The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model Lori Dwyer-Nield1, Gregory A

Published OnlineFirst August 29, 2017; DOI: 10.1158/1940-6207.CAPR-17-0050

Research Article Cancer Prevention Research The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model Lori Dwyer-Nield1, Gregory A. Hickey2,3, Micah Friedman3, Kevin Choo3, Debbie G. McArthur3, Meredith A. Tennis2, Melissa L. New2,3, Mark Geraci4,and Robert L. Keith2,3

Abstract

Prostacyclin (prostaglandin I2, PGI2) overproduction in FVB/N initiated both before (1 week) and after (6 weeks) urethane mice prevents the formation of carcinogen and tobacco smoke– exposure to better model chemoprevention studies conducted induced adenomas, and administration of the oral prostacyclin in former smokers. Neither of these dosing strategies prevented analogue iloprost to wild-type mice also prevented carcinogen- murine lung cancer; however, we did detect changes in pulmonary induced mouse lung adenoma formation. Former smokers taking inflammatory cell infiltrate and expression of CXCR4 (a chemo- oral iloprost showed improved bronchial dysplasia histology kine receptor previously shown to increase in response to tre- compared with placebo. Next-generation oral prostacyclin ana- prostinil exposure) in tumor-bearing, treprostinil-treated ani- logues, like treprostinil, were developed for the treatment of mals, indicating that the drug was bioavailable. One potential pulmonary arterial hypertension (PAH). On the basis of our prior explanation stems from iloprost and treprostinil differentially studies with iloprost, we performed preclinical studies examining activating cell surface prostaglandin receptors and intracellular the ability of treprostinil to chemoprevent urethane-induced peroxisome proliferator-activated receptors. When murine lung murine lung adenocarcinoma. We determined the MTD in chow tumor cells were treated with treprostinil, their proliferation (prior studies had delivered treprostinil by gavage), and this dose rate increased; in contrast, iloprost had no effect on prolifer- produced serum levels in the experimental animals similar to ation. Future investigations comparing these two agents will those found in PAH patients treated with treprostinil. We then provide insight into iloprost's chemopreventive mechanisms. examined the chemopreventive efficacy of treprostinil exposure Cancer Prev Res; 1–9. 2017 AACR.

Introduction and increased implementation of screening programs (3). In addition to improved early detection with low-dose CT scans, Lung cancer accounts for the most cancer-related deaths across chemoprevention is an appealing approach to reducing non– all ethnicities and genders in the United States and worldwide (1). small cell lung cancer (NSCLC) mortality because we can readily The 5-year survival rate for lung cancer has gradually increased identify high-risk populations and use regression of premalignant from 9% to just 16% in the past 40 years, even with the advent of lesions (ground glass opacities and nodules for adenocarcinoma targeted therapies (2). Lung cancer is usually diagnosed at an and endobronchial dysplasias for squamous cell carcinoma) as an advanced stage, which contributes to its high mortality rate. The intermediate endpoint. Many previous NSCLC chemoprevention greatest potential for improving the poor survival rate may lie with trials were not based on robust preclinical data and used cancer earlier detection and more effective prevention strategies. A 20% diagnosis as the primary endpoint. This resulted in large, costly improvement in lung cancer mortality was seen in the National trials, which were generally unsuccessful (4–6) and reduced Lung Screening trial with annual CT scan surveillance, and this led scientiﬁc enthusiasm for future lung cancer chemoprevention to endorsement of lung cancer screening by many organizations trials. Newer trials focusing on prevention of progression to malignant disease could potentially reduce lung cancer mortality. We previously demonstrated that selective pulmonary produc- 1 Skaggs School of Pharmacy and Pharmaceutical Science, University of Colorado tion of prostacyclin (PGI2), a product of the arachidonic acid Anschutz Medical Campus, Aurora, Colorado. 2Division of Pulmonary Sciences pathway targeted in the treatment of pulmonary arterial hyper- and Critical Care Medicine, University of Colorado Anschutz Medical Campus, 3 tension (PAH), prevented lung tumorigenesis in multiple murine Aurora, Colorado. Eastern Colorado Veterans Affairs Medical Center, Denver, lung cancer models. Mice engineered to overexpress prostacyclin Colorado. 4Department of Medicine, Indiana University, Indianapolis, Indiana. synthase (PGIS) under control of the surfactant protein C pro- Note: Supplementary data for this article are available at Cancer Prevention moter produce increased lung prostacyclin and develop fewer Research Online (http://cancerprevres.aacrjournals.org/). tumors in response to chemical carcinogens or tobacco smoke Corresponding Author: Robert L. Keith, Eastern Colorado Veterans Affairs than do their wild-type littermates (7, 8). Iloprost, an oral pros- Medical Center, 1055 Clermont Box, Denver, CO 80220. Phone: 303-399- tacyclin analogue, also prevents carcinogen (urethane)-induced 8020 ext. 3182; Fax: 303-377-5686; E-mail: [email protected] lung tumor formation in mice (9). In addition, iloprost inhibits doi: 10.1158/1940-6207.CAPR-17-0050 anchorage-independent growth of NSCLC cell lines and activates 2017 American Association for Cancer Research. peroxisome proliferator-activated receptor gamma (PPARg), a

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known inhibitor of lung cancer growth (10, 11). These preclinical and recorded twice weekly. Surviving experimental mice were findings led to a phase II clinical trial in which high-risk current sacrificed after 21 days of special diet. Animals with excessive and former smokers were randomized to either placebo or oral weight loss (>15% of their beginning weight) were removed iloprost for 6 months. Comparison of endobronchial dysplasia, a from the study and euthanized. precursor of lung squamous cell carcinoma, in baseline and follow-up lung biopsies demonstrated that former smokers Determination of bronchoalveolar inflammatory cell content fi receiving iloprost experienced signi cant improvement in endo- and prostaglandin levels bronchial dysplasia compared with those that received placebo Bronchoalveolar lavage (BAL) was performed as described pre- (12). No improvement in dysplasia was seen in current smokers. viously (18) to quantify inflammatory cells. BAL cells were har- Former smokers are currently being recruited to a trial vested by centrifugation, counted, and stained with Wright stain (NCT02237138) testing whether inhaled iloprost also results in prior to differential counting. Prostaglandin E2 (PGE2) and 6-keto lesion regression. PGF1a, the stable metabolite of prostaglandin I2 (PGI2), in BAL In an effort to build on the success of the positive phase II fluid (BALF) were measured by ELISA according to the manufac- human trial with oral iloprost and expand the pool of potential turer's instructions (Cayman Chemicals: 500141, 515211). chemopreventive agents, we investigated the chemopreventive efficacy of treprostinil. Oral iloprost is no longer manufactured, and treprostinil is a next-generation prostacyclin analogue with Treprostinil chemoprevention study – multiple delivery formulations and a longer half-life than ilo- Female FVB/N mice (8 10 weeks of age) were divided into prost. Treprostinil is currently used for PAH treatment (13), and 5 groups and fed the appropriate chow for 1 week prior to subcutaneous, intravenous, and oral formulations are FDA intraperitoneal injection with either 1 mg/g urethane (Sigma: approved in the United States for treatment of patients with WHO U2500) or 0.9% saline vehicle. Group sizes were determined group I PAH with NYHA functional class II–IV symptoms, where- using mean and variation of tumor multiplicity from previous as the inhaled form is approved for patients with functional class experiments to detect 30% differences between groups (power ¼ n ¼ III and IV symptoms (14). More recently, oral treprostinil exhib- 0.9). Group 1 mice ( 10) were fed control chow followed n ¼ ited clinical efficacy in PAH as add on therapy, and adverse safety by saline injection; group 2 mice ( 10), treprostinil contain- events were not associated with the oral formulation (15). In ing chow (28.8 mg/kg/d) followed by saline injection; group 3 n ¼ addition, treprostinil inhibits TGFb and NFkB signaling, suggest- ( 15), control chow followed by urethane (1 mg/g body n ¼ ing it may promote anticancer signaling (16, 17). Given the weight) injection; group 4 mice ( 15) treprostinil chow success of other prostacyclin analogues in the chemoprevention followed by urethane injection 1 week later [early treatment n ¼ setting and treprostinil's improved oral bioavailability/half-life, (ET) group]; and group 5 mice ( 15) fed control chow were safety, and tolerability in the treatment of PAH, we hypothesized injected with urethane, and switched to treprostinil chow that treprostinil would effectively prevent NSCLC. Our in vitro 6 weeks after urethane exposure [late treatment (LT) group]. fi and in vivo experimental results instead determined that trepros- Eighteen weeks after urethane injection, mice were sacri ced by tinil did not prevent carcinogen-induced lung tumor formation in lethal pentobarbital injection, and plasma was collected by mice and actually enhanced proliferation of mouse lung tumor– cardiac puncture. BALF was collected and differential cell derived cells. counts were performed. Blood was perfused from the lungs at thetimeofsacrifice by direct instillation of PBS through the Materials and Methods pulmonary artery. Tumors were dissected from normal appear- ing tissue (uninvolved) in 10 to 12 mice/group, counted, their Animal studies diameters measured, and flash frozen. The right lower lobe was Female FVB/N mice (4–6 weeks of age) were purchased from digested with 1 mg/mL collagenase (Sigma: C2139) at 37Cfor Harlan Laboratories and housed in microisolator cages at the 25 minutes to form a single-cell suspension for FACS analysis. Denver Veterans Administration Medical Center (DVAMC) Veter- Lungs from 3 mice per group were instilled with formalin, fixed, inary Medicine Unit according to a protocol approved by the embedded, and sectioned prior to H&E staining for histologic DVAMC Institution of Animal Care and Use Committee in accor- characterization of tumors and Ki67 IHC analyses. dance with the NIH's Guide for the Care and Use of Laboratory Animals. Mice were maintained on hardwood bedding with a Measurement of plasma treprostinil levels 12-hour light/dark cycle; food and water were provided ad libitum. Plasma was flash frozen and sent to Tandem Labs for determination of treprostinil levels. Samples were extracted with meth- MTD determination anol, and an aliquot was injected onto an ultraperformance liquid Previously, mice were given treprostinil via daily gavage, so a chromatographic system equipped with a triple quadrupole tan- dietary treprostinil MTD was determined prior to performing dem mass spectrometer (AB/MDS Sciex API-5000) detector oper- tumorigenesis studies. Twenty female FVB/N mice (5 mice/ ated in negative TurboIonSpray mode. Separation of treprostinil group) were fed AIN-76A chow containing 7.2% (14.4 mg/kg/ from extracted matrix materials was accomplished using a Water day), 14.4% (28.8 mg/kg/day), or 36.0% (72 mg/kg/day) Acquity BEH C18 (2.1 100 mm, 1.7 mm) column operated at treprostinil or control AIN-76A chow (modified to contain 65C. Mobile phase A consisted of 0.1% formic acid in water and similar maltodextrin levels to drug containing chow) for mobile phase B consisted of 0.1% formic acid in acetonitrile at a 3 weeks. Treprostinil was graciously provided by United Ther- total flow rate of 0.775 mL/minute. Calibration standards of apeutics and integrated into a chow formulation by LabDiet. treprostinil diluted in control mouse plasma were used to con- Bacon scent was added to all chow formulations to stimulate struct standard curves. Linear-weighted (1/concentration2) regres- appetite. Animal weights and activity levels were observed sion analysis of peak area ratio versus theoretical concentration

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was used to produce calibration curves. These measurements were F4/80, APC, eBioscience: 17-4801-82). Fixed cells were then conducted according to published techniques (19). subjected to FACS analysis on a BD LSRII flow cytometer. Popula- tions were determined using FlowJo software as described previ- FACS analysis ously (20). Single-cell suspensions from the collagenase lung digest were filtered twice through a 70-mm filter, and cells were harvested by CXCR4 expression centrifugation and counted. One million cells per sample were RNA was extracted with a Qiagen RNeasy Kit from mouse lung aliquoted into 96-well plates and blocked with FC Block (BD tissue harvested from control, control/treprostinil, urethane, ure- Pharmingen: 553142) prior to incubation with a fluorescently thane/early treprostinil, and urethane/late treprostinil groups. labeled lymphocyte panel of antibodies(CD3,PE, BD Pharmin- Equal amounts of RNA were reverse transcribed with a Qiagen gen: 553064; CD4, APC, BD Pharmingen: 553051; and CD8, RT2 First Strand Kit. Samples were assessed for CXCR4 expression FITC, BD Pharmingen: 553031) or macrophage/neutrophil panel by qPCR using a validated Quantitect Primer Assay (Qiagen) and (MHCII, eFluor 450, eBioscience: 48-5321-82; Ly6G, PE, Quantitect SYBR Green qPCR Master Mix. Measurements were eBioscience: 12-6931-82; CD11b, PE-Cy7, eBioscience: 25- conducted in triplicate and were normalized to 60S ribosomal 0114-82; CD11c, PerCP-Cy5.5, BD Pharmingen: 550993; and protein L13 (Bio-Rad PrimerPCR Assay).

Figure 1. Treprostinil MTD and tumor prevention study. Mice fed increasing doses of treprostinil in chow were weighed daily (A) for 3 weeks, and 28.8 mg/kg was determined to be the MTD. Treprostinil in chow (28.8 mg/kg chow) was administered 1 week before (ET) or 6 weeks after (LT) urethane (Ure) exposure, and tumor number (B), burden (C), and diameter (D) were measured in mice harvested 16 weeks after urethane exposure. Weight gain was similar between groups (E). Plasma treprostinil levels were measured in each group (F) at harvest, and consistent levels were found in each treprostinil-treated group.

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Cell culture 2 Detection Kit (MRCT523). Ki67-positive nuclei/mm2 were JF32 cells were derived from a urethane-induced tumor in an counted in each tumor and averaged within each group using an A/J mouse (21) and cultured in MEMa media (Invitrogen: 12571- Olympus BX51 microscope fitted with an Olympus DP72 digital 063) supplemented with 10% FBS (Sigma) and 1% penicillin camera and CellSens Entry software (Olympus) to calculate tumor (10,000 U/mL)/streptomycin (10,000 mg/mL)/amphotericin B area. (25 mg/mL) solution (Invitrogen: 30-004-Cl). JF32 cells were tested for mycoplasma and authenticated by IDEXX RADIL in Statistical analysis 2012. Cells used in this study were thawed from stocks frozen Differences between two groups were analyzed by Student t test, down from the tested cells and were passaged no more than 15 and differences between three or more groups by one-way times after thawing. Cells were maintained in a humidified ANOVA followed by Student Newman–Keuls post hoc analysis incubator at 37 C, 5% CO2/95% ambient air, and split twice (except where indicated). Results were considered significant at weekly. To measure proliferation, 2,000 cells per well were seeded P 0.05. EC50 values and statistical analyses were generated using into 24-well tissue culture plates and allowed to adhere overnight. GraphPad Prism (version 5.0, GraphPad Software, Inc.). Cells were serum starved for 24 hours prior to exposure to PGE2 (in DMSO, Cayman Chemicals: 14010), iloprost (in methyl Results acetate, Cayman Chemicals: 18215), or treprostinil (in DMSO, Cayman Chemicals: 10162) at the indicated concentrations for MTD studies 48 hours. Cells were harvested and counted using a Celometer The MTD for treprostinil dosing in chow was determined to be Auto T4 (Nexcelom). 28.8 mg/k/day. Animals in the 75 mg/kg/day treprostinil chow group were sacrificed after the first week because of weight loss Ki-67 proliferative index exceeding 15%. The mice in the remaining three groups (control, Ki-67 immunostaining was conducted on tumors from trepros- 14.4 and 28.8 mg/k/day) chow gained equivalent weight during tinil treated and control animals as described previously (22, 23). the remainder of the study (Fig. 1A). Treprostinil at these doses did In brief, 5-mm lung sections from the urethane, ET, and LT groups not alter BAL levels of neutrophils, lymphocytes, and macro- were deparaffinized, peroxidases blocked with 3% hydrogen phages. PGE2 and PGI2 were unchanged with treprostinil expo- peroxide, and antigen retrieval performed in boiling Diva Decloa- sure (Supplementary Fig. S1). ker (Biocare Medical, DV2004G1) reagent under pressure for 10 minutes. Sections were incubated with Ki67 primary Ab (1:25 Treprostinil chemoprevention of urethane carcinogenesis dilution, Abcam 15580) for 30 minutes at room temperature, Mouse lung adenomas were induced by a single urethane þ and Ki67 nuclei were detected using Biocare Medical's Mach exposure. To distinguish between prevention of initiation versus

Figure 2. FACS analysis of lymphocytes. Bottom right lobe digests from each lung were incubated with ﬂuorescent antibodies against CD3, CD4, and CD8. Using FlowJo software, live cells were gated and doublets excluded on forward scatter (FSC) and side scatter (SSC). CD3þ cells were separated into CD4þ and CD8þ cells (A) for the urethane and ET groups (LT results were indistinguishable from ET results). Total CD4þ and CD8þ cells/lobe were quantitated and results graphed in B. , P < 0.01 versus urethane group.

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prevention of tumor promotion, feeding of treprostinil chow neutrophil marker panels were analyzed to quantify these popula- þ (28.8 mg/kg/day) was initiated 1 week prior to carcinogen expo- tions in each group. Tissue levels of CD8 T lymphocytes were 5- to sure (ET) or 6 weeks after (LT). Neither regimen affected urethane- 6-fold higher in tumor-bearing treprostinil-treated mice compared induced tumor multiplicity, burden, or diameter (Fig. 1B–D). with those of urethane-exposed AIN-76a–fed mice and control Histologic review of H&E slides from experimental and control mice receiving either AIN76a or treprostinil chow (Fig. 2A and þ animals revealed no discernable differences in histology between B). CD4 T lymphocyte numbers increased 2- to 3-fold in trepros- groups (data not shown). Weight gain was similar between all tinil-treated, tumor-bearing mice compared with those receiving treatment groups throughout the 20-week study (Fig. 1E). Tumor urethane. Conversely, CD11bvarCD11chiF4/80hi macrophage num- incidence for urethane-exposed mice in this study was 100%, bers decreased by approximately 50% in tumor-bearing mice fed with all urethane-exposed mice developing at least two tumors. treprostinil chow (Fig. 3A–C). No significant changes wereobserved þ There were no differences in any parameters measured between in neutrophil (CD11bhi, Ly6G )orCD11bhiCD11cmidF4/80lo the ET and LT treprostinil groups. Plasma treprostinil levels interstitial/tissue macrophages (Fig. 3C). (15–20 ng/mL) from control and urethane-treated mice fed treprostinil chow were similar regardless of treatment regimen CXCR4 expression (Fig. 1F). These levels are similar to those shown to be therapeutic CXCR4 is a chemokine receptor whose expression has been for PAH in rat and human studies (19). shown to increase in response to treprostinil exposure in hematopoietic stem cells (24). CXCR4 message increased slightly, but Inflammatory cell studies insignificantly, in uninvolved tissue from treprostinil-treated, To determine whether treprostinil treatment induced differences na€ve mice and urethane-treated mice. Significant increases in in lung tissue inflammatory cell populations, lung digests were CXCR4 message were detected in the LT group (Fig. 4), indicating examined by flow cytometry. Lymphocyte and macrophage/ that treprostinil was bioavailable and that these increases

Figure 3. FACS analysis of macrophage and neutrophil populations. Bottom right lobe digests from each lung were incubated with fluorescent antibodies against MHCII, Ly6G, CD11c, CD11b, and F4/80. Using FlowJo software, live cells were gated, followed by doublet exclusion on forward scatter (FSC) and side scatter (SSC). Macrophages were identified as a CD11chiCD11bvarF4/80hi subset, including resident alveolar macrophages and recruited alveolar and interstitial/tissue macrophages and a CD11cmidCD11bhiF4/80loLy6G subset. Neutrophils were identified as CD11bhiCD11cLy6Gþ cells, urethane group (A) and urethane ET group (B; results from LT mice were indistinguishable from those of ET mice). C, Average total macrophage subsets and neutrophils/lobe were calculated for each group and results are graphically represented. , P < 0.05 versus urethane group. www.aacrjournals.org Cancer Prev Res; 2017 OF5

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that were equal in incidence (100%), multiplicity, and size to those in mice that received control chow. In spite of its attractive pharmacokinetic profile and available formulations, our preclinical testing demonstrated that treprostinil does not share iloprost's chemopreventive properties. As the levels achieved in plasma were similar to those shown to be therapeutic in rats and humans (19), and treprostinil exposure altered lung inflamma- þ þ tory cell numbers (specifically increasing CD4 and CD8 T lymphocytes and decreasing CD11chi macrophages in tumor- bearing animals) and increased CXCR4 expression in stromal tissue, we conclude that the lack of treprostinil efficacy was not due to poor bioavailability. Iloprost and treprostinil are both prostacyclin analogues, share structural similarities, and both bind and activate the single prostacyclin receptor (IP). However, similar to many Figure 4. prostaglandin analogues, each compound activates a distinct CXCR4 expression increases in lung tissue from tumor-bearing, treprostinil- treated mice. RNA was isolated from lung tissue after tumors had been removed. set of related receptors at similar physiologic concentrations. CXCR4 message was reversed transcribed and measured by qPCR, and relative Iloprost binds prostaglandin E2 receptor 1 (EP1; 0.3 nmol/L)

amounts were compared using the DDCt method. Differences were detected by and IP (0.4 nmol/L) but requires micromolar concentrations to one-way ANOVA followed by Dunnett post hoc analysis; , P < 0.05. activate EP2 (2.1 mmol/L) and DP1 (2.1 mmol/L). In contrast, treprostinil activates DP1 (0.6 nmol/L), IP (1.9 nmol/L), and EP2 (6.2nmol/L) at similar concentrations, but activates EP1 at occurred in stromal tissue. Treprostinil did not increase CXCR4 much higher levels (290 nmol/L; ref. 8). Our plasma trepros- expression in JF32 murine lung tumor–derived cells (data not tinil levels reached15 to 20 ng/mL in all of our treatment shown) regimens, indicating that EP1 receptor was not activated. Opti- mal dosing for chemoprevention studies has not been determined, and there are studies suggesting that cyclical dosing (as Effects of treprostinil exposure on tumor cell proliferation opposed to the continuous dosing required for the treatment of To further examine the effects of treprostinil on adenocarci- PAH) of prostacyclin analogues is more effective in inhibiting noma,JF32cellswereexposedtoincreasing concentrations of cancer metastasis (25). To our knowledge, this is the first study PGE , iloprost, and treprostinil for 48 hours in serum-free 2 to examine treprostinil-impregnated chow in murine lung media. Cells were harvested and counted. Iloprost had no effect cancer experiments. on cell proliferation at concentrations ranging from 0.3 nmol/L Our group has previously shown that EP2-null mice develop to 300 mmol/L (data not shown). PGE and treprostinil expo- 2 fewer urethane-induced lung tumors than their wild-type litter- sure increased JF32 cell numbers by 2.9- and 2.1-fold (respec- mates (26), so the fact that treprostinil activates the EP2 receptor tively) over control media containing the DMSO vehicle may explain its lack of chemopreventive efficacy in this preclinical (Fig. 5A and B), with EC s of 750 nmol/L (PGE )and 50 2 model. Iloprost activates PPARg independent of the IP receptor 1.1 nmol/L (treprostinil). Although iloprost did not affect cell (27), but treprostinil requires the IP receptor to activate PPARg proliferation directly, JF32 cells pretreated with Iloprost did not and even in its presence may have little effect on PPARg activity exhibit PGE or treprostinil-induced increases in proliferation 2 (28, 29). PPARg activation has been shown to prevent lung (Fig. 5C), indicating that these two prostacyclin analogues may tumors in a variety of studies (30, 31). In a study of the effects have opposing functions in lung epithelial cells. Analysis of of prostacyclin analogues on macrophage function, treprostinil tumor cell proliferation in vivo using Ki67 staining (Fig. 5D) increased phagocytosis blocks, bacterial killing, and cytokine showed no significant differences in proliferation (Fig. 5E), generation, activities more closely resembling those of the pro- indicating possible opposing effects of treprostinil activities in tumorigenic PGE than the antitumorigenic PGI (32). One or tumor and stromal cells. This supports our finding that tumor 2 2 more of these differential activities could account for the reduced sizes were similar between all groups. chemopreventive efficacy of treprostinil. Another potential explanation for differences between the two Discussion agents may be related to their differential activation of chemokine Next-generation prostacyclin analogues like treprostinil have receptor type 4 (CXCR4). CXCR4 is an a-chemokine receptor with been developed to improve the ease of administration and dosing potent chemotactic activity for lymphocytes (33) that is known to intervals that limit the utility of other prostacyclin analogues. be involved in therapeutic resistance and epithelial-to-mesenchy- Although oral iloprost improved bronchial dysplasia in our mal transition in NSCLC. Elevated CXCR4 expression correlates clinical trial, the oral formulation is currently not manufactured. with aggressive metastasis, advanced stage disease, and shorter Inhaled iloprost is currently being tested for clinical efficacy in overall NSCLC survival rates (34). A recent publication by Kazemi former smokers with bronchial dysplasia, but the delivery options and colleagues demonstrated that treprostinil (and not iloprost or and stability of treprostinil could make it an alternative treatment beraprost) increased CXCR4 expression in hematopoietic stem option. We therefore tested whether orally delivered treprostinil and progenitor cells (HSPC), enhanced CXCR4-mediated HSPC exhibited lung chemopreventive properties similar to iloprost. In migration, and improved the engraftment of HSPCs in lethally our studies, mice receiving treprostinil in two dosing regimens irradiated mice without impairing their self-renewal capacity developed urethane-induced adenomas and adenocarcinomas (24). The enhanced ability of treprostinil to boost CXCR4-related

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Figure 5. PGE2 and treprostinil increase proliferation of mouse lung adenocarcinoma cells in vitro but not in vivo. JF32 mouse lung adenocarcinoma cells were serum- starved for 24 hours prior to incubation with increasing doses of PGE2 (A) or treprostinil (B) in serum-free media. Cells were harvested by trypsinization and counted; , P < 0.05 versus control. C, Iloprost pretreatment prevented PGE2 and treprostinil-mediated increases in JF32 cell proliferation; , P < 0.01 versus PGE2 or treprostinil alone. Graphs represent two independent experiments with at least three samples/group in each experiment. D and E, Ki67þ staining (D) was used to generate a proliferative index (E). Ki67 nuclear staining (red arrowhead) was counted in each tumor. Area of each tumor was determined using CellSens Entry software, and the number of Ki67þ nuclei/mm2 tumor was determined in each group. No differences were detected between treatment groups. pathway activity may explain its lack of chemopreventive iloprost and treprostinil receptor-binding activity in epithelial, properties. dendritic, and stromal cells could elucidate the mechanism of In PGIS-overexpressing mice, PGIS transgene expression is iloprost chemoprevention and guide us toward a more effective controlled by the surfactant protein C promoter so that these chemopreventive prostacyclin-based agent in the future. mice produce excess PGI2 only in lung type II and club cells (35). This overproduction protects mice from developing lung tumors. Disclosure of Potential Conflicts of Interest These mice also show increased BAL macrophages numbers. R.L. Keith has ownership interest (including patents) in uses of prostacyclin Iloprost exposure in chow results in similar reduction in tumor analogs for cancer chemoprevention and patent (preliminary application: Identifying biomarkers to predict prostacyclin analog response for the chemo- number and increase in alveolar macrophages (9). Alveolar prevention of cancer and reversal of pre-malignancy) and is a scientific/advisory macrophage numbers in treprostinil-exposed mice were compa- board member for LUNGevity Foundation. No potential conflicts of interest rable with na€ve mice and were significantly decreased in trepros- were disclosed by the other authors. tinil-exposed tumor-bearing mice. Treprostinil's lack of chemopreventive efficacy could be a result of direct effects on stromal Authors' Contributions cells. Prostaglandin analogues (including iloprost and treprosti- Conception and design: L. Dwyer-Nield, G.A. Hickey, M. Geraci, R.L. Keith nil) have been shown to suppress lipopolysaccharide (LPS)- Development of methodology: L. Dwyer-Nield, G.A. Hickey, M. Geraci, induced TNFa expression in monocyte-derived dendritic cells R.L. Keith (38). Yeh and colleagues proved these effects were IP receptor– Acquisition of data (provided animals, acquired and managed patients, cAMP pathway mediated as EP receptor antagonists did not provided facilities, etc.): L. Dwyer-Nield, G.A. Hickey, M. Friedman, K. Choo, D.G. McArthur, M.L. New, R.L. Keith reverse the suppression. Iloprost also regulated the expression of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, costimulatory molecules CD40, HLA-DR, CD80, and CD86 (36). computational analysis): L. Dwyer-Nield, G.A. Hickey, K. Choo, M.A. Tennis, An earlier study of PGI2 analogues and proinflammatory cyto- R.L. Keith kines showed that iloprost and cicaprost, but not treprostinil, Writing, review, and/or revision of the manuscript: L. Dwyer-Nield, suppressed LPS-induced expression of CD86, CD40, and MHC G.A. Hickey, M.A. Tennis, M.L. New, M. Geraci, R.L. Keith class II molecules by bone marrow–derived dendritic cells Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.G. McArthur, M. Geraci (BMDC) and inhibited the ability of BMDCs to stimulate anti- Study supervision: R.L. Keith gen-specific CD4 T-cell proliferation and production of IL5 and IL13 (37). In addition, iloprost and treprostinil exposure differ- Grant Support entially affected murine lung adenocarcinoma cell proliferation. Each author's financial support, including the recipient name (e.g., J.A. Treprostinil promoted cell growth at a magnitude similar to that Smith), funder name, and associated grant number(s) are as follows: of PGE2 at a much lower concentration, and this effect was Lori D Dwyer-Nield–Skaggs School of Pharmacy and Pharmaceutical abrogated by iloprost pretreatment. Identifying and comparing Sciences ADR Seed Grant (L. Dwyer-Nield principal investigator), NCIP50

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Dwyer-Nield et al.

CA058187 (P. Bunn principal investigator), U.S. Dept. of Veterans Meredith A. Tennis—LUNGevity Career Development Award (M. Tennis, Affairs5101BX000382 (R. Keith, principal investigator). principal investigator) Gregory A. Hickey–Cancer League of Colorado (Robert Keith, principal Melissa L. New–U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, investigator), U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, principal principal investigator) investigator), United Therapeutics (R. Keith, principal investigator) Mark Geraci–NCIP50 CA058187 (P. Bunn, principal investigator) Micah Friedman–U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, Robert L. Keith–NCIP50 CA058187 (P. Bunn, principal investigator), principal investigator) U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, principal investiga- Kevin Choo–U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, prin- tor) Cancer League of Colorado (R. Keith, principal investigator). cipal investigator) – Debbie G. McArthur U.S. Dept. of Veterans Affairs5101BX000382 (R. Keith, Received February 28, 2017; revised July 7, 2017; accepted August 23, 2017; principal investigator) published OnlineFirst August 29, 2017.

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OF8 Cancer Prev Res; 2017 Cancer Prevention Research

Treprostinil Does Not Prevent Murine Lung Cancer

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www.aacrjournals.org Cancer Prev Res; 2017 OF9

The Second-Generation PGI2 Analogue Treprostinil Fails to Chemoprevent Tumors in a Murine Lung Adenocarcinoma Model

Lori Dwyer-Nield, Gregory A. Hickey, Micah Friedman, et al.

Cancer Prev Res Published OnlineFirst August 29, 2017.

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