Prostacyclin and EMT Pathway Markers for Monitoring ... · Research Article Prostacyclin and EMT Pathway Markers for Monitoring Response to Lung Cancer Chemoprevention Melissa L

Research Article

Prostacyclin and EMT Pathway Markers forMonitoring Response to Lung CancerChemopreventionMelissa L. New1, Collin M.White1, Polly McGonigle1, Debbie G. McArthur2,Lori D. Dwyer-Nield3, Daniel T. Merrick4, Robert L. Keith1,2, and Meredith A.Tennis1

Abstract

Lung cancer is the leading cause of cancer deathworldwide andglobal burden couldbe reduced throughtargeted application of chemoprevention. The develop-ment of squamous lung carcinomahas been linkedwithpersistent, high-grade bronchial dysplasia. Bronchialhistology improved in former smokers in a chemopre-vention trial with the prostacyclin analogue iloprost.Prostacyclin acts through peroxisome proliferator-acti-vated receptor gamma (PPARg) to reverse epithelial tomesenchymal transition and promote anticancer sig-naling. We hypothesized that the prostacyclin signalingpathway and EMT could provide response markers forprostacyclin chemoprevention of lung cancer. Humanbronchial epithelial cells were treated with cigarettesmoke condensate (CSC) or iloprost for 2 weeks, CSCfor 16 weeks, or CSC for 4 weeks followed by 4weeks of CSC and/or iloprost, and RNA was extracted.

Wild-type or prostacyclin synthase transgenicmicewereexposed to 1week of cigarette smoke or one injection ofurethane, and RNA was extracted from the lungs.We measured potential markers of prostacyclin andiloprost efficacy in these models. We identified apanel of markers altered by tobacco carcinogens andinversely affected by prostacyclin, including PPARg ,15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMEN-TIN,CRB3,MIR34c, andMIR221. These data introduce apanel of potential markers for monitoring interceptionof bronchial dysplasia progression during chemopre-ventionwith prostacyclin. Chemoprevention is a prom-ising approach to reduce lung cancermortality in a high-risk population. Identifying markers for targeted use iscritical for success in future clinical trials of prostacyclinfor lung cancer chemoprevention. Cancer Prev Res; 11(10);643–54. �2018 AACR.

IntroductionThere are more cancer-related deaths from lung cancer

than any other malignancy worldwide (1). Lung cancerchemoprevention aims to reduce mortality from lungcancer by treating patients at high risk of developing lungcancer while they are still at the premalignant stage. Pros-tacyclin and its downstream target peroxisome prolifera-tor-activated receptor gamma (PPARg) play an importantrole in lung cancer and in chemoprevention. Decreasedexpression of PPARg in lung tumors is associatedwith poorprognosis, whereas a retrospective clinical study showed a

33% reduction in lung cancer among patients with diabe-tes using rosiglitazone, a PPARg agonist (2, 3). Severalpreclinical studies have shown a similar protective effectagainst the development of lung cancer from increasedpulmonary prostacyclin and the prostacyclin analog ilo-prost (4, 5). A phase II clinical trial of oral iloprost in high-risk patients demonstrated regression of endobronchialpremalignant histology in former smokers (6). Histologicregression of dysplastic lesions is an accepted endpoint foreffective chemoprevention, however intermediate biomar-kers of response (potentially measured in noninvasivespecimens) could indicate if prostacyclin is generating aresponse independent of visible histologic changes.The loss of epithelial features and gain of mesenchymal

properties, known as the epithelial-to-mesenchymal tran-sition (EMT), has been associated with an invasive ormetastatic phenotype in lung cancer and poor clinicaloutcomes, including increased cancer recurrence anddecreased survival (7). EMT is also relevant at the earlieststages of lung cancer development (8). Cigarette smokealtersmarkers of EMT, includingECADHERIN,VIMENTIN,and FIBRONECTIN (9). Conversely, activation of PPARg innon–small cell lung cancer inhibits EMT by increasing

1Division of Pulmonary Sciences and Critical Care Medicine, Department ofMedicine, University of Colorado Denver, Anschutz Medical Campus, Aurora,Colorado. 2Eastern Colorado Veterans Affairs Medical Center, Aurora, Colorado.3Department of Pharmaceutical Sciences, University of Colorado Denver,Anschutz Medical Campus, Aurora, Colorado. 4Department of Pathology, Uni-versity of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado.

Corresponding Author: Meredith A. Tennis, University of Colorado AnschutzMedical Campus, 12700E 19thAvenue, RC2BoxC272, Aurora, CO80045. Phone:3037246073; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-18-0052

�2018 American Association for Cancer Research.

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expression of ECADHERIN and inhibiting expression ofCOX-2 and the transcription factor SNAIL (10–12). Earlystages of lung premalignancy are also regulated bymiRNA,making them potentially relevant to prostacyclin chemo-prevention (8, 13). Although very little is known aboutprostacyclin or PPARg and miRNA, we demonstrated thatreduction ofMIR31 occurs with prostacyclin treatment andmay be linked to expression of the presumed iloprostreceptor in the lung epithelium (14, 15).We hypothesized that downstream targets of prostacy-

clin are affected by cigarette smoke and could act asmarkers of response to prostacyclin chemoprevention. Wetested this hypothesis using several models, includingin vitro in human bronchial epithelial cells (HBEC), in vivoin murine models of lung cancer carcinogenesis, and incultured primary dysplastic bronchial epithelial cells.Across these models, we identified markers that are alteredby cigarette smoke carcinogens and are inversely affectedby prostacyclin. These markers could be used to monitorresponse to iloprost chemoprevention and their identifi-cation is a critical step towards providing effective, person-alized lung cancer chemoprevention.

Materials and MethodsCell cultureHBEC (a gift in 2013 fromDr. JohnMinna, University of

Texas Southwestern, Dallas, TX) were cultured in Kerati-nocyte Serum Free Medium (GIBCO) at 37�C in a humid-ified 5% CO2 incubator and passaged twice per week.Morphology of cells lines was verified twice weekly. Cellswere tested for mycoplasma and fingerprinted uponreceipt. All long-term HBEC cultures were passaged onceafter thawing before beginning experiments and weregrown and handled in a dedicated incubator. Indepen-dently passaged, biologic triplicates were performed foreach HBEC experiment.

Drug and cigarette smoke condensate exposureCigarette smoke condensate (CSC; Murty Pharmaceuti-

cals) was applied to HBEC at 5 mg/mL and iloprost (Cay-man Chemicals) to HBEC or primary cells at 10 mmol/L incell growth media. DMSO or methyl acetate were thevehicle controls. Cells were allowed 24 hours for recoveryafter plating before drug or carcinogen application. In the8-week smoke model, HBEC were treated with CSC orvehicle every 72 hours for 4 weeks, then CSC was removedor continued with or without iloprost treatment every 72hours for another 4 weeks (Fig. 1B). HBEC with CSC andiloprost exposure were carried as individual cell lines intriplicate with twice a week passaging for 2 to 16 weeks.

Mouse modelsAll procedures were approved by the Denver Veterans

Administration Medical Center Institutional Animal Careand Use Committee. Mouse tissues used in this study were

frozen in RNALater (Qiagen). For urethane studies in wild-type and prostacyclin synthase transgenic (Pgistg) FVBmice, one 1 mg/g urethane intraperitoneal injection wasgiven, and mice were sacrificed 20 weeks later (5). Eachgroup includedfive animals. Tumors andvisible adenomaswere dissected from the lungs and uninvolved tissue wassaved for RNA extraction. For the 1-week smoke exposurestudy, wild-type and Pgistg FVB mice were exposed towhole body cigarette smoke for 6 hours/day for 5 days/week in a TE-10 smoking machine (Teague Enterprises) at80 mg/m3 (4). Each group included five mice. Mice weresacrificed after 1 week and whole lung tissue was removedfor RNA extraction. All experiments were conducted inaccordance with AAALAC International guidelines and theUnited State Animal Welfare Act.

Primary dysplastic cellsThe University of Colorado Cancer Center SPORE in

Lung Cancer Tissue Bank and Biomarkers Core (TissueBank) provides publicly available, deidentified samplesfor small studies. The Colorado Multiple InstitutionalReview Board approves all the human experimental pro-tocols that generate specimens for the Tissue Bank andcollects written, informed consent from all subjects. Threeprimary dysplastic lung cell cultures were isolated fromsquamous dysplasia biopsies at theUniversity of ColoradoCancer Center and were obtained from the Tissue Bank in2018. Cultures were taken from tissue adjacent to thebiopsy site used for histologic grading. After thawing, thecultures were grown in Bronchial Epithelial Basal Media(Lonza) at 37�C in a humidified 5% CO2 incubator for 2weeks and treated every 48 hours with 10 mmol/L iloprostor vehicle. Cells were treated with iloprost in triplicate, buttypically only two replicates survived to the end of treat-ment for RNA extraction.

Quantitative PCRRNA was extracted from HBEC and primary cells using

the AllPrep Universal Kit (Qiagen) and from mousetissue using the AllPrep DNA/RNA Kit (Qiagen) on theQiacube automated processor (Qiagen). mRNA wasreverse transcribed using the ABI HC cDNA Kit (ThermoFisher Scientific) and miRNA using the miScript II RTKit (Qiagen). qPCR primers for human and mouse inclu-ded: PPARg , 15PGDH, CES1, COX-2, (PTGS2), SNAIL,VIMENTIN, ECADHERIN, CRB3, 18sRNA, and RPL13(Bio-Rad) GAPDH and MIR34c, MIR221, and RNU6(Qiagen). qPCR was conducted using standard protocolsfor Sso Advanced SYBR Green Master Mix (Bio-Rad) orthe miScript SYBR Green PCR Kit (Qiagen) on a CFX96Touch (Bio-Rad). Genes for normalization of mousemodel PCR were determined by screening eight geneswith the Mouse Housekeeping Genes PCR Array (Qiagen;PAMM-000Z). 18sRNA was used for the urethane studyand RPL13 for the smoke studies. Unless indicated, PCRanalysis was conducted in triplicate.

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Statistical analysisFor single comparisons, a two-sided t test was used to

calculate P values. For multiple comparisons, ANOVA wasused. P values are indicated in figure legends. All statisticalanalysis was done using GraphPad Prism software.

ResultsExpression changes in prostacyclin targets and EMT

genes are associated with CSC and iloprost exposure inHBECcells.WeusedHBEC to investigatemRNAmarkers ofCSC exposure that are reversed by iloprost treatment aspotential markers of iloprost response during a chemo-prevention treatment protocol. In HBEC exposed to 16weeks of continuous CSC, PPARg , 15PGDH, and CES1expression decreased, whereas Cox-2 expression increased,suggesting that these targets of prostacyclin in the lungepitheliumcouldbe affectedby exposure to tobacco smoke(Fig. 1A). In HBEC, we modeled current and former smo-kers to parallel the oral iloprost clinical trial. We exposedcells to 4 weeks of CSC or control and then removed orcontinued CSC for another 4 weeks (Fig. 1B). We addediloprost exposure alone at 4 weeks or after CSC wasremoved in former smoke cells (Fig. 1B). At 8 weeks, RNAwas extracted from all cells for qPCR analysis.

PPARg expression increased with iloprost comparedwith control and was lower than control with current orformer CSC exposure (Fig. 1C). When iloprost was addedafter CSC was removed (former smoke with iloprost),PPARg level increased over any CSC exposure, almostreaching control level. 15PGDH is a prostaglandin path-way enzyme that degrades tumor-promoting PGE2, isinversely associated with COX-2, and decreased inlung cancer (16). 15PGDH expression increased with ilo-prost alone or with iloprost after CSC exposure butremained at control level with continued CSC exposure(Fig. 1C). CES1 is an enzyme involved with drug metab-olism shown to have increased expression in prostacyclinsynthase transgenic mice compared with wild type(17, 18). In ourHBECmodel,CES1 increasedwith iloprostbut remained low with any CSC exposure, suggestingincreased length of iloprost treatment may be necessaryto overcome CSC induced repression (Fig. 1C). COX-2expression is reduced by PPARg activation and has beenassociated with worse outcome in for NSCLC, whereas itsinhibition in combination with chemotherapy increasesoverall survival for advanced NSCLC (19, 20). COX-2expression decreased with iloprost and significantlyincreased with continued CSC exposure. When CSC was

Figure 1.

CSC and iloprost alter expression of PPARg and prostacyclin targets in HBEC.A, HBEC cells were treated in triplicate with 5 mg/mL CSC or vehicle for 16 weeks. � , vs.control, P value � 0.05. mRNA expression was measured by qPCR in triplicate, normalized to GAPDH, and compared to vehicle control. B, In the current andformer in vitro smoke model, HBEC cells were treated in triplicate with 5 mg/mL CSC or vehicle for 4 weeks. CSC was removed or continued and/or iloprostwas added for an additional 4 weeks. C, In the HBEC smokemodel, mRNA expressionwasmeasured by qPCR in triplicate, normalized toGAPDH, and compared withvehicle control and is represented as fold change. � , vs. control; þ, vs. former smoke, P-value � 0.05.

Monitoring Prostacyclin Lung Cancer Chemoprevention

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removed, COX-2 expression decreased, an effect that wasnot significantly augmented by the addition of iloprost(Fig. 1C).EMT changes occur early in the development of lung

cancer and EMT is targeted by PPARg , so we also measureda panel of EMT-related genes (11, 21). After 16 weeks ofCSC exposure in HBEC, mesenchymal markers SNAILand VIMENTIN increased compared with control whereasepithelial markers ECADHERIN and CRB3 decreased(Fig. 2A). PPARg inhibits EMT in part by suppressingSNAIL and VIMENTIN expression and increasing Ecad-herin in NSCLC (10, 11, 22). CRB3 is essential for normalepithelial polarity, is destabilized by Snail, and suppressesepithelial tumor progression (23, 24). In the 8-week HBECmodel (depicted in Fig. 1B), 4 weeks of iloprost decreasedSNAIL expression and increased ECADHERIN and CRB3(Fig. 2B). Current smoke increased SNAIL and VIMENTIN,while decreasing ECADHERIN and CRB3. When CSCwas removed (former smoke), expression of SNAILand VIMENTIN returned to control levels, whereasECADHERIN and CRB3 remained low. When 4 weeksof iloprost was added to former smoke cells, SNAILand VIMENTIN remained at control levels, whereasECADHERIN and CRB3 increased over former smoke.Prostacyclin and tobacco carcinogens lead to changes in

prostacyclin targets and EMT gene expression in murinemodels of lung cancer chemoprevention. Preclinical mod-els are essential to generating evidence for chemopreven-tion clinical trials and we interrogated two models forprostacyclin marker data. In the urethane mouse modelof lung cancer harvested 20 weeks after carcinogen expo-sure, FVB mice develop multiple adenomas (comparableto very early lesions in humans) but are protected byincreased levels of prostacyclin produced in Pgistg mice(5, 18). We removed visible lesions and analyzed geneexpression in the remaining whole lung to determine

whether early lesion development resulted in markerchanges. With urethane exposure, ECADHERIN, CRB3,PPARg ,CES1, and15PGDHdecreasedwhereasVIMENTIN,SNAIL, and COX-2 increased, suggesting a switch toward amesenchymal state and progression toward malignancy(Fig. 3A–H). The level of prostacyclin in the Pgistgmice resulted in rescue of urethane induced changes inECADHERIN, PPARg , CRB3, 15PGDH, COX-2, SNAIL, andVIMENTIN (Fig. 3A–H). In the 1-week smoke exposedmodel, mice are harvested after 1 week of smoke exposureor ambient air. After 1 week of smoke, FVBmice have earlygene expression changes associatedwith lung cancer but donot yet have lesions (14). With smoke exposure, SNAIL,VIMENTIN, and COX-2 increase, whereas ECADHERIN,CRB3, and PPARg , decrease (Fig. 3A–H). Pgistg mice withsmoke exposure reversed this trend for ECADHERIN,PPARg , CRB3, 15PGDH, CES1, VIMENTIN, SNAIL, andCOX-2 (Fig. 3A–H). Gene expression analysis in wholelung from these mouse models of chemopreventionrevealed a trend toward decreased epithelial markers,increased mesenchymal markers, and decreased PPARgsignaling with carcinogen exposure. Increased prostacyclinreversed this trend.Tobacco carcinogens and prostacyclin alter expression of

MIR34c and MIR22 in vitro and in vivo. We investigatedMIR34c and MIR221 as potential markers of iloprostactivity based on our initial screens and previous studieslinking their dysregulation to lung cancer (14, 25–29). InHBEC treated for 2 weeks, MIR34c expression increasedwith iloprost but didnot changewithCSC (Fig. 4A). LongerCSC exposure was required to observe a decrease inMIR34c expression (Fig. 4B).MIR221 expression increasedwith CSC at both 2-week and 16-week time points anddecreased with 2 weeks of iloprost treatment (Fig. 4Aand B). In our 8-week HBEC model of iloprost and smokeexposure (depicted in Fig. 1B), iloprost alone increased

Figure 2.

EMTmarkers are altered byCSC and iloprost exposure in HBEC.A,HBEC cellswere treated in triplicatewith 5mg/mLCSC or vehicle for 16weeks. � , vs. control,P value� 0.05. mRNA expression was measured by qPCR in triplicate, normalized to GAPDH, and compared with vehicle control. B, In the HBEC smoke model(depicted in Fig. 1), mRNA expression was measured by qPCR in triplicate, normalized to GAPDH, and compared with vehicle control and is represented as foldchange. � , vs. control, P-value �0.05.

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MIR34c expression while current exposure to smokedecreased expression (Fig. 4C). Removing CSC increasedMIR34c expression and adding iloprost to former smokecells led to an additional small increase. There was a largeincrease inMIR221with CSC exposure that decreased withremoval of CSC and further decreased with iloprost treat-ment (Fig. 4D).We also examined MIR34c and MIR221 expression in

our in vivo lung cancer models using PGIStg mice. A verysmall decrease in MIR34c expression occurred with ure-thane in the tumor model, but a significant increaseoccurred with Pgis overexpression (Fig. 5A). However, inurethane exposed mice, Pgis overexpression did notincrease MIR34c expression. miR-34c decreased with 1week of smoke exposure, increased in Pgistg mice, and

was slightly elevated with increased prostacyclin oversmoke exposure alone (Fig. 5B). Reinforcing the observedinverse expression pattern compared with MIR34c,MIR221 increased with urethane and smoke exposure andwas lower with increased prostacyclin (Fig. 5C and D).Results from both in vitro and in vivo models indicateMIR34c and MIR221 may be markers for prostacyclinchemopreventive activity.Gene expression changes in human primary dysplastic

cells parallel in vitro and in vivomodels. To expand markeranalysis to primary human samples, we identified threedysplastic cell bronchial epithelial cultures from biopsysiteswith high grade dysplasias. These types of higher gradebiopsies have been associated with an increased riskof developing invasive squamous lung carcinoma (30).

Figure 3.

Expression of EMT markers andprostacyclin targets is altered in vivoby exposure to prostacyclin andtobacco carcinogens. A–H, qPCR wasused to measure gene expression inRNA extracted from murine wholelung samples. Twenty-week urethanemice (urethane) were exposed toone injection of urethane or salineand sacrificed 20 weeks later(N¼ 5/group). One-week smokemice(Smoke) were exposed to 1 week ofcigarette smoke particulate orambient air (N ¼ 5/group). A,ECADHERIN; B, PPARg ; C, CRB3;D, 15PGDH; E, CES1; F, SNAIL;G, VIMENTIN (VIM); H, COX-2. Targetswere measured in triplicate,normalized to 18sRNA (20-weekurethane) or RPL13 (1-week smoke),and compared with saline or ambientair controls. � , vs. control; #, vs.urethane or smoke, P � 0.05.






To determine the effects of iloprost on dysplastic cells,we cultured the biopsies and treated them with iloprostfor 2 weeks. We then used qPCR to measure expressionof our panel of iloprost activity markers. In general, thethree iloprost treated dysplastic cultures demonstratedsimilar expression patterns to our in vitro and in vivomodels. Fig. 6A–C show expression data with SEM for thereplicate treatments within each culture, whereas Fig. 6Dshows average expression data of the three dysplasias foreach marker. Expression of prostacyclin targets associatedwith antitumor signaling increased with iloprost, whileCOX-2 decreased (Fig. 6A). EMT markers trended towarddecreased mesenchymal marker expression and increasedepithelial marker expression with iloprost (Fig. 6B). Agree-ing with the inverse expression pattern identified in ourother models, MIR34c increased slightly with iloprostwhereas MIR221 decreased (Fig. 6C). In Fig. 6D, the datafrom dysplasias is displayed in aggregate for each target tocompare average expression across all markers and tohighlight those with the strongest marker potential. Datafrom these primary cultures suggest that iloprost reversesgene expression changes associated with cigarette smoke

exposure, likely leading to interception of dysplasia pro-gression, and support the potential of these markers forclinical application.

DiscussionThe prostacyclin analogue iloprost is an agent with

strong potential as lung cancer chemoprevention. Signif-icant preclinical data supported moving this agent toclinical trials (4, 5, 14, 15, 31). Former smokers treatedwith iloprost in a phase II clinical trial had improvedendobronchial dysplasia, a premalignant lesion linkedwith development of squamous cell carcinoma (6, 30).This trial demonstrated that intermediate endpoints in thelung canmeasure the effectiveness of chemoprevention.Ofthe 48 patients who received iloprost in the chemopreven-tion trial, 23 had regressive histology and 25 had stableor progressive histology, pointing to the potential for aprecision medicine approach (6). Identification of predic-tive markers could improve future clinical trials of iloprostby limiting patients enrolled to those likely to respond,thereby reducing the trial cost, sample size, duration,

Figure 4.

Expression of MIR34c and MIR221 change in response to prostacyclin and tobacco carcinogens. A–E, qPCR was used to measure miRNA expression in RNAextracted from HBEC. A, HBEC were treated in triplicate with 5 mg/mL CSC, 10 mmol/L iloprost, or vehicle for 2 weeks. B, HBEC were treated in triplicatewith 5 mg/mL CSC for 16 weeks. � , vs. control P-value ¼ 0.02. C and D, HBEC cells were treated in triplicate with 5 mg/mL CSC or vehicle for 4 weeks.CSC was removed or continued and/or iloprost was added for an additional 4 weeks. Ilo, Iloprost; Csmoke, current smoke; Fsmoke, former smoke; Fsmokeþ Ilo, former smoke with iloprost. C, MIR34c. �, vs. control, P-value ¼ 0.06; ^, vs. control, P-value ¼ 0.09; #, vs. ilo, P-value

and improving detection of a clinical effect. Reducing theinvasiveness of the treatment protocol by replacingbronchoscopies with blood or sputum iloprost activitymarker assays would improve interest, compliance, andcost of future chemoprevention trials. Even though pros-tacyclin is the subject of one of very few successful phase IIlung cancer chemoprevention trials, iloprost as a chemo-prevention agent requires refinement of its application andimproved understanding of its mechanisms. This refine-ment may come in the form of delivery (oral vs inhaled),drug choice (iloprost vs. next-generation prostacyclin ana-logues), mechanism (manipulating drug receptors or tar-gets), or biomarkers (response predicting or treatmentmonitoring).Prostacyclin activates PPARg in lung epithelial cells

(15, 31). PPARg has been investigated for its role inmany aspects of lung cancer, including as chemopreven-tion or treatment (32). PPARg inhibits lung cancer pro-liferation, promotes sensitivity to targeted therapy, andsensitizes cells to chemotherapy (33–35). In lung epi-thelial cells, PPARg is activated by WNT7a bindingto its receptor FZD9, leading to decreased transformedgrowth, a pathway also stimulated by iloprost (15, 22).

FZD9 is the presumed receptor for iloprost in the lungepithelium and its expression is reduced in HBEC by1 week of CSC exposure (14, 15). We identified FZD9 asa potential marker for predicting response to iloprost inhigh-risk subjects and are investigating its role in themechanism of iloprost activity (14, 15). Although wefound decreased PPARg expression in response to tobac-co carcinogens in this study, some lung tumors retainexpression of PPARg (36, 37). Another study foundincreased PPARg expression with KRAS/p53 alterationspremalignant HBEC in vitro, but decreased expressionwhen tumorigenic clones of these cells were grown in vivo(20). In addition, activation of PPARg in macrophagesmay stimulate metastasis after primary tumor develop-ment (38). PPARg expression during lung cancer pro-gression is likely context and time dependent, so it willbe important to establish its expression and activity inthe earliest stages of lung lesions for chemopreventionapproaches that activate PPARg .With our in vitro and in vivo chemoprevention models

and primary cells, we tested markers across a range oftobacco-carcinogen and prostacyclin exposures. In a tar-geted approach, we selected activity markers to test based

Figure 5.

MIR34c andMIR221 expression changeswith exposure to prostacyclin and tobacco carcinogens in vivo. qPCRwas used tomeasure gene expression in RNA extractedfrom murine whole lung samples. A and C, 20-week urethane mice were exposed to one injection of urethane or saline and sacrificed 20 weeks later(N ¼ 5/group). B and D, Mice were exposed to 1 week of mainstream cigarette smoke before lungs were harvested (N ¼ 5/group). A and B, MIR34cexpression. C and D, MIR221 expression. Targets were measured in triplicate, normalized to RNU6, and compared with saline controls. � , vs. control; ^, vs. PGIS;#, vs. smoke P-value �0.05.






on known prostacyclin pathways. PPARg expressionincreased with iloprost treatment, suggesting it couldbe an activity marker for iloprost and that iloprost mayuse a positive feedback loop to increase response. 15pgdh,COX-2, and CES1 are all known targets of prostacyclinand we demonstrated that their expression is alsoaltered by iloprost (4). EMT pathways are targeted byprostacyclin through PPARg , offering another set ofpotential activity markers (11). We found that SNAIL,VIMENTIN,ECADHERIN, andCRB3 are alteredby iloprostafter exposure to tobacco carcinogens, suggesting thatreversal of EMT may be an important mechanism foriloprost chemoprevention activity.As biomarkers, miRNA have strong potential because

they are highly stable and exhibit tissue specificity.They have been proposed as markers in tissue andliquid biopsies for diagnosis, prognosis, and therapeuticapproaches (39). miRNA expression has been associatedwith smoke exposure, stages of lung cancer progression,lung tumor aggressiveness, and treatment resistance(8, 39, 40). Animal models have demonstrated that

chemopreventive agents, such as N-acetyl-l-cysteineor phenethyl isothiocyanate, can modulate miRNAin vivo in the context of smoke exposure (41). However,little is known about miRNA targets of prostacyclin orPPARg . We previously identified MIR31 as associatedwith iloprost binding to FZD9, suggesting that miRNAdo play an important role in iloprost signaling (14).A growing understanding of these master regulatorsin lung cancer and their suitability as biomarkers war-rants a thorough investigation of miRNA in lung cancerchemoprevention.Low expression of lung or circulating MIR34 family

members is associated with poor prognosis, metastasis,and relapse (26, 27). MIR34 directly targets PDL1, sug-gesting potential interactions between iloprost chemo-prevention and current immunotherapies (42). MIR221is included in profiles for lung cancer detection and hasan oncogenic role in lung tumor progression (29, 43)Also, MIR221 expression is inhibited by prostacyclin invascular cells (44). In this study, we found MIR34cexpression was decreased with exposure to tobacco

Figure 6.

Expression of EMT markers and prostacyclin targets are affected by exposure to iloprost in primary dysplastic cells. Primary dysplastic cells were treated with10 mmol/L iloprost for 2 weeks. Targets were measured by qPCR, normalized to GAPDH, and compared with vehicle-treated controls. A, prostacyclin targets;B, EMT markers; C, miRNA; D, aggregate data for Dysp1, Dysp2, and Dysp3 across all markers. Dysp, dysplastic culture. �, vs. control, P � 0.05.

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carcinogens and increased with iloprost or prostacyclinoverexpression, whereas MIR221 demonstrated inversechanges. In our mouse models, we observed thatincreased prostacyclin led to increased MIR34c only ina model of very early lesions and not in a tumor model.These data suggest that after carcinogen exposure, pros-tacyclin may only increaseMIR34c in the context of earlylesions and thatMIR34cmay be resistant to prostacyclin-induced changes once tumors develop. Changes inMIR221, however, were observed in both the tumor andpremalignant mouse models, suggesting that MIR221may be involved continuously in lesion progression andcould reflect prostacyclin activity throughout progres-sion. Decreases inMIR221 in urethane or smoke exposedPGIS mice were modest but might be stronger in a smokemodel with longer exposure.In a previous qPCR study, expression of MIR34c

progressively decreased from normal epithelium of non-smokers to invasive squamous bronchial lesions ofsmokers (25). Examination of MIR34c in biopsies fromthe oral iloprost chemoprevention trial also found thatexpression was inversely correlated with histologic gradeand with current smoke exposure (45). This supportsour observation of a significant decrease in MIR34cexpression in HBEC after 16 weeks of CSC and a slightincrease with removal of CSC. MIR34c was associatedwith response in both treatment and placebo arms inthis study on oral iloprost clinical trial samples, so theauthors did not find that MIR34c predicts response toiloprost or function as an intermediate endpoint marker(45). Because of design limitations from the previousstudy and based on our preclinical data, further studiesare warranted to determine whether MIR34c could serveas an activity marker for iloprost chemoprevention. Thisalso points to the importance of confirming that che-moprevention drugs target the lesions most likely toprogress to carcinoma, rather than lesions that wouldhave regressed on their own. In small clinical studies,molecular testing results could be confounded bypatients with low-grade lesions more likely to sponta-neously regress. Ongoing studies are characterizingbronchial dysplasias as likely to progress or regress andthis data can be combined with predictive markers toprecisely apply chemoprevention (30, 46).Our current murine chemoprevention studies are lim-

ited by lack of access to oral iloprost, however, similar-ities in mechanism between prostacyclin and iloprost,identified by comparing cells treated with iloprost toPgistg mice, support continued studies using the Pgistgmouse. We were unable to directly associate markerchanges with reduced dysplasia in our models, althoughin the mouse urethane model, increased prostacyclin andchanges in marker expression are associated withdecreased tumor burden (18). Significance of our in vitroexperiments was limited, however, the data does fit a

trend supporting the concept that in HBEC, iloprostactivity is associated with increased expression of anti-cancer markers and decreased expression pro-cancermarkers. Dysplastic cultures could only be treated withiloprost for 2 weeks, which also may not be enough timeto see significant changes across all markers in thesepremalignant cells. Ongoing work will measure potentialresponse prediction and activity markers in the oraliloprost chemoprevention trial, which can be validatedin samples from the ongoing inhaled iloprost chemo-prevention trial.Over half of lung cancers are diagnosed in former

smokers, patients who have already made the mostimportant change for lung cancer prevention but remainat high risk (1, 47). In this population, likely to havemultiple areas of premalignant, mildly altered lung cells,interception of progression with chemoprevention mayhave a stronger impact on mortality than treatment ofhighly mutated, established lung cancers. High-gradebronchial dysplasias associated with squamous carcino-ma are most likely to be identified by bronchoscopy andbiopsy. However, patients may develop carcinomas atsites distant from biopsies, highlighting the need forsystemic chemoprevention, as opposed to localized ther-apy (30). Implementation of chemoprevention for lungcancer has been challenged by multiple negative trialsand by the economic and health risks inherent in treatinglarge, at-risk populations for extended periods of time.Improved identification of the highest risk populations,and the highest risk lesions, will allow for more effectiveclinical trial design and targeted chemoprevention. Arecent search of Clinicaltrials.gov revealed that for everyone lung cancer chemoprevention trial there are over 150lung cancer therapy trials. Even with this focus on treat-ment, 5-year survival for lung cancer has remainedaround 15% for decades (1). To significantly reduce lungcancer mortality, we must improve application of exist-ing strategies, such as prostacyclin, and increase thedevelopment of new prevention approaches.

Disclosure of Potential Conflicts of InterestR.L. Keith has ownership interest (including stock, patents, etc.) in

U.S. Patent No. 8,623,917 (issued January 7, 2014). Uses of prosta-cyclin analogs for cancer chemoprevention. No potential conflicts ofinterest were disclosed.

Authors' ContributionsConception and design: M.L. New, C.M. White, D.T. Merrick,R.L. Keith, M.A. TennisDevelopment of methodology: R.L. Keith, M.A. TennisAcquisition of data (provided animals, acquired and managedpatients, provided facilities, etc.): M.L. New, P. McGonigle,R.L. Keith, M.A. TennisAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): M.L. New, C.M. White,L.D. Dwyer-Nield, R.L. Keith, M.A. Tennis






Writing, review, and/or revision of the manuscript: M.L. New,L.D. Dwyer-Nield, R.L. Keith, M.A. TennisAdministrative, technical, or material support (i.e., reportingor organizing data, constructing databases): D.G. McArthur,D.T. Merrick

AcknowledgmentsM.A. Tennis was supported by a LUNGevity Career Development

Award and NCI 1R01CA214531. R.L. Keith, L. Nield, and D.G.McArthur were supported by VAMerit 2 I01 BX000382-05. This workused the Protein Production/MoAB/Tissue Culture Shared Resource,

which is supported by the University of Colorado Cancer Centersupport grant P30CA046934 (to R. Schulick).

The costs of publication of this article were defrayed in part by thepayment of page charges. This articlemust therefore be herebymarkedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Received February 6, 2018; revised June 2, 2018; accepted July 16,2018; published first July 25, 2018.

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2018;11:643-654. Published OnlineFirst July 25, 2018.Cancer Prev Res Melissa L. New, Collin M. White, Polly McGonigle, et al. Lung Cancer ChemopreventionProstacyclin and EMT Pathway Markers for Monitoring Response to

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