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Cancer Therapy: Clinical

A Novel Pharmacologic Activity of Ketorolac forTherapeutic Benefit in Ovarian Cancer PatientsYuna Guo1,2, S. Ray Kenney3, Linda Cook2,4, Sarah F. Adams2,5, Teresa Rutledge2,5,Elsa Romero1, Tudor I. Oprea2,6, Larry A. Sklar1,2, Edward Bedrick2,4, Charles L.Wiggins2,4,Huining Kang2,4, Lesley Lomo1,2, Carolyn Y. Muller2,5, Angela Wandinger-Ness1,2, andLaurie G. Hudson2,3

Abstract

Purpose: We previously identified the R-enantiomer of ketor-olac as an inhibitor of the Rho-family GTPases Rac1 and Cdc42.Rac1 and Cdc42 regulate cancer-relevant functions, includingcytoskeleton remodeling necessary for tumor cell adhesion andmigration. This study investigated whether administration ofracemic (R,S) ketorolac after ovarian cancer surgery leads toperitoneal distribution of R-ketorolac, target GTPase inhibitionin cells retrieved from the peritoneal cavity, and measureableimpact on patient outcomes.

Experimental Design: Eligible patients had suspectedadvanced-stage ovarian, fallopian tube or primary peritonealcancer. Secondary eligibility was met when ovarian cancer wasconfirmed and optimally debulked, an intraperitoneal port wasplaced, and there were no contraindications for ketorolac admin-istration. R- and S-ketorolac were measured in serum andperitoneal fluid, and GTPase activity was measured in peritoneal

cells. A retrospective study correlated perioperative ketorolac andovarian cancer–specific survival in ovarian cancer cases.

Results: Elevated expression and activity of Rac1 and Cdc42was detected in ovarian cancer patient tissues, confirming targetrelevance. Ketorolac in peritoneal fluids was enriched in the R-enantiomer and peritoneal cell GTPase activity was inhibited afterketorolac administration when R-ketorolac was at peak levels.After adjusting for age, AJCC stage, completion of chemotherapy,and neoadjuvant therapy, women given perioperative ketorolachad a lower hazard of death (HR, 0.30; 95% confidence interval,0.11–0.88).

Conclusions: Ketorolac has a novel pharmacologic activityconferred by the R-enantiomer and R-ketorolac achieves sufficientlevels in the peritoneal cavity to inhibit Rac1 and Cdc42, poten-tially contributing to the observed survival benefit in womenwhoreceived ketorolac. Clin Cancer Res; 21(22); 5064–72. �2015 AACR.

IntroductionOvarian cancer is the leading cause of death from gynecologic

malignancies and the second most common gynecologic cancer(1). Five-year patient survival remains less than 50% and the

mortality rate has not changed appreciably in two decades (1).The majority of women are diagnosed with metastatic disease,and although a substantial proportion of women respond toinitial treatment, recurrence is common (2). Despite concertedefforts, identification of effective targeted therapies has remainedelusive in this disease (3). There remains a great need to identifynew strategies to treat and manage ovarian cancer.

The Ras-homologous (Rho) family of small GTPases (Rac,Cdc42, and Rho) are key regulators of cancer-relevant cellularfunctions, including actin reorganization, cell motility, cell–cell,and cell–extracellular matrix (ECM) adhesion and invasion (4–6). In many human tumors (including colon and breast), there isclinical and experimental evidence that aberrant Rho-familysignaling contributes to tumor growth, survival, invasion, andmetastasis (4, 7–10). On the basis of these functions, Rac1 andCdc42 have been recognized as attractive therapeutic targets (11,12) and inhibitors are effective in experimental systems (13–19),but specific inhibitors of Rac1 or Cdc42 have not been translatedto clinical use.

A Cdc42-selective inhibitor effectively blockedmigration of twoovarian tumor cell lines (19, 20), suggesting that Rho-familyGTPases may be potential therapeutic targets in ovarian cancer.Using findings obtained from a high-throughput screen of thePrestwick library of off patent, FDA-approved drugs and chemin-formatics approaches, we identified the R-enantiomers of a limitednumber of non-steroidal anti-inflammatory drugs (NSAIDs) asinhibitors of Rac1 and Cdc42. The corresponding S-enantiomers

1Department of Pathology, University of New Mexico School of Med-icine, Albuquerque, New Mexico. 2Cancer Center, University of NewMexico, Albuquerque, New Mexico. 3Department of PharmaceuticalSciences, University of New Mexico College of Pharmacy, Albuquer-que, New Mexico. 4Division of Epidemiology, Biostatistics, and Pre-ventive Medicine, Department of Internal Medicine, University of NewMexico School of Medicine, Albuquerque, New Mexico. 5Division ofGynecologic Oncology, Department of Obstetrics and Gynecology,University of New Mexico School of Medicine, Albuquerque, NewMexico. 6Division of Translational Informatics, Department of InternalMedicine, University of NewMexico School of Medicine, Albuquerque,New Mexico.

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

Y. Guo and S.R. Kenney share first authorship.

Current Address for E. Bedrick: University of ColoradoDenver-AnschutzMedicalCampus, 2AMCA Anschutz Administration, Aurora, Colorado.

Corresponding Author: S. Ray Kenney, University of NewMexico, 2703 FrontierAvenue, Albuquerque, NM 87131. Phone: 505-272-3962, Fax: 505-272-0704;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0461

�2015 American Association for Cancer Research.

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are considered the active component in racemic drug formulationsacting as NSAIDs with selective activity against cyclooxygenase(COX). One candidate, R-ketorolac, inhibited ovarian tumor cellmigrationand adhesionwithout causing cytotoxicity (21–24).Ourdata indicate that the clinically administered racemic ketorolac(Toradol) has two distinct pharmacologic activities; thewell-estab-lished inhibition ofCOX1 and 2 by S-ketorolac serving as the basisof the FDA-approved indication for pain management, and apreviously unrecognized property of Rac1 and Cdc42 inhibitionconferred by the R-enantiomer. Cell-based measurement ofGTPase activity demonstrated that R-ketorolac specifically inhibitsEGF-stimulated Rac1 and Cdc42 activation at low micromolarconcentrations. TheGTPase inhibitoryeffects ofR-ketorolac in cellsmimic those of established Rac1 (NSC23766) and Cdc42(CID2950007/ML141)-specific inhibitors (19, 24).

In this study, we report that R-ketorolac achieved an effectiveconcentration in peritoneal fluids and inhibited Rac1 and Cdc42activity in cells retrieved from the peritoneal compartment ofpost-surgical ovarian cancer patients following administration ofthe racemic drug for postoperative pain management. A medicalrecord review to compare the ovarian cancer–specific survival ofovarian cancer patients who did or did not receive ketorolac forpostoperative analgesia revealed increased survival of patientsreceiving ketorolac. This observation is in keepingwith reports forimproved clinical outcomes associated with ketorolac usage, ascompared with other NSAIDs, in breast cancer patients (25–27).Although it has been long recognized that R-enantiomers ofNSAIDs are poor inhibitors of COX activity (28–30), potentialpharmacologic activities or benefits of the R-enantiomers haveremained largely unexplored. Our findings show that Rac1 andCdc42 are unrealized therapeutic targets in ovarian cancer anduseof ketorolac may benefit ovarian cancer patients.

Patients and MethodsImmunohistochemical analyses of GTPase targets

Immunohistochemical staining was performed using standardprocedures (see Supplementary Methods). Rac1 was stained with

mAb (clone 102; BD Biosciences, 10155-1-AP) and Cdc42 wasstained with rabbit pAb (Protein Tech Group). A VectastainReady-to-Use (RTU) ABC-peroxidase kit and ImmPact DAB(SK-4105) were used to visualize primary antibody labeling withhematoxylin nuclear counterstain (H-3401) for tissue stainingand samples were mounted in VectaMount (H-5000; all fromVector Laboratories).

For large-scale ovarian tumor profiling, tissue microarrayswere purchased from U.S. BioMax, Inc. (cat# OV1005 061 andOV8010 009). In total, 180 unique tissue samples were includedin the evaluation; ranging from stage I–IV and grades 1–3 (seeSupplementary Table S1). All tumor types were validated andstaining was scored by a pathologist with gynecologic pathologyspecialty (Lesley Lomo, MD, Dept. of Pathology, University ofNew Mexico) and evaluated for location (nuclear and cyto-plasmic), as well as intensity of positive staining. Scoring wasbased on the product of the percentage of cells stained and theintensity of the staining in each localization (3þ: strong, 2þ:intermediate, 1þ:weakand0:no staining), resulting inaminimumof 0 (100% cells� 0) and a maximum of 300 (100% cells � 3þ).

Quantitative PCR of ovarian cancer cDNA arraysQuantitative PCR (qPCR) analysis of Rho family GTPases was

performed using Tissuescan Ovarian Cancer cDNA microarraysfromOrigene (cat#HORT301,HORT302,HORT303)and standardtechniques (see SupplementaryMethods and Supplementary TableS2). qPCR amplification usedQiagenQuantitect primers forCdc42(QT01674442), Rac1 (QT00065856), and RhoA (QT00044723),and b-actin (Origene), and custom Rac1b primers from Invitrogen.Primer information for Rac1b, methods for PCR product validationand additional information on the Ovarian Cancer cDNA arrays ispresented in SupplementaryMethods and Supplementary Table S2.Analysis of serous cancer only is shown in Supplementary Fig. S1.Ct values were obtained using iCycler software. Relative expressionlevels were determined using DDCt values.

Patients, study design, and treatmentA phase 0 trial investigating the use of postoperative ketorolac

was reviewed and approved by the University of New MexicoHealth Sciences Center Human Research Review Committee (clin-icaltrials.gov, NCT01670799). Patients presenting with a newdiagnosis of ovarian, fallopian tube or primary peritoneal cancerwere screened for eligibility. Eligible womenwere at least 18-years-old, had an ECOG Performance Status <2 and had consented to aplanned debulking surgery—Consent was obtained before surgeryif primary eligibility was met. Secondary eligibility after surgeryincluded confirmed histologic diagnosis of epithelial ovarian,fallopian tube, or primary peritoneal cancer; optimal cytoreductionand placement of an intraperitoneal port for planned chemother-apy; adequate renal function and no postoperative complicationsprohibiting ketorolac use. Patients with known bleeding disordersor other contraindications to NSAID use were excluded.

Subjects received a single IV dose of Toradol (15 or 30mg basedonthepatient ageandcreatinine clearance)within thefirst 72hoursof surgerywhenall clinical safetyparametersweremet.Use of otherNSAIDs during the trial were not permitted; however, narcoticregimens were allowed for postoperative pain management. Allstudy protocols were reviewed by an independent data and safetymonitoring board. Baseline ascites samples were obtained at sur-gery. Subsequent peritoneal fluid samples were collected from theintraperitoneal port before dosing and at 1, 6, and 24 hours after

Translational Relevance

Clinically administered racemic ketorolac has two distinctpharmacologic properties: inhibition of COX by S-ketorolacand a previously unknown inhibitory activity against the smallGTPases Rac1 and Cdc42 by R-ketorolac. Rac1 and Cdc42 arehighly relevant tumor targets lacking clinically available selec-tive inhibitors. In this study, we provide the first demonstra-tion that ketorolac administration inhibits Rac1 and Cdc42 incells retrieved from the peritoneal cavity of post-surgicalovarian cancer patients. A novel phase 0 trial design allowedreal time sampling of fluids and cells from the peritonealcavity. Measurement of each enantiomer within the peritonealfluids revealed greater concentration of the R- versus S-enan-tiomer, thereby accounting for the GTPase inhibition. Further-more, a retrospective study identified improved ovarian can-cer–specific survival in patients who received ketorolac. Thiswork provides evidence that targeting the tumor-relevant Rho-family GTPases Rac1 and Cdc42 through repositioning ofketorolac may benefit ovarian cancer patients.

Racemic Distribution and Peritoneal Bioactivity of Ketorolac

www.aacrjournals.org Clin Cancer Res; 21(22) November 15, 2015 5065




single-dose ketorolac administration. Peripheral bloodwas collect-ed at the same time points. Serum and peritoneal fluid wereseparated from cellular material via low speed centrifugation.Tumor cells were further purified on Ficoll gradients to removered blood cells and negative selection with anti-CD45 beads toremove lymphocytes. The resulting tumor cell fractions were ana-lyzed by flow cytometry for EpCAM and MUC16/CA125 (seeSupplementary Fig. S2 for representative purity).

High-performance liquid chromatographyR- and S-enantiomers of ketorolac were analyzed by high-

performance liquid chromatography (HPLC) using publishedprocedures (31, 32). See Supplementary Methods and Supple-mentary Fig. S3 for additional information. The R value for thestandard curve of total ketorolac was 0.9997 and represented aconcentration range that spanned established human serumconcentrations (0.092–6.0 mg/mL).

Analysis of GTPase activityTwomethods were used to assess GTPase activity in cells based

on effector binding. Commercial GLISA kits from Cytoskeleton,Inc., analyzed Rac1 (cat# BK-128), Cdc42 (BK-127), or RhoA (BK-124) per the manufacturer's instructions. Alternatively, Rac1,Cdc42, andRhoA activitiesweremeasured using aflow cytometriceffector–binding assay (24, 33). Briefly, active Rac1 and Cdc42GTPases in prepared cell lysates were quantified individuallybased on binding to GST-PAK1-PBD from Millipore (cat#14-864) immobilized on GSH beads and use of specific antibodiesfor bound GTPase detection. Antibodies used to quantify theamount of active (GTP-bound)GTPases captured on thebeads arelisted in Supplementary Methods. Fluorescence intensity (meanchannel fluorescence, MCF) was measured by flow cytometry(Accuri C6).GTPase activitywas calculated by the (MCFof samplegroup—MCF of unstimulated group)/MCF of stimulated group.Equal amounts of protein were used for each assay.

Retrospective patient outcomes reviewA medical record review was conducted under Institutional

Review Board approval with a waiver of patient consent. Ovariancancer patients were identified from the New Mexico TumorRegistry (NMTR), a member of the population-based Surveil-lance, Epidemiology, and End Results (SEER) Program of theNational Cancer Institute (34). Inclusion criteria were as follows:invasive, epithelial ovarian cancer (any histology), ages 40 to 79years at diagnosis, years of diagnosis 2004 to 2006, and receipt ofsurgery at an Albuquerque, New Mexico hospital (only threehospitals in the metropolitan area provide this level of surgery).Diagnosis years of 2004 to 2006 ensured at least 6 years follow-up(mortality followed through Dec 31, 2012) for each patient. Weabstracted the surgical medical records for all analgesics andanesthesia medications used before hospital admission, duringsurgery and hospital stay, and given at discharge. Of the 138potential cases, 6 women did not undergo surgery because ofadvanced disease/severe comorbidities or desired palliative careonly, 1 woman had her surgery in another state, 2 women diedbefore surgery, andmedical recordswerenot located for 6women,leaving 123 women in the final analysis.

Statistical analysisqPCR findings for GTPase expression levels were analyzed

using one-way ANOVA followed by Dunnett multiple compar-isons test to determine differences between ovarian cancer grade.

IHC data were analyzed using one-way ANOVA followed by theTukey post-test to determine significant differences betweengroups. Data obtained from patient fluid and cell samples wereanalyzed as a repeated measure ANOVA followed by the Dunnettmultiple comparisons test to determine significant differencesbetween groups. For the retrospective medical record review,information fromthemedical recordwasmergedwith informationfrom the NMTR for final analysis. Clinical and treatment charac-teristics of patients who did and did not receive perioperativeketorolac were comparedwith c2 tests and t tests. In a preliminary,crude analysis the Kaplan–Meier method was used to estimate thesurvival probabilities. The difference in survival based on receipt ofperioperative ketorolac was examined using the stratified log-ranktest to adjust the effect of a single categorical factor such as agegroup, AJCC stage, etc. Because thiswas anobservational study andnot a randomized controlled trial, the final analysis was based on aCox proportional hazards model to adjust for clinical and treat-ment characteristics thatmay have differed between thosewho didand did not receive perioperative ketorolac. We estimated thehazard ratio (HR) for ovarian cancer–specific mortality comparingthose who did and did not receive perioperative ketorolac whileadjusting for age at diagnosis (<50, 50–64,�65) AJCC stage (I, II,III, and IV), completion of chemotherapy as planned (yes and no),and receipt of neoadjuvant chemotherapy (yes and no). On thebasis of the Cox proportional hazards regression, an examplesurvival plot is presented in Fig. 5. Additional survival plots arepresented in Supplementary Figs. S4–S7.

ResultsExpression of Rac1 and Cdc42 in ovarian cancer

On the basis of the established functions of Rho-familyGTPases, ovarian cancer metastasis is predicted to be stronglydependent on Rac1/Cdc42–regulated pathways for exfoliation,formation of multicellular aggregates, mesothelial adhesion, andlocalized invasion into the interstitial collagen-rich submesothe-lial matrix (2, 3, 35). To test whether these GTPases are dysregu-lated in ovarian cancer, we examined grade-dependent expressionof Cdc42 and Rac1 protein by immunohistochemical staining ofhuman tumor samples (Fig. 1) and GTPase mRNA using quan-titative PCR analysis of ovarian cancer tissue cDNA arrays (Fig. 2and Supplementary Fig. S1). Cdc42 protein overexpression levelswere highly significant for malignant, high-grade tumors (P <0.001) compared with lower-grade tumors without an apparentincrease in mRNA levels. In contrast, there was little evidence ofincreased expression of Rac1 protein with increasing grade (Fig.1). However, significantly elevated expression of a constitutivelyactive splice variant Rac1b (36–38) was detected in ovariantumors (Fig. 2). These findings provide evidence for aberrantexpression of Rac1 and Cdc42 in ovarian cancer and suggest thatthey may represent therapeutic targets for this disease.

Study design and patient populationIn previous work, we identified R-ketorolac as an inhibitor of

Rac1 and Cdc42 at low micromolar concentrations (21–24). R-ketorolac is inactive against the enzyme targets of S-ketorolac,COX-1, and COX-2 (28–30), and the inhibitory concentration(IC)50 values for inhibition of Rac1 and Cdc42 by S-ketorolacwere more than 100-fold greater than R-ketorolac (24). Thus, theracemic (R,S) ketorolac possesses two distinct pharmacologicactivities andourfindings identify R-ketorolac as a novel inhibitorof Rac1 and Cdc42.

Guo et al.

Clin Cancer Res; 21(22) November 15, 2015 Clinical Cancer Research5066




In this "phase 0" feasibility study, ketorolac was administeredfor its FDA-approved indication for postoperative pain manage-ment. Eligible patients had suspected advanced-stage ovarian,fallopian tube, or primary peritoneal cancerwithplannedoptimalcytoreductive efforts. Secondary eligibility was met if the patienthad confirmed ovarian cancer, was optimally cytoreduced andhad an intraperitoneal port placed for planned peritoneal che-motherapy. The patients had no active postoperative bleeding,did not require therapeutic anticoagulation, and had good post-operative organ function. Forty-two patients met primary eligi-bility, and considering secondary eligibility requirements, 20patient samples were collected at surgery. Samples of blood andperitoneal fluids after ketorolac administration were obtainedfrom 13 patients. Pathologic analysis of the 20 surgical samplesconfirmed fifteen patients (75%) had stage III or IV disease.Histologically, 100% had high-grade carcinoma; one was a carci-nosarcoma, 16 of 19 were pure serous carcinomas, one wasprimary peritoneal and one fallopian tube primary, and the restovarian primary tumors. The average agewas 60.8 (range, 33–81),race and ethnic distribution was 85% Caucasian (29.4% Hispan-

ic), 5% American Indian, and 10% Black or African American.Ketorolac dosages administered on the basis of clinical indica-tionswere 15mg (33%of patients) or 30mg (77%of patients). Asillustrated in Fig. 3A, blood and peritoneal fluid were obtained atT¼0, 1, 6, and24hours after administrationof the recommendeddose of ketorolac.

Distribution of R- and S-ketorolac in peritoneal fluidsSerum and peritoneal fluid samples were analyzed by HPLC to

resolve and quantify R- and S-ketorolac enantiomers to determineenantiomeric ratios and distribution over time (Fig. 3B and C).Clinical-grade ketorolac tromethamine is a 1:1 mixture of R- andS-enantiomers (Supplementary Fig. S3); however, the racemicdistribution favors the R-form in both serumandperitoneal fluidsat each time point. This is in keeping with the established shorterhalf-life of S-ketorolac in human serum based on differences inpharmacokinetic parameters for each enantiomer (28, 30, 39).Ketorolac distributes to the peritoneum within 1 hour after IVadministration, and ketorolac levels in the peritoneal fluids arenearly equivalent to those present in the serum at 6 hours and

Figure 1.Overexpression of Rac1 and Cdc42 protein in ovarian cancer specimens. A to C, representative images of ovarian serous cancer tissue are shown. Clinicalcharacteristics of the samples in the array are provided in Supplementary Table S1; magnification �200; scale bar 20 mm. A, hematoxylin and eosin staining(H&E). B and C, samples were stained with antibodies against Rac1 or Cdc42 and avidin/biotin horseradish peroxidase enzyme complex. Controls and tissuesamples were developed for identical times. D and E, tissue pathology and staining evaluated by board certified pathologist with gynecologic pathologyspecialization (Lesley Lomo,MD) and statistical analyses by statistician (EdBedrick, PhD, University of ColoradoDenver, AnshutzMedical Campus). For Rac1 onewaynonparametric ANOVA (P¼ 0.0087) and Tukey post-test shows normal stroma versus intermediate to high-grade carcinoma (P < 0.05) with all other comparisonsnonsignificant. For Cdc42 one way nonparametric ANOVA (P ¼ 0.0001) and Tukey post-test shows normal stroma and benign to borderline tumor versusintermediate to high-grade carcinoma (P < 0.05) with all other comparisons nonsignificant.






decline dramatically by 24 hours in both serum and peritonealfluids. Our results represent the first evidence of ketorolac distri-bution to peritoneal fluids.

The IC50 values for Rac1 and Cdc42 by R-ketorolac are 0.57 and1.07 mmol/L, respectively, whereas the IC50 values for S-ketorolacfor these targets was >100 mmol/L (24). The concentrations of R-and S-ketorolac in the peritonealfluidswere 0.98 and 0.32mmol/L,respectively, 6 hours after IV ketorolac administration. Thus, R-ketorolac achieved concentrations in the peritoneal fluids at orabove the IC50values forRac1andCdc42and ispredicted to inhibitthese GTPase targets in cells obtained from this compartment.

Analysis of patient-derived cellsTumor cell–enriched fractions were prepared from ascites

samples obtained at the time of cytoreductive surgery fromovarian cancer patients and post-surgery immediately prior and1, 6, and 24hours post IV ketorolac administration (Fig. 3A). BothRac1 and Cdc42 were highly activated in freshly isolated tumorcells from ascites and the activity level declinedwithin 48 hours inculture medium (Fig. 4A), suggesting that the ovarian tumorenvironment fosters Rac1 and Cdc42 GTPase activation. Post-surgery, weobserved a statistically significant decrease inRac1 andCdc42 activity with time after ketorolac administration (Fig. 4Band C). In contrast, RhoA activity was insensitive to ketorolac(Supplementary Fig. S8), further affirming the selectivity of thedrug. R-ketorolac predominates in the peritoneal fluids and theS-enantiomer is virtually undetectable at 24 hours (Fig. 3C),indicating that the R-enantiomer is bioactive and accounts forthe observed inhibition of the GTPases in vivo.

Retrospective patient outcomes reviewPerioperative ketorolac was used in 14% of the 123 women in

the study. Younger women (<50 years) were more likely thanolder women to receive perioperative ketorolac (P < 0.05); allother clinical and treatment characteristics were similar betweenthe two groups. At 60 months of follow-up, 3 of 17 ketorolac-treated patients (18%) and 40 of 92 nontreated patients (43%)had died of ovarian cancer. Stratified log-rank tests for categoricalfactors such as age group, AJCC stage, completion of chemother-apy as planned, and receipt of neoadjuvant chemotherapy ascoded in Table 1, showed a consistent ketorolac survival benefit ineach strata (see Supplementary Figs. S4–S7). The better survival inwomen treated with ketorolac consistently found in the stratifiedanalysis was also evident in the proportional hazards analysiswhen we adjusted for age at diagnosis, AJCC stage, completion ofchemotherapy as planned, and receipt of neoadjuvant chemo-therapy. The adjusted HR for ovarian cancer–specific mortalityassociatedwithperioperative ketorolac (yes vs. no)was 0.30 [95%confidence interval (CI), 0.11–0.88; Table 1]. On the basis of theproportional hazards model, an example survival plot is shownin Fig. 5 for women who had AJCC stage III cancer, were 50 to 60years at diagnosis, did not receive neoadjuvant therapy, andcompleted post-surgery chemotherapy as planned. Other survivalplots are shown in Supplementary Figs. S4–S7, and althoughthese plots highlight the results for women who completed theirpost-surgery chemotherapy as planned, all combinations ofwom-en defined by stage of disease, age at diagnosis, neoadjuvanttherapy, and post-surgery chemotherapy showed a consistentlybetter survival with ketorolac versus without. These preliminary

Figure 2.Expression of constitutively activeRac1b mRNA is elevated in ovariancancer specimens. Tissuescan ovariancancer cDNA microarrays (Origene)were amplified using primers againstRac1, Rac1b, Cdc42, RhoA, and b-actinas described in Patients and Methods.As per themanufacturer's description,patients with endometriosis,leiomyoma of myometrium, follicularcysts, abscesses, or secretoryendometrium, but otherwise healthyovarian tissue, were considerednormal (n¼ 19). Tissues defined as lowgradehaveaFIGO score of 1 (n¼ 19) or2 (n ¼ 32). Tissues considered highgrade have a FIGO score of 3 (n¼ 60)or 4 (n¼ 9). The cDNAs of 10 patientswere excluded due to a lack of gradeinformation. Clinical characteristics ofthe samples in the array are providedin Supplementary Table S2. Groupswere compared with normal using atwo-tailed t test, and significantincrease in Rac1b was detected;� , P < 0.05. Serous only, analysis bygrade is reported in SupplementaryFig. S1.

Guo et al.





findings suggest that perioperative ketorolac reduces ovariancancer–specific mortality.

DiscussionIn many human cancers, aberrant Rho-family GTPase activity

or downstream signaling pathways are associated withincreased aggressiveness and poor patient prognosis (4, 6, 8,9, 11, 12). The specific mechanisms by which Rho-familyGTPases modulate tumor development and progression remainunder investigation (4, 5, 7–9, 14); however, experimentalevidence places Rac1 and Cdc42 within the metastatic cascade.Little is known regarding Rac1 and Cdc42 expression in ovariancancer. We demonstrated elevated expression of Rac1 andCdc42 in human ovarian cancer specimens and high activityof these GTPases in freshly isolated tumor cells from ascitesobtained at surgery (Figs. 1 and 4). In a recent study, high Rac1protein expression in ovarian cancer was associated with earlyrecurrence and poor prognosis (40). Furthermore, partialsilencing of Rac1 by shRNA decreased tumor cell proliferation,migration and invasion in culture, and decreased growth ofsubcutaneous ovarian cancer xenografts in vivo (40). We findthat R-ketorolac inhibits adhesion and invasion of primaryhuman ovarian tumor cells from patient ascites (24), therebyindicating that pharmacologic inhibition of Rac1 and Cdc42also blocks these tumor-relevant functions. These observationsin conjunction with inhibition of ovarian tumor cell migrationby a Cdc42-specific inhibitor (19) indicate the potential valueof targeting Rac1 and Cdc42 in ovarian cancer.

In the present study, we show evidence that Rac1 and Cdc42inhibition can be achieved in ovarian cancer patients followingadministration of racemic ketorolac (Toradol). Ketorolac is a 1:1racemic mix of the R- and S-enantiomers. The S-form inhibitsCOX enzymes, which confers the drug's anti-inflammatory activ-ities. The COX inhibitory action of S-ketorolac supports its indi-cation for postoperative pain management, but also limits itslong-term use due to COX-related toxicity (28–30). R-ketorolachas little activity against COX (28–30), and therefore is notfunctional as an NSAID, but is bioactive and inhibits Rac1 andCdc42 (24). Importantly, the levels of R-ketorolac within theperitoneal fluidswere sufficient to inhibit Rac1 andCdc42 activityin cells obtained from the peritoneal cavity following ketorolacadministration. The innovative phase 0 clinical trial designenabled real-time sampling of fluids and cells from the peritonealcavity. Direct demonstration of the difference in racemic distri-bution of ketorolac enantiomers illustrates the value of a studydesign that allows direct testing of drug and cell activities withinperitonealfluids rather than extrapolation fromserumdrug levels.

Furthermore, the peritoneal bioactivity of ketorolac is shown tohave benefit for ovarian cancer patient outcomes. We found thatperioperative use of ketorolac reduces ovarian cancer–specificmortality (Fig. 5). There is precedence in the literature thatketorolac usage in the perioperative period is associated withimproved cancer outcomes. The first observation was made forbreast cancer patients in 2010 (25). In this study, ketorolac usewas associated with a decrease risk of breast cancer relapse(HR, 0.37; 95% CI, 0.0–0.79). Follow-up articles noted that thisrelapse reduction was most pronounced in the first 24 months

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Figure 3.Ketorolac distributes to peritoneal fluids and is enriched in the R-enantiomer. A, ascites samples were obtained at cytoreductive surgery and 1 to 3 days aftersurgery patients received a single dose of either 15 or 30 mg of clinical grade racemic ketorolac. Blood and peritoneal fluid from patients were collected beforedosing (T¼0), and at 1, 6, and 24 hours after dosing as depicted by the arrows. B andC, ketorolac enantiomers (R and S)weremeasured in blood andperitoneal fluidsusing HPLC. B, total ketorolac levels in sera and peritoneal fluids. C, the levels of each ketorolac enantiomer (R or S) at each time point in sera and peritonealfluids were measured. Concentration conversion to micromolar in serum and peritoneal fluids is provided in Supplementary Table S3. Administered drug isa 1:1 ratio of R toS (Supplementary Fig. S3), but S-ketorolac is eliminatedmore rapidly thanR-ketorolac leading to a ratio favoring theR-enantiomer in both serumandperitoneal fluids. The R value for the standard curve used to calculate the ketorolac concentrations was 0.9997 and represented a concentration range thatspanned established human serum concentrations (0.092–4.0 mg/mL; 3.0 mg/mL ¼ 10 mmol/L).






post-surgery (26, 27). No change in breast cancer recurrence wasnoted in patients who received sufentanil, clonidine, ketamine, orother intraoperative analgesics. Lung cancer patients receivingketorolac displayed improved overall survival as well (41). Theauthors hypothesize that the benefit is due to the anti-inflamma-tory actions of ketorolac, particularly on the extravasation ofcirculating tumor cells in the transient inflammatory environment

stimulated by surgery (42). Ketorolac appears to have morepronounced positive outcomes than other NSAIDs (42), and thismay be based on the combined impact of anti-inflammatoryactivity by the S-enantiomer and R-enantiomer effects on Rac1and Cdc42 leading to decreased adhesion and implantation ofcirculating or residual tumor cells. Ketorolac is not cytotoxic toovarian tumor cells (24), but predicted decreases in establishmentor further development of micrometastases due to Rac1 andCdc42 inhibition would be expected to improve response tosubsequent chemotherapy, which cannot be initiated untilpatients have recovered from cytoreductive surgery.

Collectively, our findings support the potential repositioningof ketorolac as an addition to current ovarian cancer therapy. Ourwork demonstrates that the R-enantiomer of ketorolac acts as afirst-in-class drug for inhibition of the cancer-relevant targets Rac1and Cdc42 (23, 24) and provides the first evidence that thesetherapeutic targets can be inhibited in humans using an approveddrug. There is precedence for pharmacologic activities dictated byR-enantiomers of specific NSAIDs against novel (non-COX) tar-gets (43–45). For example, R-etodolac and its analogues SDX-301and SDX-308 display antitumor activity in chronic lymphocyticleukemia and activity against multiple myeloma in cell andanimal models (44, 46–49). R-etodolac also significantly sup-pressed tumors in a colitis-relatedmousemodel colon cancer (43)and retarded tumor development and metastasis in a transgenicmouse model of prostate cancer (45). These examples and othersdemonstrate that R-enantiomers of NSAIDs can possess unantic-ipated anti-cancer activities based on interactions with non-COXtargets. Further evidence that targeting Rac1 may provide thera-peutic benefit in ovarian cancer was recently reported (50).Zoledronic acid is a nitrogen containing bisphosphonate thatinhibits prenylation of smallGTPases. Administration of this drugdecreased growth of ovarian cancer peritoneal xenografts throughinhibition of angiogenesis driven by a Rac1-mediated pathway

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Figure 4.GTPases are activated in patient ascites and inhibited by ketorolac administration in vivo. A, GTPase activity and target inhibition in patient-derived cells. GLISA PAK-effector binding was used to individually detect activated Rac1-GTP or Cdc42-GTP in tumor cells isolated from ovarian cancer patient ascites. PurifiedGTP-loaded GTPases were used to calculate ng GTP-bound GTPase in the patient sample. Unpaired two-tailed t tests showed samples in culture for 48 hours werestatistically different from fresh ascites samples for both Rac1 and Cdc42 (P ¼ 0.0109). The levels of active GTPase declined sharply with 48 hours in culture,indicating that soluble factors in the ascites serve to upregulate Rac1 and Cdc42 GTPase activities. B and C, Rac1 GTPase target inhibition following administration ofracemic ketorolac to ovarian cancer patients post-surgery. Cells isolated from patient ascites samples post-surgery were assayed for active Rac1 or Cdc42using a flow-based effector–binding assay. Patient diagnoses were all stage III, high-grade ovarian serous or papillary serous carcinoma, with one mixed serousendometrioid carcinoma and one suspected primary peritoneal carcinoma (Pt 20, 24, 35, 39, 43). Fluorescence readings were normalized to the 0-hourtime point drawn immediately before ketorolac administration. For Rac1, one way nonparametric ANOVA (P¼ 0.0009) and Bonferroni multiple comparisons test,� indicates P < 0.05, �� indicates P < 0.01: 0 versus 6 hours (��); 0 versus 24 hours (��); 1 versus 6 hours (�); 1 versus 24 hours (��). Differences between 0 versus1 hour and 6 versus 24 hours were nonsignificant. For Cdc42, one way nonparametric ANOVA (P ¼ 0.0250) and Bonferroni multiple comparisons test,� indicates P < 0.05: P < 0.05 for 0 versus 24 hours (�) was significant and all others were nonsignificant. RhoA was not responsive to ketorolac (SupplementaryFig. S8).

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Figure 5.Example survival plot among ovarian cancer patients with and withoutperioperative ketorolac. Cox proportional hazards regression was used toestimate ovarian cancer–specific survival probabilities for womenwho did (dashed line, 17 women) and did not (solid line, 92 women) receiveketorolac among ovarian cancer cases with AJCC stage III cancer,50 to 60 years of age at diagnosis, no neoadjuvant chemotherapy, andcompleted chemotherapy as planned (overall adjusted HR, 0.30; 95% CI,0.11–0.88, likelihood ratio test P ¼ 0.013).

Guo et al.





(50). Collectively, our results suggest that racemic ketorolac mayprovide a survival benefit to ovarian cancer patients throughinhibition of COX enzymes by the S-enantiomer and inhibitionof the small GTPases Rac1 and Cdc42 by the R-enantiomer.Additional studies to determine whether clinical benefit can beobserved in ovarian cancer patients through perioperative admin-istration of ketorolac in a placebo-controlled clinical trial are inprocess.

Disclosure of Potential Conflicts of InterestT.I. Oprea is an employee of Sunset Molecular Discovery LLC and reports

receiving commercial research grants from Givaudan Flavors Corporation. L. A.Sklar has ownership interest (including patents) in IntelliCyt. A. Wandinger-Ness, L.A. Sklar, T.I. Oprea, and L.G. Hudson are listed as co-inventors on apendingUS patent application entitled "Modulators of GTPases and Their Use,"which is owned by the University of New Mexico and its technology transfer

office, STC.unm, and is under an option to license. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Guo, L. Cook, T.I. Oprea, L.A. Sklar, C.Y. Muller,A. Wandinger-Ness, L.G. HudsonDevelopment of methodology: Y. Guo, S.R. Kenney, L.A. Sklar, E. Bedrick,C.Y. Muller, A. Wandinger-Ness, L.G. HudsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Guo, S.R. Kenney, L. Cook, S.F. Adams, T. Rutledge,E. Romero,C.L.Wiggins, L. Lomo,C.Y.Muller, A.Wandinger-Ness, L.G.HudsonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Guo, S.R. Kenney, L. Cook, T.I. Oprea, E. Bedrick,H. Kang, C.Y. Muller, A. Wandinger-Ness, L.G. HudsonWriting, review, and/or revision of the manuscript: Y. Guo, S.R. Kenney,L. Cook, S.F. Adams, T. Rutledge, T.I. Oprea, E. Bedrick, C.L. Wiggins, H. Kang,L. Lomo, C.Y. Muller, A. Wandinger-Ness, L.G. HudsonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Guo, E. Romero, T.I. Oprea, A.Wandinger-NessStudy supervision: L. Cook, C.Y. Muller, A. Wandinger-Ness, L.G. Hudson

AcknowledgmentsThis work is dedicated to the memory of Austin Hudson-LaPore.

Grant SupportThis study is supported by DOD OC110514 W81XWH-11-OCRP-TEA, UNM

Science and Technology Corporation Gap Fund, Core Facility support (FlowCytometry Shared Resource, Fluorescence Microscopy Shared Resource andHuman Tissue Repository) from the University of New Mexico Cancer Researchand Treatment Center (P30 CA118100). Focus Interactive Group grants fromUNMCancer Center (0990MD; 0990Q8 to A.Wandinger-Ness and L.G. Hudson;1146 to L. Cook). Contract HHSN261201300010I from the National CancerInstitute. Center for Molecular Discovery grants from the NIHMolecular LibrariesProgram (MH074425 and MH084690 to L.A. Sklar). NCI R25CA153825 (pre-doctoral fellowship to Y. Guo); INBRE NCRR 5P20RR016480 (predoctoralfellowship to S.R. Kenney).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 26, 2015; revised May 29, 2015; accepted June 2, 2015;published OnlineFirst June 12, 2015.

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Table 1. Hazard ratios (HRs) for ovarian cancer specific mortality for eachcharacteristic adjusted for the other characteristics in the table

Characteristic Number HR (95% CI) Pb

Perioperative ketorolacNo 106 1.00 (reference) 0.011Yes 17 0.30 (0.11–0.88)

AJCCa stageI 26 1.00 (reference) 0.050II 11 0.52 (0.11–2.47)III 57 2.17 (0.97–4.86)IV 29 1.56 (0.65–3.74)

Age, y<50 36 1.00 (reference) 0.04550–60 55 2.50 (1.17–5.35)60þ 32 1.85 (0.80–4.29)

Completion of post-surgery chemotherapy as plannedNo 78 1.00 (reference) 0.557Yes 45 1.19 (0.67–2.14)

Neoadjuvant chemotherapyNo 101 1.00 (reference) 0.050Yes 22 1.94 (1.03–3.66)

aAJCC, American Joint Committee.bLikelihood ratio test P value.






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Guo et al.




2015;21:5064-5072. Published OnlineFirst June 12, 2015.Clin Cancer Res Yuna Guo, S. Ray Kenney, Linda Cook, et al. Benefit in Ovarian Cancer PatientsA Novel Pharmacologic Activity of Ketorolac for Therapeutic

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