Cyclooxygenase-derived proangiogenic metabolites of epoxyeicosatrienoic acids Amy A. Randa,b, Bogdan Barnycha,b, Christophe Morisseaua,b, Tomas Cajkac, Kin Sing Stephen Leea,b, Dipak Panigrahyd,e, and Bruce D. Hammocka,b,1 aDepartment of Entomology and Nematology, University of California, Davis, CA 95616; bUC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616; cWest Coast Metabolomics Center, UC Davis Genome Center, University of California, Davis, CA 95616; dCenter for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; and eDepartment of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115 Contributed by Bruce D. Hammock, March 2, 2017 (sent for review October 12, 2016; reviewed by James F. Callahan and A. Daniel Jones) Arachidonic acid (ARA) is metabolized by cyclooxygenase (COX) which suppress the formation of prostaglandins (16). Compared and cytochrome P450 to produce proangiogenic metabolites. with sEH inhibitors, COX inhibitors (i.e., the COX-2 selective Specifically, epoxyeicosatrienoic acids (EETs) produced from the inhibitor celecoxib) suppress angiogenesis and tumor growth, P450 pathway are angiogenic, inducing cancer tumor growth. A whereas sEH inhibitors alone tend to stimulate these same re- – previous study showed that inhibiting soluble epoxide hydrolase sponses in animals on a high omega-6 fat diet (15). It was (sEH) increased EET concentration and mildly promoted tumor therefore surprising that administration of a dual sEH and COX growth. However, inhibiting both sEH and COX led to a dramatic inhibitor led to an antiangiogenic and anticancer response more decrease in tumor growth, suggesting that the contribution of pronounced than administration of these inhibitors alone (15). Similarly, when a COX (celecoxib) and sEH inhibitor (trans-4- EETs to angiogenesis and subsequent tumor growth may be [4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid; t-AUCB) attributed to downstream metabolites formed by COX. This study were administered simultaneously to mice, tumor growth was explores the fate of EETs with COX, the angiogenic activity of much more inhibited than administration of the same two inhibitors the primary metabolites formed, and their subsequent hydrolysis alone, as shown in Fig. 1B (15). These results indicate that coin- by sEH and microsomal EH. Three EET regioisomers were found to cubation with a COX and sEH inhibitor produces a synergistic be substrates for COX, based on oxygen consumption and product effect, better at inhibiting primary tumor growth and metastasis formation. EET substrate preference for both COX-1 and COX-2 through the suppression of cancer angiogenesis. This suggests that were estimated as 8,9-EET > 5,6-EET > 11,12-EET, whereas 14,15- EETs, whose levels are controlled by sEH, are metabolized by EET was inactive. The structure of two major products formed COX to form angiogenic, protumorigenic products that enhance from 8,9-EET in this COX pathway were confirmed by chemical the procancer response of EETs. Inhibiting COX decreases their synthesis: ct-8,9-epoxy-11-hydroxy-eicosatrienoic acid (ct-8,9-E- formation and activity, as observed in a previous study (16). 11-HET) and ct-8,9-epoxy-15-hydroxy-eicosatrienoic acid (ct-8,9- The link between the COX and sEH pathways that stimulates E-15-HET). ct-8,9-E-11-HET and ct-8,9-E-15-HET are further metab- angiogenesis and tumor progression may be explained by a rel- olized by sEH, with ct-8,9-E-11-HET being hydrolyzed much more atively unexplored pathway associated with EET metabolism. slowly. Using an s.c. Matrigel assay, we showed that ct-8,9-E-11- Although EETs are primarily metabolized by sEH, a few studies HET is proangiogenic, whereas ct-8,9-E-15-HET is not active. This have observed that the 5,6-EET and 8,9-EET are substrates for study identifies a functional link between EETs and COX and COX-1 (17, 18) and COX-2 (19). The COX metabolites of EETs ct have largely undefined biological activity, yet Homma et al. de- identifies -8,9-E-11-HET as an angiogenic lipid, suggesting a phys- ct iological role for COX metabolites of EETs. termined that the -8,9-epoxy-11-hydroxy-eicosatrienoic acid (ct-8,9-E-11-HET) metabolite is a renal vasoconstrictor and potent mitogen, approximately three orders of magnitude more omega-6 fatty acids | epoxyeicosatrienoic acids | metabolism | potent than its parent, 8,9-EET (20). This provides evidence that cyclooxygenase | angiogenesis EETs undergo metabolism by COX, forming metabolites that have greater activity than their parent compounds. If these COX rachidonic acid (ARA) is an omega-6 fatty acid that is metabolites are also angiogenic, this activity may be mitigated Ametabolized by three major classes of enzymes, cyclo- with COX inhibitors. oxygenases (COXs), lipoxygenases, and cytochrome P450s (CYPs), to produce an array of biologically active metabolites (1–3). The Significance CYP pathway transforms ARA into four epoxyeicosatrienoic acids (EETs) in addition to hydroxylated metabolites (1). EETs have This study furthers our understanding of epoxyeicosatrienoic several biological actions, and are considered antihypertensive, acid metabolism by cyclooxygenase (COX) enzymes as a phys- antiinflammatory, neuroprotective, cardioprotective, and analgesic iologically relevant metabolic pathway, producing signaling (4). EETs also play a role in angiogenesis (5–10), the formation of molecules that are angiogenic. It explains, in part, why inhib- new blood vessels from preexisting vessels that is important for many iting the soluble epoxide hydrolase (sEH) in some systems is physiological and pathological processes, including cancer (11). angiogenic whereas combining sEH inhibition with COX in- EETs can enhance tumor growth and metastasis through their hibition is dramatically antiangiogenic, which in turn may angiogenic activity (12); however, this activity is transient due to suppress tumor growth. their metabolic instability. EETs are further metabolized by epoxide hydrolases (EHs), primarily soluble epoxide hydrolase Author contributions: A.A.R., B.B., C.M., D.P., and B.D.H. designed research; A.A.R., B.B., (sEH), to their corresponding diols (4, 13), which are generally C.M., T.C., and D.P. performed research; B.B., T.C., and K.S.S.L. contributed new reagents/ not biologically active (14) (Fig. 1A). Inhibition of sEH prolongs analytic tools; A.A.R., C.M., and B.D.H. analyzed data; and A.A.R., B.B., C.M., T.C., K.S.S.L., EET biological activity, potentiating their angiogenic activity, D.P., and B.D.H. wrote the paper. leading to increased tumor growth and metastasis in some sys- Reviewers: J.F.C., GlaxoSmithKline; and A.D.J., Michigan State University. tems (12, 15) (Fig. 1B). The authors declare no conflict of interest. ARA can also be metabolized by COX to form proangiogenic 1To whom correspondence should be addressed. Email: [email protected]. and proinflammatory prostaglandins (16). Angiogenesis stimulated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. from exposure to ARA can be mediated using COX inhibitors, 1073/pnas.1616893114/-/DCSupplemental. 4370–4375 | PNAS | April 25, 2017 | vol. 114 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1616893114 Downloaded by guest on September 27, 2021 t P > K ABArachidonic literature values (independent test, 0.05) (21). Likewise, M Acid (ARA) *** values for 8,9-EET with COX-1 and COX-2 are not significantly CYP 8000 different from the ARA KMs, indicating that both substrates have ) V 3 similar affinities for both enzymes. All values obtained for m 6000 also showed no significant difference. 11,12-EET gave rates that 5,6 EET 5,6-epoxy-PGH2 * COX were so low that the kinetic constants could not be calculated 8,9 EET 4000 based on the response curves, and 14,15-EET showed no apparent ct-8,9-E-11-HET reaction with COX-1 or COX-2 (SI Appendix, Figs. S3 and S4). 11,12 EET 2000 With respect to the enzymatic mechanism, epoxides placed at Tumor Volume (mm the 11,12 and 14,15 positions may alter the substrate orientation 14,15 EET ct-8,9-E-15-HET within the enzyme, limiting apparent reactivity. COX first cata- Epoxyeicosatrienoic 0 Acids (EETs) lyzes oxygenation by abstracting the substrate’s 13-proS-hydro- trol sEH Con gen, generating a radical with maximal electron density at C-11 Celecoxib Dihydroxy sEHi t-AUCB and C-15, where oxygen then adds to form a peroxyl radical Eicosatrienoic (22). Epoxides placed in either of these locations may limit the Acids (DHETs) t-AUCB + Celecoxib initial abstraction rate and addition of oxygen, compared with Fig. 1. ARA is metabolized by CYP to form metabolites that enhance an- epoxides placed further from the initial catalytic C-13 (i.e., 5,6- giogenesis and tumor growth. (A) EETs are metabolized by sEH to less active and 8,9-EET). 14,15-EET was least active compared with the dihydroxyeicosatrienoic acids and by COX to hydroxy-EETs (ct-8,9-E-11-HET other regioisomers, possibly due to the epoxide proximity to the and ct-8,9-E-15-HET) and prostaglandin analogs (5,6-epoxy-PGH2), regulators ω-end of the fatty acid. Because the ω-end resides within a of mitogenesis, vasoactivity, and angiogenesis. sEH inhibition preserves and channel at the top of the COX active site, substrates with sub- increases EET levels, thought responsible for angiogenesis and increased stituents closer to the ω-end may further limit COX activity tumor growth, yet COX inhibition blocks this activity. (B) Tumor volume is through added steric hindrance within this channel. For example, altered by the selective COX-2 inhibitor celecoxib and an sEH inhibitor (sEHi adding bulky substitutions within this channel has been shown to t-AUCB).