Cyp2c44 regulates prostaglandin synthesis, lymphangiogenesis, and metastasis in a mouse model of breast cancer

Rushendhiran Kesavana, Timo Frömela,b, Sven Zukunfta,b, Hebatullah Labana,b, Alexandra Geyera, Zumer Naeema, Juliana Heidlerb,c, Ilka Wittigb,c, Eiman Elwakeeld, Bernhard Brüned, Andreas Weigertd, Rüdiger Poppa,b, and Ingrid Fleminga,b,1

aInstitute for Vascular Signalling, Centre for Molecular Medicine, Goethe-University, 60590 Frankfurt am Main, Germany; bGerman Centre for Cardiovascular Research (DZHK), Partner Site RheinMain, 60590 Frankfurt, Germany; cFunctional Proteomics, Sonderforschungsbereich (SFB) 815 Core Unit, Faculty of Medicine, Goethe University, 60590 Frankfurt am Main, Germany; and dInstitute of Biochemistry I, Goethe-University, 60590 Frankfurt am Main, Germany

Edited by Tak W. Mak, University of Toronto, Toronto, Canada, and approved February 4, 2020 (received for review December 13, 2019) epoxides generated by (CYP) metalloproteinases, as well as down-regulation of the anti-angiogenic have been linked to increased tumor growth and factor thrombospondin-1 and the antimetastatic CD82 and metastasis, largely on the basis of overexpression studies and the nm-23 (6, 7). Importantly, however, some of these data were application of exogenous epoxides. Here we studied tumor obtained using animals that overexpressed CYP enzymes (partic- − − growth and metastasis in Cyp2c44 / mice crossed onto the polyoma ularly CYP2C8 or CYP2J2) or were treated with an inhibitor of middle T oncogene (PyMT) background. The resulting PyMT2c44 mice the soluble epoxide (sEH) to markedly increase EET developed more primary tumors earlier than PyMT mice, with levels or with high concentrations of 14,15-EET. Little information increased lymph and lung metastasis. Primary tumors from is available on the consequences of physiological levels of CYP Cyp2c44-deficient mice contained higher numbers of tumor- activity on tumor development. associated , as well as more lymphatic endothelial The aim of this investigation was to study the consequences of cells than tumors from PyMT mice. While epoxide and diol levels interfering with the CYP pathway in tumor growth and metas- CELL BIOLOGY were comparable in tumors from both genotypes, prostaglandin Δ2c44 tasis without the overexpression of CYP enzymes or exogenous (PG) levels were higher in the PyMT tumors. This could be application of high concentrations of epoxides or pharmacolog- accounted for by the finding that Cyp2c44 metabolized the PG ical inhibitors. Cyp2c44 was targeted in mice, given that it was precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic reported to have a role in tumor growth and angiogenesis (8, 9), acid (12-HHT), thus effectively reducing levels of effector PGs and more recently, Cyp2c44 expression was reported to be al- (including PGE ). Next, proteomic analyses revealed an up- 2 tered during cancer initiation at a point when arachidonic acid regulation of WD repeating domain FYVE1 (WDFY1) in tumors Δ2c44 metabolism was dysfunctional (10). Therefore, breast cancer from PyMT mice, a phenomenon that was reproduced in − − development was studied in Cyp2c44 / mice crossed with mice Cyp2c44-deficient macrophages as well as by PGE2.Mechanisti- cally, WDFY1 was involved in Toll-like receptor signaling, and its expressing the polyoma middle T (PyMT) oncogene under the down-regulation in human attenuated the LPS- control of the mouse mammary tumor virus promoter (11). induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 pro- Significance tects against tumor growth and metastasis by preventing the

synthesis of PGE2. The latter eicosanoid influenced macrophages The bioactive eicosanoid prostaglandin (PG) E2, which is gener- at least in part by enhancing Toll-like receptor signaling via the ated from PGG2 and PGH2 by prostaglandin E synthases, is up-regulation of WDFY1. known to alter the phenotype of macrophages in the tumor microenvironment. Here, we show that a cytochrome P450 en-

cytochrome P450 | WDFY1 | metastasis | lymphangiogenesis zyme, Cyp2c44, interferes with this pathway by decreasing PGG2 and PGH2 levels. The deletion of Cyp2c44 in a genetic mouse ytochrome P450 (CYP) enzymes can metabolize polyunsat- model of breast cancer resulted in increased tumor growth and Curated fatty acids (PUFAs) to biologically active signaling metastases that were associated with increased PGE2 levels, mediators. For example, CYP enzymes can convert arachidonic lymphangiogenesis, and the alternative polarization of macro- acid to hydroxyeicosatetraenoic acids and epoxyeicosatrienoic phages. Moreover, inflammatory Toll-like receptor signaling in acids (EETs), which have been implicated in intracellular sig- Cyp2c44-deficient macrophages was enhanced, at least partly naling events in numerous cell types. Perhaps most is known because of the upregulation of WD repeating domain FYVE1. about the biological actions of the EETs, which possess vasodi- Author contributions: I.F. designed research; R.K., T.F., S.Z., H.L., A.G., Z.N., J.H., I.W., E.E., lator, anti-inflammatory, and angiogenic properties (for review, A.W., and R.P. performed research; R.K., T.F., S.Z., I.W., B.B., A.W., and I.F. analyzed data; see ref. 1). Many compounds that promote angiogenesis have and I.F. wrote the paper. also been implicated in tumor development, and the same is true The authors declare no competing interest. for the PUFA epoxides. In fact, several different tumor cell types This article is a PNAS Direct Submission. have been shown to express CYP and to generate Published under the PNAS license. EETs, which in turn accelerate tumor cell proliferation in vitro α – Data deposition: The mass spectrometry proteomics data have been deposited to the and protect against TNF -induced apoptosis (2 5). In addition ProteomeXchange Consortium via the PRIDE partner repository (identifiers PXD017336 to potentially promoting tumor angiogenesis and growth, EETs and PXD017303). have also been linked to increased tumor metastasis (6, 7). A 1To whom correspondence may be addressed. Email: [email protected]. number of mechanisms have been proposed to explain this re- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ lationship, including the up-regulation of vascular endothelial doi:10.1073/pnas.1921381117/-/DCSupplemental. cell growth factor (VEGF), CD44, and prometastatic matrix First published March 2, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1921381117 PNAS | March 17, 2020 | vol. 117 | no. 11 | 5923–5930 Downloaded by guest on October 1, 2021 Results Cellular Composition of Primary Tumors from PyMT and PyMT2c44 Mice. Δ Tumor Development and Metastases in PyMT and PyMT 2c44 Mice. Given that metastatic potential has been partly attributed to the Palpable tumors developed in adult PyMT mice globally lack- lymphatic system in tumors, the formation of capillaries and lymph Δ ing Cyp2c44 (so-called PyMT 2c44 mice) from 13 wk of age and capillaries was assessed in primary tumors. Using immunohistochem- istry, it was possible to demonstrate that the vascular characteristics of increased in number and size during the observation period (max- the tumors differed significantly (i.e., primary tumors from Δ + + imum 20 wk). However, when compared with the PyMT mice, the PyMT 2c44 mice contained more Lyve1 cells than endomucin cells, number of tumors that developed, as well as their size, was signif- indicating a dominance of lymph capillaries; Fig. 2A). A similar icantly greater in animals lacking Cyp2c44 (Fig. 1 A–D). This in phenomenon was detected when FACS was used to identify vascular + + + itself was unexpected, as the inhibition/down-regulation of Cyp2c44 endothelial cells (CD31 , CD146 ,Ly-6C cells) and lymph endo- + − − has been proposed as a strategy to treat cancer (9). thelial cells (CD31 ,CD146 ,Ly-6C cells) present in primary tumor Increasing CYP epoxide levels by inhibition or deletion of the digests (Fig. 2B). Consistent with these data, the expression of the sEH has been linked to escape from tumor dormancy and increased lymph markers VEGFR3 and Prox1 was high in PyMT2c44 tumors. metastasis (7). Therefore, the numbers of metastases that de- Although the vascular angiogenesis markers VEGFR1, VEGFR2, Δ2c44 veloped in axillary lymph node and lungs from the affected 20-wk- EphB4, and Sox7 were also higher in PyMT than in PyMT tu- C old animals was determined. Significantly more metastases mors, the differences were relatively subtle (Fig. 2 ). + + (cytokeratin , CD326 cells) were detected in lymph nodes from The tumors also displayed differences in inflammatory cell in- Δ2c44 filtration as more tumor-associated macrophages were detected in PyMT than from PyMT mice (Fig. 1E). The situation was not Δ tumors from PyMT 2c44 mice (Fig. 3A), whereas numbers of resident asclearinthelung,asthetotalnumberofmetastaseswasnot F4/80 high macrophages were comparable in tumors from both F different between the two genotypes (Fig. 1 ). However, metastases groups of animals. In addition, more tumor-associated macrophages Δ2c44 Δ detected in the PyMT mice were markedly larger than those were detected in the PyMT 2c44 group (Fig. 3B). One of the hallmarks found in the PyMT group, in which only micrometastases were of tumor-associated macrophages is the suppression of adaptive im- − detected. munity (12); accordingly, the numbers of CD11b lymphocytes, as well

Δ Fig. 1. Consequences of Cyp2c44 deletion on primary tumor growth and metastases. (A) Time-dependent increase in tumor number in PyMT and PyMT 2c44 (Δ2c44) mice from weeks 13 to 20. (B) Tumor volume per animal/week from weeks 13 to 20. A&B n = 13 mice per group (ANOVA for repeated measures and Newman-Keuls test). (C) Total tumor weight at week 20. (D) Tumor burden (total tumor weight normalized to body weight) at week 20. (CandD) n = 16 PyMT and n = 17 PyMTΔ2c44 mice (Student’s t test). (E) Metastatic tumor cells identified using cytokeratin (red) and CD326 (green) in axillary lymph nodes from PyMT and PyMTΔ2c44 (Δ2c44) mice. (Lower) Magnifications of the areas marked by boxes. (Scale bar = 100 μm; Upper,20μm.) (Lower) n = 17 to 18 mice per Δ group (Student’s t test). (F) Breast cancer metastases (H&E staining), in lungs from PyMT and PyMT 2c44 mice. (Scale bar = 500 μm.) (Insets) Magnifications of the areas marked by boxes; n = 16 animals per group with 5 sequential slides evaluated per sample (Student’s t test). *P < 0.05; **P < 0.01; ***P < 0.001.

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Δ Fig. 2. Angiogenesis and lymphangiogenesis in primary tumors. Primary tumors were removed from 20-wk-old PyMT and PyMT 2c44 (Δ2c44) mice. (A) Vascular endothelial cells; endomucin+ (EM, green) and lymph endothelial cells (Lyve-1+, red). (Lower) Magnifications of the areas marked by boxes, n = 10 Δ PyMT and n = 12 PyMT 2c44 mice with 5 sequential slides evaluated per tumor. (Scale bars = 50 μm.) (B) FACS analysis of primary tumor digests for endothelial + + + + − − Δ cells (CD31 , CD146 , Ly-6C ) and lymph endothelial cells (CD31 , CD146 , Ly-6C ); n = 9PyMTandn = 13 PyMT 2c44 mice. (C) Expression of lymphatic endothelial cell markers (VEGFC, VEGFR3, and Prox1) and blood endothelial cell markers (VEGFA, VEGFR1, VEGFR2, SOX7, EphB4) in primary tumors; n = 6 animals per group. ns = not significant (Student’s t test). *P < 0.05; **P < 0.01; ***P < 0.001.

+ + + + + as both T cells (CD3 CD4 ) and NKT cells (CD8 CD19 NK1.1 ), in PGES3, whereas the expression of PGES2 was even signifi- Δ Δ and T cell markers were decreased in tumors from PyMT 2c44 mice cantly decreased in PyMT 2c44 tumors (SI Appendix, Fig. S3C). versus PyMT mice. Thus, the deletion of Cyp2c44 in PyMT mice The increase in PG production was specific to the tumors and was increased metastasis at the same time as promoting the immuno- not reflected by generally increased plasma PG levels (SI Appen- suppressive effects of the tumor microenvironment. dix,Fig.S3D). There is a precedent for crosstalk between the CYP and COX PUFA-Derived Lipid Metabolite Profiles in Primary Tumors from PyMT pathways, as at least one CYP (i.e., CYP2S1) was previously 2c44 ω ω and PyMT Mice. Next, the concentrations of -6 and -3 PUFA foundtometabolizePGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E- epoxides and diols were assessed in primary tumors from PyMT Δ trienoic acid (12-HHT), thus attenuating the generation of PGE2 and PyMT 2c44 mice. Although some subtle but significant differ- (15). Cyp2c44 exerted similar effects, as 12-HHT levels were lower ences in epoxide or diol levels were detected, the knockout of in tumors from PyMT2c44 mice versus their PyMT counterparts ω SI Cyp2c44 could not be linked to a clear decrease in specific -3 ( (Fig. 4A). Similarly, PGE2 levels were higher and 12-HHT levels Appendix,Fig.S1)orω-6 (SI Appendix,Fig.S2) PUFA epoxides. −/− Δ lower in liver microsomes from Cyp2c44 versus wild-type mice However, primary tumors from PyMT 2c44 mice contained signif- (Fig. 4B). The reverse was the case when Cyp2c44 was overex- icantly more prostaglandin (PG) E2,PGD2,PGF2α,6-ketoPGF1α, pressed together with the cytochrome reductase in Sf9 cells, where A and PGE1 than primary tumors from PyMT mice (Fig. 4 ). the addition of PGH2 resulted in a decrease in PGE2 but an in- Prostaglandins are produced in a two-step process involving the crease in 12-HHT production (Fig. 4C). The importance of the Δ2c44 initial generation of PGG2 and PGH2 by cyclooxygenase (COX) COX pathway for the enhanced tumor growth in PyMT mice enzymes, followed by an additional metabolic step involving spe- was confirmed by the fact that treating animals with celecoxib de- cific prostaglandin synthases to generate the effector PG (13). creased tumor growth to levels detectable in the PyMT mice. COX expression is known to be elevated in breast cancer (14), and Moreover, the decrease in PG levels was concomitant with a de- both COX1 and COX2 were detectable in all the tumors tested crease in TAMs, as well as angiogenesis and lymph angiogenesis (SI (SI Appendix,Fig.S3A). Although COX RNA levels were elevated Appendix Δ ,Fig.S4). in tumors from PyMT 2c44 versus PyMT mice, there were no consistent differences in expression (SI Appendix,Fig. Consequences of Cyp2c44 Deletion on the PyMT Tumor Proteome. To S3B). Proteomic analyses of similarly sized primary tumors from gain insight into the molecular mechanism or mechanisms un- Δ PyMT and PyMT 2c44 mice (Dataset S1) also revealed no change derlying the differences observed in tumor growth, proteomic

Kesavan et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 5925 Downloaded by guest on October 1, 2021 Δ withtheincreaseofWDFY1inthePyMT 2c44 versus PyMT tu- mors, levels of NRP2 were also increased.

Consequences of PGE2 on WDFY1 Expression in Macrophages. To assess whether a change in the PG profile could be responsible for altered WDFY1 protein expression, similarly sized primary tumors were isolated from 16- to 20-wk old PyMT mice and PyMT mice lacking PGES1, and thus unable to generate PGE2. Although WDYF1 was clearly detectable in samples from the PyMT group, its expression was significantly reduced in tumors from animals lacking mPGES1 (Fig. 6A). Moreover, although the addition of PGE2 to dissociated PyMT tumors increased WDFY1 expression (Fig. 6B), it was unable to increase WDFY1 expression in cultured PyMT cells (Fig. 6C). Given that tumor-associated mac- rophages were highly abundant in the PyMT2c44 tumors and the tumor microenvironment influences the phenotype of macrophages, the consequences of Cyp2c44 deletion on the polarization of bone − − marrow-derived macrophages from wild-type and Cyp2c44 / mice were assessed. WDFY1 and NRP2 were detectable in macrophages − − from wild-type and Cyp2c44 / mice, but their expression was markedly increased in Cyp2c44-deficient macrophages treated with

Fig. 3. Lymphocyte infiltration into primary tumors. Primary tumors were Δ removed from 20-wk-old PyMT and PyMT 2c44 (Δ2c44) mice. (A) F4/80 + (green) and CD11b (red) staining, bar = 20 μm; n = 8 to 12 mice per group with 5 sequential slides evaluated per tumor (Student’s t test). (B)FACS- based quantification of primary tumor digests for monocytes, neutrophils, + resident macrophages (Res. MO), CD45 cells, tumor-associated macro- phages (TAMs), lymphocytes, T cells, and NKT cells relative to the total cell count (total); n = 6 to 14 animals per group (Student’s t test). **P < 0.01; ***P < 0.001.

analyses were performed using similarly sized primary tumors Δ from PyMT and PyMT 2c44 mice (Fig. 5A and Dataset S1) (16). Δ The most altered in PyMT 2c44 versus PyMT mice were the hemoglobin β2 chain, which fits with the increased perfusion of the tumors, vitamin K epoxide reductase (165-fold change), and WD Repeat and FYVE domain containing 1 (WDFY1, 187- fold change vs. background in primary tumor lysates; Fig. 5B). The latter protein was also identified as the most altered protein when microsomes from the tumors were studied (159-fold increase in Δ PyMT 2c44 vs. PyMT tumor microsomes; Dataset S2)(17).A comparison of the expression of different CYP enzymes in these samples revealed no marked differences (i.e., there did not seem to Fig. 4. Cyp2c44 and the prostaglandin profile. (A) Tumors were isolated be a compensatory increase in the expression of another CYP en- from 20-wk-old PyMT and PyMTΔ2c44 (Δ2c44) mice and prostaglandin (PG) = ’ zyme to compensate for the loss of Cyp2c44). WDFY1 is also a levels assessed by LC-MS/MS; n 7 mice per group (Student s t test). (B) PGE2 −/− downstream effector of neuropilin-2 (NRP2), and both proteins and 12-HHT levels in liver microsomes from wild-type (WT) and Cyp2c44 −/− = have been implicated in the endocytic transport of cell surface ( ) mice, n 6 independent experiments, each performed in triplicate (Student’s t test). (C) PGE2 and 12-HHT levels in microsomes from Cyp2c44- epidermal (18, 19). It was possible to con- deficient (CTL) and Cyp2c44-expressing (2c44) Sf9 cells in the presence of C firm the elevated expression of WDYF1 mRNA (Fig. 5 )and PGH2. n = 6 independent experiments, each performed in triplicate (Stu- 2c44 protein (Fig. 5 D and E) in tumors from PyMT mice. Consistent dent’s t test). *P < 0.05; **P < 0.005; ***P < 0.001.

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Δ Fig. 5. Link between Cyp2c44 deletion and WDFY1. (A) Volcano blot showing the proteins most altered in tumors from PyMT 2c44 (Δ2c44) versus PyMT mice. (B) WDFY1 expression (Log2 LFQ intensity) from the proteomics data set; n = 7 animals per group (Student’s t test). (C) WDFY1 mRNA expression from an independent series of tumors; n = 6 animals per group (Student’s t test). (D) WDFY1 and NRP protein levels normalized to nonmuscle myosin (NMM) in similarly sized primary tumors from PyMT and PyMTΔ2c44 mice; n = 6 animals per group (Student’s t test). (E) WDFY1 and NRP protein levels in similarly sized Δ primary tumors from PyMT and PyMT 2c44 mice. (Insets) Magnifications of the areas marked by boxes. (Scale bar = 5 μm; Upper,bar= 2 μm.) (Lower) n = 12 animals per group (Student’s t test). *P < 0.05; ***P < 0.001.

LPS and IFN-γ (to elicit M1 polarization). Alternative activation was increased in the both M1 and M2 polarized macrophages − − − − (M2 polarization) of wild-type and Cyp2c44 / macrophages exerted from Cyp2c44 / mice (SI Appendix, Fig. S6B). This is consistent a weaker effect (Fig. 6D). The increase in WDFY1 and NRP2 with the view that tumor-associated macrophages, which are expression could be reproduced in macrophages from wild-type expanded in primary PyMT2c44 tumors, show markers of both M1 mice after incubation with PGE2 (Fig. 6E). Moreover, treat- and M2 macrophages (20). −/− ing Cyp2c44 macrophages with diclofenac prevented the Recently, endogenous PGE2 production in macrophages was M1 polarization-induced increase in WDFY1 and Nrp2 expression reported to amplify IL-33 production (21), which in turn can (Fig. 6F). polarization itself had marked effects on affect the numbers of myeloid-derived suppressor cells and their Cyp2c44 levels, as although the enzyme was clearly expressed in generation of immunosuppressive TGF-β (22, 23). Certainly, the bone marrow-derived macrophages from wild-type mice, levels were expression of TGF-β and IL-33 was higher in macrophages significantly decreased after M1 or M2 polarization (SI Appendix, lacking Cyp2c44 (SI Appendix, Fig. S6C). This profile is consis- Fig. S5 A and B). In keeping with this finding, macrophage polari- tent with the lack of Cyp2c44 favoring polarization toward zation also increased WDFY1 expression in cells from wild-type tumor-associated macrophages. Functionally, WDFY1 was mice (SI Appendix,Fig.S5C), although the effects were less pro- reported to recruit the signaling adaptor TIR-domain-containing − − nounced than in the Cyp2c44 / macrophages. adapter-inducing IFN-β (TRIF) to Toll-like receptor (TLR) 3 Similar to the isolated tumors, -derived macrophages and TLR4 to potentiate their signaling (24). Indeed, in human − − from Cyp2c44 / mice generated more PG and less 12-HHT monocyte-derived macrophages, preventing the up-regulation of than cells from wild-type mice, after polarization with LPS and WDFY1 using a siRNA approach prevented the LPS- as well as γ IFN- (Fig. 7). Looking at markers of macrophage activation, the the PGE2-induced phosphorylation of IFN regulatory factor 3 expression of interleukin (IL)-1β, IL-10, TNFα, and the inducible (IRF3) and nuclear factor (NF)-κB (Fig. 8). nitric oxide synthase (iNOS) were all significantly increased in − − M1 polarized macrophages from Cyp2c44 / mice versus wild- Discussion type littermates (SI Appendix, Fig. S6A). Interestingly, the ex- The present study was aimed at determining the consequences of pression of Fizz1 (also referred to as RELMα), c-Myc, and early targeting Cyp2c44 on tumor growth and metastasis in a genetic growth response protein 2 were also increased in M2 polarized model of breast cancer. The results revealed that in mice lacking − − macrophages from Cyp2c44 / mice while arginase expression Cyp2c44, primary tumors contained more tumor-associated

Kesavan et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 5927 Downloaded by guest on October 1, 2021 Fig. 6. Role of PGE2 in the regulation of WDFY1 expression. (A) WDFY1 protein expression relative to nonmuscle myosin (NMM) in similarly sized primary − − tumors from PyMT and PyMTxmPGES1 / mice; n = 5 animals per group (Student’s t test). (B) WDFY1 protein expression relative to NMM in dissociated

tumors, treated with solvent (Sol) or PGE2 (100 μmol/L) for 24 h. The blot shown is representative of 3 additional samples. (C) WDFY1 and NMM in cultured PyMT tumor cells, treated with solvent (Sol) or PGE2 (100 μmol/L) for 24 h. The blot shown is representative of 5 additional samples. (D) Expression of WDFY1 and NRP2 mRNA in bone marrow-derived macrophages from wild-type (+/+) and Cyp2c44−/− (−/−) mice treated with solvent (M0), LPS and IFN-γ (M1), IL-4 (M2),

or PGE2 (E2; 100 μmol/L) for 24 h; n = 10 animals per group (two-way ANOVA and Newman-Keuls). (E) WDFY1 and NRP2 mRNA expression in M0 macrophages from wild-type mice treated with solvent or PGE2 (100 μmol/L); n = 6 mice per group (Student’s t test). (F) WDFY1 (red) and NRP2 (green) expression in M0, M1, −/− and M2 polarized bone marrow-derived macrophages from Cyp2c44 mice treated with solvent (Sol) or diclofenac (Diclo, 40 μmol/L) or PGE2 (E2; 100 μmol/L). (Scale bar = 10 μm.) n = 6 mice per group (two-way ANOVA and Newman-Keuls). *P < 0.05; **P < 0.01; ***P < 0.001.

macrophages, and demonstrated marked lymphangiogenesis as lymphangiogenesis, there were also differences in inflammatory well as elevated expression of WDFY1, which is involved in TLR cell infiltration, and significantly more tumor-associated macro- Δ signaling. The effects went hand in hand with increased lymph phages were detected in tumors from PyMT 2c44 mice. The latter node metastasis and pulmonary metastatic growth. Mechanisti- population was of particular interest, as these macrophages cally, the consequences of Cyp2c44 deletion could be attributed generate VEGFC (21) and can transdifferentiate into lymphatic to an increase in PG production, as the enzyme was found to endothelial cell clusters that join existing lymphatic vessels (22, 23). + + metabolize PGH2 to 12-HHT, thus attenuating the production of Indeed, Lyve1 /CD11b macrophages have been proposed to rep- effector PG’s, including PGE2. Further downstream, tumors resent a lymphatic endothelial progenitor cell population (29) and lacking Cyp2c44 demonstrated a more inflammatory phenotype associate with aberrant lymphatics in murine models of ovarian and elevated expression of WDFY1, which has been implicated cancer (30), thus contributing to metastasis (31). Moreover, tumor- in TLR signaling. associated macrophages can suppress T cell responses, and the When the study was initially designed, it was expected that its numbers of T cells were clearly decreased in tumors from Δ main focus would be on a potential benefit of Cyp2c44 down- PyMT 2c44 mice versus PyMT mice. Gender differences have been regulation in the PyMT model. This was assumed on the basis of reported in Cyp2c44-deficient mice, at least for the sensitivity to reports linking CYP-derived EETs to angiogenesis (25, 26), tu- hypoxia-induced pulmonary hypertension (32). However, it was not mor growth (2–5), and tumor metastasis (6). Also, sEH deletion possible to make any statement regarding gender, as our study fo- and higher EET levels were associated with the escape from cused on a model of spontaneous breast cancer in which male mice tumor dormancy in several different cancer models (7). Added to develop tumors much more slowly than females (33). that, Cyp2c44 down-regulation in endothelial cells was reported to Given the role of CYP enzymes in metabolizing PUFAs, the attenuate proliferation and angiogenesis and decrease tumor vas- next step was to identify alterations in the fatty acid epoxide and cularization (8). However, exactly the opposite was observed, and diol profile that could influence inflammatory cells and tumor Δ tumor growth was accelerated in PyMT 2c44 compared with PyMT growth. There was no clear effect of Cyp2c44 deletion on the mice. Moreover, there was a clear increase in lymph node metas- PUFA epoxides or diols assayed, a finding that is consistent with a tases, as well as in the development of pulmonary metastases. The recent report looking at the role of Cyp2c44 in the lung and heart Δ2c44 more aggressive expansion of tumors in PyMT mice was cor- (32). Given the well-established links among PGE2, lymphangio- related with a marked increase in lymphangiogenesis, an effect that genesis (34), tumor growth, and metastases (34, 35), particular at- may, at least partially, underlie the effects on the lymph nodes and tention was paid to alterations in the PG profile and PG-generating lungs, as lymphatics have been linked with metastatic potential enzymes. Interestingly even though there were no consistent (for reviews, see refs. 27 and 28). In addition to the effects on changesinCOX,PGESexpressionandlevelsofPGG2,PGH2,and

5928 | www.pnas.org/cgi/doi/10.1073/pnas.1921381117 Kesavan et al. Downloaded by guest on October 1, 2021 Δ clear differences in infiltrating cells in the tumors from PyMT 2c44 versus PyMT mice indicated that the effects observed may be at- tributable to tumor-associated macrophages. Certainly, Cyp2c44 may be relevant for tumor-associated macrophages, as it has al- ready been allocated a role in the resolution of inflammation (41). Indeed, macrophages lacking Cyp2c44 favored polariza- tion toward tumor-associated macrophages and expressed more WDFY1 and NRP2 than cells isolated from wild-type mice, an effect that could be prevented by COX inhibition and mim- icked by PGE2. The latter observation likely accounts for the marked decrease in WDFY1 expression in tumors from PyMT x − − mPGES1 / mice. Also in macrophages, WDFY1 was linked to TLR signaling as the down-regulation of WDFY1 in macrophages attenuated the LPS-induced phosphorylation of IRF3 and NFκ-B. In the human macrophages studied, PGE2 wasalsoabletoinitiate the phosphorylation of IRF3 and NFκ-B, while at first sight this was unexpected; PGE2 has been reported to elicit the phosphor- ylation of NFκ-B in gastric cancer cells via an EP2 receptor- dependent mechanism (42). Cyp2c44 was chosen for this study because of previous reports indicating that the inhibition/down-regulation of Cyp2c44 may be a strategy to treat cancer (9). However, in the PyMT model studied, exactly the opposite was the case, as tumor growth, as well as metastases, were markedly increased in animals lacking Cyp2c44. A role for Cyp2c44 in cancer certainly fits with a recent study focusing on a woodchuck hepatitis virus/c-Myc model of Fig. 7. Prostaglandin generation by bone marrow-derived macrophages. hepatocellular carcinoma (10). In the latter model, Cyp2c44 +/+ −/− −/− Bone marrow-derived macrophages from wild-type ( ) and Cyp2c44 ( ) expression was found to be inversed during the critical period of γ CELL BIOLOGY mice were incubated in the presence of solvent (M0), LPS and IFN- (M1) or cancer initiation, at a point when inflammatory markers were IL-4 (M2) for 24 h. Thereafter, PGD2, PGE2,PGF2α, PGH2, and 12-HHT levels in the cell supernatant were assessed by LC-MS/MS, n = 10 mice per group (two high and arachidonic acid metabolism was dysfunctional (10). way ANOVA and Newman-Keuls). *P < 0.05; **P < 0.01; ***P < 0.001. The authors of the latter study even proposed that Cyp2c44 is involved in the critical transition from inflammation to carci- noma. The results of the present study certainly support such a the downstream products PGF2α,PGE2,andPGD2 were all in- critical role of decreased Cyp2c44 expression in activated mac- Δ2c44 creased in primary tumors from PyMT mice versus the PyMT rophages, which boosts PGE2 production and cancer develop- group. There was, however, a significant decrease in levels of 12- ment, and highlight that the lipid microenvironment within HHT, which can be generated from PGH2 by some CYP enzymes including the thromboxane A2 synthase (36) and CYP2S1 (15, 37, 38). Cyp2c44 was found to function in a similar manner, as 12-HHT was clearly generated from PGH2 by microsomes derived from Cyp2c44-expressing Sf9 cells. The consequence of the generation of 12-HHT by Cyp2c44 was that PGE2 production was attenuated. It follows that a decrease in Cyp2c44 expression would be expected to result in the shunting of PGH2 into PG production, which fits with the marked increase in PG production in Cyp2c44-deficient ani- mals. Indeed, the finding that PGE2 levels were increased in tumors Δ from PyMT 2c44 mice goes a long way to accounting for the ob- served phenotype, including the increase in lymph angiogenesis. For example, PGE2 has been reported to activate the EP4 receptor on lymphatic endothelial cells and promote lymphangiogenesis Δ2c44 (34). Preventing the increase in PGE2 levels in PyMT mice with celecoxib also effectively prevented tumor tumor growth and infiltration with tumor-associated macrophages, as well as lym- phangiogenesis. Interestingly, consistent with previous reports that celecoxib is metabolized by CYP enzymes, and can thus prevent the metabolism of endogenous substrates (39), we found that treating animals with the COX inhibitor also decreased epoxide generation as well as that of 12-HHT. To further specify the anti-tumor role of Cyp2c44, protein expression was compared in similarly sized tumors from PyMT Fig. 8. Consequences of preventing WDFY1 up-regulation on TLR signaling Δ and PyMT 2c44 mice. One of the proteins most affected by in human monocyte-derived macrophages. Human peripheral blood-derived Cyp2c44 deletion was WDFY1, which plays a role in autophagy macrophages were transfected with a control siRNA or siRNA directed (18, 40) and in the potentiation of TLR3 and TLR4 signaling against WDFY1 (siWDFY1) for 24 h before stimulation with LPS (100 ng/mL) or PGE2 (100 μmol/L) for 60 min. The blots and graphs summarize the ef- (21). WDFY1 was identified as a downstream effector of NRP2 fectiveness of WDFY1 down-regulation and the consequences of WDFY1 (18), and it was possible to confirm that the tumors from down-regulation on the phosphorylation of IRF3 on Ser396 and NFκBon Δ2c44 PyMT mice expressed much higher levels of both proteins. Ser536. NMM = non muscle myosin as loading control; n = 6 independent WDFY1 is known to be expressed in cancer cells (19, 40), but the experiments (one-way ANOVA & Newman-Keuls). **P < 0.01; ***P < 0.001.

Kesavan et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 5929 Downloaded by guest on October 1, 2021 tumors plays a determinant role in immune cell function as well Data Availability. While most of the data are available in the manuscript as the balance between angiogenesis and lymphangiogenesis to and supporting information, the mass spectrometry proteomics data have support metastasis. been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (43), with the dataset identifiers PXD017336 and Materials and Methods PXD017303. All animal experiments were approved by the local government authorities (Regierungspräsidium Darmstadt: FU_1072, FU_1095). Information on ani- ACKNOWLEDGMENTS. The authors are indebted to Katharina Engel-Herbig, Mechtild Pipenbrock, and Jana Meisterknecht for expert technical assistance. mal monitoring, sample collection immunohistochemistry, tumor digestion This work was supported by the Else Kröner-Fresenius-Stiftung (Else Kröner- and FACS analysis, RNA isolation and quantitative real-time PCR, insect cell Fresenius-Graduiertenkolleg stipend to R.K.), the Deutsche Forschungsgemein- (Sf-9) culture and assay of Cyp2c44 activity, monocyte isolation, small in- schaft [SFB-TR 23/3 A06 and SFB 1039/2 A06 (to I.F.), B04 (to B.B.), B06 (to A.W.), terfering RNA, immunoblotting, UPLC-MS/MS-based fatty acid metabolite SFB 815/Z1 (to I.W.); GRK 2336, and the Cardio-Pulmonary Institute (CPI), EXC profiling, proteomics, and statistical analyses is provided in SI Appendix. 2026, Project ID: 390649896], and the Deutsche Krebshilfe (109599).

1. I. Fleming, The pharmacology of the cytochrome P450/soluble epoxide axis in the 24. Y.-H. Hu et al., WDFY1 mediates TLR3/4 signaling by recruiting TRIF. EMBO Rep. 16, vasculature. Pharmacol. Rev. 66, 1106–1140 (2014). 447–455 (2015). 2. T. Yokose et al., Immunohistochemical study of cytochrome P450 2C and 3A in human 25. U. R. Michaelis et al., Cytochrome P450 2C9-derived epoxyeicosatrienoic acids induce non-neoplastic and neoplastic tissues. Virchows Arch. 434, 401–411 (1999). angiogenesis via cross-talk with the epidermal growth factor receptor (EGFR). FASEB 3. J. G. Jiang et al., Cytochrome P450 2J2 promotes the neoplastic phenotype of carci- J. 17, 770–772 (2003). noma cells and is up-regulated in human tumors. Cancer Res. 65, 4707–4715 (2005). 26. U. R. Michaelis, I. Fleming, From endothelium-derived hyperpolarizing factor (EDHF) 4. C. Chen et al., Selective inhibitors of CYP2J2 related to terfenadine exhibit strong to angiogenesis: Epoxyeicosatrienoic acids (EETs) and cell signaling. Pharmacol. Ther. activity against human cancers in vitro and in vivo. J. Pharmacol. Exp. Ther. 329, 908– 111, 584–595 (2006). 918 (2009). 27. Y. Chen et al., A meta-analysis of the relationship between lymphatic microvessel 5. C. Chen et al., Cytochrome P450 2J2 is highly expressed in hematologic malignant density and clinicopathological parameters in breast cancer. Bull. Cancer 100,1–10 diseases and promotes tumor cell growth. J. Pharmacol. Exp. Ther. 336, 344–355 (2013). (2011). 28. G. L. Semenza, Cancer-stromal cell interactions mediated by hypoxia-inducible factors 6. J. G. Jiang et al., Cytochrome p450 promotes human cancer metastasis. promote angiogenesis, lymphangiogenesis, and metastasis. Oncogene 32, 4057–4063 – Cancer Res. 67, 6665 6674 (2007). (2013). 7. D. Panigrahy et al., Epoxyeicosanoids stimulate multiorgan metastasis and tumor 29. S. Ran, K. E. Montgomery, Macrophage-mediated lymphangiogenesis: The emerging – dormancy escape in mice. J. Clin. Invest. 122, 178 191 (2012). role of macrophages as lymphatic endothelial progenitors. Cancers (Basel) 4, 618–657 8. A. Pozzi et al., The anti-tumorigenic properties of peroxisomal proliferator-activated (2012). α – receptor are arachidonic acid epoxygenase-mediated. J. Biol. Chem. 285, 12840 30. B. H. Jeon et al., Profound but dysfunctional lymphangiogenesis via vascular endo- 12850 (2010). thelial growth factor ligands from CD11b+ macrophages in advanced ovarian cancer. 9. N. Skrypnyk et al., PPARα activation can help prevent and treat non-small cell lung Cancer Res. 68, 1100–1109 (2008). cancer. Cancer Res. 74, 621–631 (2014). 31. E. Y. Lin et al., Macrophages regulate the angiogenic switch in a mouse model of 10. M. Li et al., Dysfunction of PLA2G6 and CYP2C44-associated network signals immi- breast cancer. Cancer Res. 66, 11238–11246 (2006). nent carcinogenesis from chronic inflammation to hepatocellular carcinoma. J. Mol. 32. S. R. Joshi et al., Cyp2c44 disruption exacerbated pulmonary hypertension and Cell Biol. 9, 489–503 (2017). heart failure in female but not male mice. Pulm. Circ. 6, 360–368 (2016). 11. E. Y. Lin et al., Progression to malignancy in the polyoma middle T oncoprotein mouse 33. C. T. Guy, R. D. Cardiff, W. J. Muller, Induction of mammary tumors by expression of breast cancer model provides a reliable model for human diseases. Am. J. Pathol. 163, polyomavirus middle T oncogene: A transgenic mouse model for metastatic disease. 2113– 2126 (2003). Mol. Cell Biol. 12, 954–961 (1992). 12. R. Noy, J. W. Pollard, Tumor-associated macrophages: from mechanisms to therapy. 34. P. Nandi et al., PGE2 promotes breast cancer-associated lymphangiogenesis by acti- Immunity 41,49–61 (2014). vation of EP4 receptor on lymphatic endothelial cells. BMC Cancer 17, 11 (2017). 13. P. Patrignani, C. Patrono, Aspirin and cancer. J. Am. Coll. Cardiol. 68, 967–976 (2016). 35. T. J. Kochel, J. C. Reader, X. Ma, N. Kundu, A. M. Fulton, Multiple drug resistance- 14. E. P. Chen, E. M. Smyth, COX-2 and PGE -dependent immunomodulation in breast 2 associated protein (MRP4) exports prostaglandin E2 (PGE2) and contributes to me- cancer. Prostaglandins Other Lipid Mediat. 96,14–20 (2011). tastasis in basal/triple negative breast cancer. Oncotarget 8, 6540–6554 (2017). 15. T. Frömel et al., Cytochrome P4502S1: A novel monocyte/macrophage fatty acid ep- 36. M. Hamberg, J. Svensson, B. Samuelsson, Prostaglandin endoperoxides. A new con- in human atherosclerotic plaques. Basic Res. Cardiol. 108, 319 (2013). cept concerning the mode of action and release of prostaglandins. Proc. Natl. Acad. 16. I. Wittig, I. Fleming, Consequences of Cyp2c44 deletion in the polyoma middle T – (PyMT) breast cancer tumor proteome. PRIDE repository. https://www.ebi.ac.uk/pride/ Sci. U.S.A. 71, 3824 3828 (1974). archive/projects/PXD017336. Deposited 30 January 2020. 37. P. Bui, P. Solaimani, X. Wu, O. Hankinson, 2,3,7,8-Tetrachlorodibenzo-p-dioxin 17. I. Wittig, I. Fleming, Consequences of Cyp2c44 deletion on proteome of the micro- treatment alters eicosanoid levels in several organs of the mouse in an aryl hydro- – somal fraction of primary polyoma middle T (PyMT) tumors. PRIDE repository. https:// carbon receptor-dependent fashion. Toxicol. Appl. Pharmacol. 259, 143 151 (2012). www.ebi.ac.uk/pride/archive/projects/PXD017303. Deposited 28 January 2020. 38. T. W. Madanayake, T. P. Fidler, T. M. Fresquez, N. Bajaj, A. M. Rowland, Cytochrome 18. S. Dutta et al., NRP2 transcriptionally regulates its downstream effector WDFY1. Sci. P450 2S1 depletion enhances cell proliferation and migration in bronchial epithelial Rep. 6, 23588 (2016). cells, in part, through modulation of prostaglandin E(2)synthesis.Drug Metab. Dispos. 19. S. Dutta et al., Neuropilin-2 regulates endosome maturation and EGFR trafficking to 40, 2119–2125 (2012). support cancer cell pathobiology. Cancer Res. 76, 418–428 (2016). 39. Y. A. Siu, M. H. Hao, V. Dixit, W. G. Lai, Celecoxib is a substrate of CYP2D6: Impact on 20. J. A. Van Ginderachter et al., Classical and alternative activation of mononuclear celecoxib metabolism in individuals with CYP2C9*3 variants. Drug Metab. Pharma- phagocytes: Picking the best of both worlds for tumor promotion. Immunobiology cokinet. 33, 219–227 (2018). 211, 487–501 (2006). 40. M. J. Stanton et al., Angiogenic growth factor axis in autophagy regulation. Auto- 21. D. Moussai et al., The human cutaneous squamous cell carcinoma microenvironment phagy 9, 789–790 (2013). is characterized by increased lymphatic density and enhanced expression of 41. D. W. Gilroy et al., CYP450-derived oxylipins mediate inflammatory resolution. Proc. macrophage-derived VEGF-C. J. Invest. Dermatol. 131, 229–236 (2011). Natl. Acad. Sci. U.S.A. 113, E3240–E3249 (2016). 22. K. Maruyama et al., Inflammation-induced lymphangiogenesis in the cornea arises 42. S. Lian et al., Prostaglandin E2 stimulates urokinase-type plasminogen activator re- from CD11b-positive macrophages. J. Clin. Invest. 115, 2363–2372 (2005). ceptor via EP2 receptor-dependent signaling pathways in human AGS gastric cancer 23. S. El-Chemaly et al., Abnormal lymphangiogenesis in idiopathic pulmonary fibrosis cells. Mol. Carcinog. 56, 664–680 (2017). with insights into cellular and molecular mechanisms. Proc. Natl. Acad. Sci. U.S.A. 106, 43. Y. Perez-Riverol et al., The PRIDE database and related tools and resources in 2019: 3958–3963 (2009). Improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

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