S4C; Fig. 4C; ref. 27). Finally, our work here introducesanother independent and novel aspect of the TGFb–RUNX3axis wherein RUNX3 loss promotes further ROS accumulationduring TGFb signaling, thus triggering genomic instability. Insupport, genomic analyses of TCGA lung adenocarcinomadatasets showed that mutation load and copy number altera-tions of tumors harboring a high-TGFb signature increasedfollowing RUNX3 inactivation.

The observation that RUNX3 levels reduce following TGFbexposure might seem at odds with the genome protection rolefor RUNX3 identified in this study. It is noteworthy that despitethe reduction in RUNX3 levels, TGFb-treated cells do not accu-mulateDNAdamage, implying that the lower levels of RUNX3 are


Krishnan et al.




Figure 6.

RUNX3 depletion induces cellular senescence in an ATM/ATR dependent, but p53-independent manner. A, A549 cells were subjected to CONT-KD, RUNX3-KD,or RUNX3/p53-DKD for 3 days. As indicated, cells were pretreated with either vehicle (DMSO), KU-55933 (10 mmol/L), or VE-821 (2 mmol/L) for 2 hours,following which, they were exposed to TGFb for 48 hours in the presence of the small-molecule inhibitors. Coimmunofluorescence staining was performed withantibodies recognizing pSQ/TQ sites and gH2AX. Zoomed (200%) image of the inset is shown on the right. Percent cells expressing greater than 5 gH2AXfoci are indicated within the parentheses. Scale bar, 50 mm. B, For the experiment described in A above, cells were fixed and stained for SA-b-gal enzymeovernight at 37�C. Scale bar, 100 mm. C, SA-b-gal–positive cells (blue) were scored and plotted. Graphs show mean � SD. Asterisks represent significantdifferences. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; n.s, not significant.






Figure 7.

Exacerbation of genomic instability and radiosensitization upon RUNX3 or HMOX1 depletion in the presence of TGFb. A, Lung adenocarcinomas were stratified onthe basis of TGFb signature and RUNX3 expression levels utilizing median expression values. Copy number alterations are shown in the scatter plot with bar(mean� SEM).B, Lung adenocarcinomaswere stratified on the basis of TGFb signature andRUNX3 expression levels utilizingmedian expression values. Mutations permegabase of the genome are shown in the scatter plot with bar (mean� SEM). C, Kaplan–Meier survival curve of the indicated cohorts is shown. n¼ 181, P¼ 0.0955,HR¼ 1.845. Statistical analysiswasdonewith theMann–WhitneyU test.D,A549cellswere subjected to control orRUNX3-KDand exposed tovehicle control or TGFb for48 hours. Cells were exposed to increasing doses of X-irradiation in clonogenic assay. E, For experiment described in D, percent survival was plotted relativeto radiation-untreatedcontrols.F,A549cellswere subjected tocontrol orHMOX1-KDandexposed toTGFborvehicle control. Forty-eighthours followingTGFb addition,cells were exposed to increasing doses of X-irradiation. G, For experiment described in F, percent survival was plotted relative to radiation-untreated controls.H, RUNX3 provides defense against TGFb-dependent promotion of cancer progression. Top, TGFb signaling induces ROS production, which participates in redoxsignaling and promotes TGFb-mediated EMT. However, RUNX3 mitigates the accumulation of excessive ROS via the transcriptional upregulation of the HMOX1and thus curtails tumor progression. Under these conditions, where RUNX3 levels are intact, TGFb-mediated EMT is associated with low levels of DNA damage andgenomic instability. Bottom, in the absence of RUNX3, low HMOX1 levels perpetrate high levels of ROS, which promotes cancer progression by the induction of DNAdamage and senescence. Cellular senescence, in turn, fuels the production of inflammatory cytokines that further amplify the pro-oncogenic state. The reinstatement ofHMOX1 expression in RUNX3-depleted cells restored cellular ROS, rescued DNA damage and premature senescence in the presence of TGFb. Thus, HMOX1downregulation seems to be one of the major mechanisms by which genomic instability is brought about by the TGFb exposure of RUNX3-deficient cells.


Krishnan et al.




competent in conferring genome protection. Interestingly, arecent seminal study has showed that expression levels of Runx3respond to and combine with Smad4 status to regulate thebalance between cell division and dissemination in pancreaticcancers (15). Indeed, data mining of ChIP-sequencing datasetsreveal that theRUNX3promoter harbors elements that bind toTFsdownstream of TGFb pathway like SMAD4, SOX2, FOXO1,FOXC1, and EZH2 (44). We speculate that the binding of oneof these TFs to the RUNX3 promoter might represent a feedbackmechanism to alter RUNX3 levels during TGFb signaling

In previous studies, ROS levelswere shown to elevate between8and 16hours following TGFb exposure and subside to basal levelswithin 48 hours, suggesting temporal regulation of ROS produc-tion (20, 45). Our observations that HMOX1 is transcribed in aRUNX3-dependent manner within 8 hours, suggests that cellshave evolved this mechanism to mitigate the accumulation oftumor-promoting ROS during TGF-b pathway. It is currentlyunclear if HMOX1 induction by RUNX3 is via the direct bindingof RUNX3 to the HMOX1 promoter or through indirect means.Interestingly, genome-wide RUNX3-CHIP data have revealed thebinding of RUNX3 to a super enhancer region located -3.5 kbupstream of the HMOX1 promoter (Encode consortium). How-ever, the indirect regulation ofHMOX1 transcription by RUNX3 isalso plausible, because the DNA binding mutant, R178-RUNX3was also capable of rescuing DNA damage accumulation. Inearlier studies, it has been shown that RUNX2 binds and recruitsCEBPd to promoter enabling transcription and precluding therequirement for direct DNA binding (46). Alternatively, sinceRUNX3 is known to establish multiple protein-interacting part-ners, it is possible that RUNX3 might control HMOX1 transcrip-tion indirectly through interaction with transcription factorsknown to regulate HMOX1 levels (47).

Aside from being a major regulator of HMOX1 expression, wespeculate that RUNX3 has additional roles in genome mainte-nance upon TGFb exposure. This is based on the observation thatthe gH2AX foci generated by RUNX3 loss were higher in numberand more distinct as compared to the more diffuse and lessergH2AX signals generated by HMOX1 knockdown. One possibleexplanation is that HMOX1 depletion creates oxidative stress andsingle-stranded breaks that may not be efficiently converted inDSBs. Given that RUNX proteins have added roles in DNA repair,we speculate that the absence of RUNX3 might additionallydisrupt repair of oxidative stress–mediatedDNAdamage (48, 49).

Our work raises novel possibilities of exploiting ROS-depen-dent genomic instability for the therapeutic targeting of cancersexposed to pro-oncogenic TGFb. This is based on the rationalethat increasing ROS to very high levels has the potential to blocktumor progression by inducing cancer cell senescence or apopto-sis (41, 50). Accordingly, our studies (Fig. 7F and G) suggest that

triggering high levels of ROS through HMOX1 inhibition canpromote greater radiosensitivity following TGFb exposure.

In summary, our studies imply intriguing collaborationbetween an intrinsically inactivated tumor suppressor, RUNX3,and an extrinsically acting cytokine, TGFb in the promotion ofDNA damage and genomic instability. Dissecting how genomicinstability is exacerbated through a crosstalk between cancer cell–autonomous and nonautonomous factors is likely to create newtherapeutic targets and immune-related approaches to tackleTGFb-driven tumors.

Disclosure of Potential Conflicts of InterestJ.P. Thiery is a visiting professor at the University Of Bergen and is a

consultant/advisory board member for Aim Biotech Singapore and Actge-nomics Taipei. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: V. Krishnan, M.D. Kulkarni, J.P. Thiery, Y. ItoDevelopment of methodology: V. Krishnan, M.D. Kulkarni, L.S. Tay,A. GanesanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Krishnan, Y.L. Chong, M.D. Kulkarni, M.B. BinRahmat, D.S. JokhunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Krishnan, T.Z. Tan, M.D. Kulkarni, D.S. Jokhun,D. Chih-Cheng Voon, GV Shivashankar, J.P. ThieryWriting, review, and/or revision of the manuscript: V. Krishnan, Y.L. Chong,M.D. Kulkarni, L.S.H. Chuang, Y. ItoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):Study supervision: Y. Ito

AcknowledgmentsThe authors would like to thank Ms. Shu Ying from the Confocal facility,

National University of Singapore, for help with image analysis and the Cyto-genetic Facility of the Genome Institute of Singapore for Karyotyping services.We would like to thank Ms. Charell Lim, Ms. Charmaine Nai and Ms. Sound-harya Ravindran for their contributions during the initial phases of this work.The research is supported by the New Investigator Grant (CBRG-NIG; providedto V. Krishnan) from the National Medical Research Council (NMRC), Singa-pore (grant number-NMRC/BNIG/2024/2014) and by the National ResearchFoundation (NRF) and the Singapore Ministry of Education under its ResearchCentres of Excellence initiative and by the NRF under its Translational andClinical Research Flagship Programme (provided to Y. Ito; grant number-NMRC/TCR/009-NUHS/2013).

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 April 25, 2017; revised September 11, 2017; accepted October 23,2017; published OnlineFirst October 26, 2017.

References1. Negrini S, Gorgoulis VG,Halazonetis TD.Genomic instability–an evolving

hallmark of cancer. Nat Rev Mol Cell Biol 2010;11:220–8.2. Tubbs A,Nussenzweig A. EndogenousDNAdamage as a source of genomic

instability in cancer. Cell 2017;168:644–56.3. Massague J. TGFbeta in cancer. Cell 2008;134:215–30.4. David CJ, Huang YH, Chen M, Su J, Zou Y, Bardeesy N, et al. TGF-beta

tumor suppression through a lethal EMT. Cell 2016;164:1015–30.5. Hasegawa Y, Takanashi S, Kanehira Y, Tsushima T, Imai T, Okumura K.

Transforming growth factor-beta1 level correlates with angiogenesis,

tumor progression, and prognosis in patients with nonsmall cell lungcarcinoma. Cancer 2001;91:964–71.

6. Yu JR, Tai Y, Jin Y,HammellMC,Wilkinson JE, Roe JS, et al. TGF-beta/Smadsignaling through DOCK4 facilitates lung adenocarcinoma metastasis.Genes Dev 2015;29:250–61.

7. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymaltransitions in development and disease. Cell 2009;139:871–90.

8. Moustakas A, Heldin CH. Induction of epithelial-mesenchymal transitionby transforming growth factor beta. Semin Cancer Biol 2012;22:446–54.






9. Kirshner J, Jobling MF, Pajares MJ, Ravani SA, Glick AB, Lavin MJ, et al.Inhibition of transforming growth factor-beta1 signaling attenuates ataxiatelangiectasia mutated activity in response to genotoxic stress. Cancer Res2006;66:10861–9.

10. Wang M, Saha J, Hada M, Anderson JA, Pluth JM, O'Neill P, et al.Novel Smad proteins localize to IR-induced double-strand breaks:interplay between TGFbeta and ATM pathways. Nucleic Acids Res2013;41:933–42.

11. Comaills V, Kabeche L, Morris R, Buisson R, Yu M, Madden MW, et al.Genomic instability is induced by persistent proliferation of cellsundergoing epithelial-to-mesenchymal transition. Cell Rep 2016;17:2632–47.

12. Ito Y. RUNX genes in development and cancer: regulation of viral geneexpression and the discovery of RUNX family genes. Adv Cancer Res2008;99:33–76.

13. Yano T, Ito K, Fukamachi H, Chi XZ, Wee HJ, Inoue K, et al. The RUNX3tumor suppressor upregulates Bim in gastric epithelial cells undergoingtransforming growth factor beta-induced apoptosis. Mol Cell Biol2006;26:4474–88.

14. Chi XZ, Yang JO, Lee KY, Ito K, Sakakura C, Li QL, et al. RUNX3 suppressesgastric epithelial cell growth by inducing p21(WAF1/Cip1) expression incooperation with transforming growth factor {beta}-activated SMAD.MolCell Biol 2005;25:8097–107.

15. Whittle MC, Izeradjene K, Rani PG, Feng L, Carlson MA, DelGiorno KE,et al. RUNX3 controls a metastatic switch in pancreatic ductal adenocar-cinoma. Cell 2015;161:1345–60.

16. Redon CE, Weyemi U, Parekh PR, Huang D, Burrell AS, Bonner WM.gamma-H2AX and other histone post-translational modifications in theclinic. Biochim Biophys Acta 2012;1819:743–56.

17. Harper JW, Elledge SJ. The DNA damage response: ten years after. Mol Cell2007;28:739–45.

18. Lindahl T. Instability and decay of the primary structure of DNA. Nature1993;362:709–15.

19. Ryoo IG, Ha H, Kwak MK. Inhibitory role of the KEAP1-NRF2 pathway inTGFbeta1-stimulated renal epithelial transition to fibroblastic cells: amodulatory effect on SMAD signaling. PLoS One 2014;9:e93265.

20. Jain M, Rivera S, Monclus EA, Synenki L, Zirk A, Eisenbart J, et al.Mitochondrial reactive oxygen species regulate transforming growth fac-tor-beta signaling. J Biol Chem 2013;288:770–7.

21. Hubackova S, Kucerova A, Michlits G, Kyjacova L, Reinis M, Korolov O,et al. IFNgamma induces oxidative stress, DNA damage and tumor cellsenescence via TGFbeta/SMAD signaling-dependent induction of Nox4and suppression of ANT2. Oncogene 2016;35:1236–49.

22. Bakin AV, Stourman NV, Sekhar KR, Rinehart C, Yan X, Meredith MJ,et al. Smad3-ATF3 signaling mediates TGF-beta suppression of genesencoding Phase II detoxifying proteins. Free Radic Biol Med 2005;38:375–87.

23. Liu RM, Choi J, Wu JH, Gaston Pravia KA, Lewis KM, Brand JD, et al.Oxidative modification of nuclear mitogen-activated protein kinase phos-phatase 1 is involved in transforming growth factor beta1-induced expres-sion of plasminogen activator inhibitor 1 in fibroblasts. J Biol Chem2010;285:16239–47.

24. Gatza ML, Lucas JE, Barry WT, Kim JW, Wang Q, Crawford MD, et al. Apathway-based classification of human breast cancer. Proc Natl Acad SciU S A 2010;107:6994–9.

25. Huang RY, Wong MK, Tan TZ, Kuay KT, Ng AH, Chung VY, et al. AnEMT spectrum defines an anoikis-resistant and spheroidogenic inter-mediate mesenchymal state that is sensitive to e-cadherin restorationby a src-kinase inhibitor, saracatinib (AZD0530). Cell Death Dis2013;4:e915.

26. Tan TZ, Miow QH, Huang RY, Wong MK, Ye J, Lau JA, et al. Functionalgenomics identifies five distinct molecular subtypes with clinical relevanceand pathways for growth control in epithelial ovarian cancer. EMBO MolMed 2013;5:983–98.

27. Voon DC, Wang H, Koo JK, Nguyen TA, Hor YT, Chu YS, et al. Runx3protects gastric epithelial cells against epithelial-mesenchymal transi-tion-induced cellular plasticity and tumorigenicity. Stem Cells 2012;30:2088–99.

28. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin isan antioxidant of possible physiological importance. Science 1987;235:1043–6.

29. Zarjou A, Agarwal A. Heme oxygenase-1 as a target for TGF-beta in kidneydisease. Semin Nephrol 2012;32:277–86.

30. Okita Y, Kamoshida A, Suzuki H, Itoh K, Motohashi H, Igarashi K, et al.Transforming growth factor-beta induces transcription factors MafK andBach1 to suppress expression of the heme oxygenase-1 gene. J Biol Chem2013;288:20658–67.

31. Lavrovsky Y, Schwartzman ML, Levere RD, Kappas A, Abraham NG.Identification of binding sites for transcription factors NF-kappa B andAP-2 in the promoter region of the human heme oxygenase 1 gene. ProcNatl Acad Sci U S A 1994;91:5987–91.

32. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL. Nrf2, aCap'n'Collar transcription factor, regulates induction of the heme oxyge-nase-1 gene. J Biol Chem 1999;274:26071–8.

33. Igarashi K, Sun J. The heme-Bach1 pathway in the regulation of oxidativestress response and erythroid differentiation. Antioxid Redox Signal2006;8:107–18.

34. Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad thingshappen to good cells. Nat Rev Mol Cell Biol 2007;8:729–40.

35. Davalos AR, Coppe JP, Campisi J, Desprez PY. Senescent cells as a source ofinflammatory factors for tumor progression. Cancer Metastasis Rev2010;29:273–83.

36. Acosta JC, O'Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, et al.Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell2008;133:1006–18.

37. Kang C, Xu Q, Martin TD, Li MZ, Demaria M, Aron L, et al. The DNAdamage response induces inflammation and senescence by inhibitingautophagy of GATA4. Science 2015;349:aaa5612.

38. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, Orr AI, et al.Identification and characterization of a novel and specific inhibitor of theataxia-telangiectasia mutated kinase ATM. Cancer Res 2004;64:9152–9.

39. Reaper PM,GriffithsMR, Long JM,Charrier JD,Maccormick S,CharltonPA,et al. Selective killing of ATM- or p53-deficient cancer cells throughinhibition of ATR. Nat Chem Biol 2011;7:428–30.

40. Lee YS, Lee JW, Jang JW, Chi XZ, Kim JH, Li YH, et al. Runx3 inactivation is acrucial early event in the development of lung adenocarcinoma. CancerCell 2013;24:603–16.

41. Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as ananticancer strategy. Nat Rev Drug Discov 2013;12:931–47.

42. Fang J, Sawa T, Akaike T, Greish K, Maeda H. Enhancement of chemo-therapeutic response of tumor cells by a heme oxygenase inhibitor,pegylated zinc protoporphyrin. Int J Cancer 2004;109:1–8.

43. Ito Y, Bae SC, Chuang LS. The RUNX family: developmental regulators incancer. Nat Rev Cancer 2015;15:81–95.

44. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z,et al. Enrichr: a comprehensive gene set enrichment analysis web server2016 update. Nucleic Acids Res 2016;44:W90–7.

45. Thannickal VJ, Fanburg BL. Activation of an H2O2-generating NADHoxidase in human lung fibroblasts by transforming growth factor beta1. J Biol Chem 1995;270:30334–8.

46. Gutierrez S, Javed A, Tennant DK, van Rees M, Montecino M, Stein GS,et al. CCAAT/enhancer-binding proteins (C/EBP) beta and delta activateosteocalcin gene transcription and synergize with Runx2 at the C/EBPelement to regulate bone-specific expression. J Biol Chem 2002;277:1316–23.

47. Chuang LS, Ito K, Ito Y. RUNX family: Regulation and diversificationof roles through interacting proteins. Int J Cancer 2013;132:1260–71.

48. Wang CQ, Krishnan V, Tay LS, Chin DW, Koh CP, Chooi JY, et al.Disruption of Runx1 and Runx3 leads to bone marrow failure andleukemia predisposition due to transcriptional and DNA repair defects.Cell Rep 2014;8:767–82.

49. Krishnan V, Ito Y. A regulatory role for RUNX1, RUNX3 in themaintenanceof genomic integrity. Adv Exp Med Biol 2017;962:491–510.

50. Glasauer A, Chandel NS. Targeting antioxidants for cancer therapy.Biochem Pharmacol 2014;92:90–101.


Krishnan et al.




2018;78:88-102. Published OnlineFirst October 26, 2017.Cancer Res Vaidehi Krishnan, Yu Lin Chong, Tuan Zea Tan, et al.

Promotes Genomic Instability after Loss of RUNX3βTGF

Updated version

10.1158/0008-5472.CAN-17-1178doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2017/10/26/0008-5472.CAN-17-1178.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/1/88.full#ref-list-1

This article cites 50 articles, 15 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/78/1/88.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/1/88To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-1178

http://cancerres.aacrjournals.org/content/suppl/2017/10/26/0008-5472.CAN-17-1178.DC1

http://cancerres.aacrjournals.org/content/78/1/88.full#ref-list-1

http://cancerres.aacrjournals.org/content/78/1/88.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/1/88


molecular cell biology - cancer research · molecular cell biology tgfb promotes genomic...

Documents