Nune Markosyan

Deletion of cyclooxygenase 2 in mouse mammary epithelial cells delays breast cancer onset through augmentation of type 1 immune responses in tumors

Inhibition of cyclooxygenase (COX) 2, which is associated with >40% of breast cancers, decreases the risk of tumorigenesis and breast cancer recurrence. To study the role of COX-2 in breast cancer, we engineered mice that lack selectively mammary epithelial cell (MEC) COX-2 (COX-2 KO(MEC)). Compared with wild type (WT), MEC from COX-2 KO(MEC) mice expressed >90% less COX-2 messenger RNA (mRNA) and protein and produced 90% less of the dominant pro-oncogenic COX-2 product, prostaglandin (PG) E(2). We confirmed COX-2 as the principle source of PGE(2) in MEC treated with selective COX-2 and COX-1 inhibitors. Tumors were induced in mice using medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene. Breast cancer onset was significantly delayed in COX-2 KO(MEC) compared with WT (P = 0.03), equivalent to the delay following systemic COX-2 inhibition with rofecoxib. Compared with WT, COX-2 KO(MEC) tumors showed increased mRNA for Caspase-3, Ki-67 and common markers for leukocytes (CD45) and macrophages (F4/80). Analysis of multiple markers/cytokines, namely CD86, inducible nitric oxide synthase (iNOS), interleukin-6, tumor necrosis factor α (TNFα) and Tim-3 indicated a shift toward antitumorigenic type 1 immune responses in COX-2 KO(MEC) tumors. Immunohistochemical analysis confirmed elevated expression of CD45, F4/80 and CD86 in COX-2 KO(MEC) tumors. Concordant with a role for COX-2 in restraining M1 macrophage polarization, CD86 and TNFα expression were offset by exogenous PGE(2) in bone marrow-derived macrophages polarized in vitro to the M1 phenotype. Our data reveal the importance of epithelial COX-2 in tumor promotion and indicate that deletion of epithelial COX-2 may skew tumor immunity toward type 1 responses, coincident with delayed tumor development.

Mammary Carcinoma Cell Derived Cyclooxygenase 2 Suppresses Tumor Immune Surveillance by Enhancing Intratumoral Immune Checkpoint Activity

IntroductionSystemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreasesthe risk of breast cancer and its recurrence. However, the biology of COX-2 in themulticellular tumor microenvironment is poorly defined.MethodsMammary tumor onset and multiplicity were examined in ErbB2 transgenic mice thatwere deficient in mammary epithelial cell COX-2 (COX-2MECKO) comparedto wild type (WT) mice.Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, forproliferation, apoptosis, angiogenesis and immune microenvironment. LentiviralshRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breastcancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenicrecipient mice, with or without depletion of CD8+ immune cells.ResultsMammary tumor onset was delayed, and multiplicity halved, in COX-2MECKOmice compared to WT. COX-2MECKO tumors showed decreased expression ofKi67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2,endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumorvascularization. COX-2MECKO tumors contained more CD4+ Thelper (Th) cells and CD8+ cytotoxic immune cells (CTL)consistent with increased immune surveillance. The ratio of Th markersTbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla,a M2 macrophage marker, lower, in COX-2MECKO tumor infiltratingleukocytes compared to WT, suggesting a prevalence of pro-immune Th1over immune suppressive Th2 lymphocytes, and reduced macrophagepolarization to the immune suppressive M2 phenotype. Enhanced immune surveillancein COX-2MECKO tumors was coincident with increased intratumoral CXCL9,a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitoryreceptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was alsodecreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumorsin immune competent mice was markedly suppressed. However, robust growth ofCOX-2KD tumor cells was evident when recipients were depleted of CD8+ cells.ConclusionsThe data strongly support that, in addition to its angiogenic function, tumor cellCOX-2 suppresses intratumoral cytotoxic CD8+ immune cell function,possibly through upregulation of immune checkpoints, thereby contributing to tumorimmune escape. COX-2 inhibition may be clinically useful to augment breast cancerimmunotherapy.

Myeloid Cell COX-2 deletion reduces mammary tumor growth through enhanced cytotoxic T-lymphocyte function

Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8(+) cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8(+), but not CD4(+) cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8(+) CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy.

Heterodimerization With the Prostacyclin Receptor Triggers Thromboxane Receptor Relocation to Lipid Rafts

Objective—Prostacyclin and thromboxane mediate opposing cardiovascular actions through receptors termed IP and TP, respectively. When dimerized with IP, the TP shifts to IP-like function. IP localizes to cholesterol-enriched membrane rafts, but TP and IPTP heterodimer localization is not defined. We examined these receptors’ membrane localization and the role of rafts in receptor function. Methods and Results—Microdomain distribution of IP, TP, and IPTP heterodimers was examined in COS-7 cells by measuring energy transfer from renilla luciferase-fused receptors to fluorescently labeled rafts. IP raft association was confirmed. TP was raft excluded, but redistributed to rafts upon dimerization with IP. Signaling of the IP and IPTP heterodimer, but not TP alone, was suppressed after raft disruption by cholesterol depletion. Cholesterol enrichment also selectively suppressed IP and IPTP function. Native IP and IPTP signaling in smooth muscle cells and macrophages were similarly sensitive to cholesterol manipulation, whereas macrophages from hypercholesterolemic mice displayed suppressed IP and IPTP function. Conclusion—IP and TP function within distinct microdomains. Raft incorporation of TP in the IPTP heterodimer likely facilitates its signaling shift. We speculate that changes in IP and IPTP signaling after perturbation of membrane cholesterol may contribute to cardiovascular disease associated with hypercholesterolemia.

Estrogen promotes luteolysis by redistributing prostaglandin F2α receptors within primate luteal cells.

Prostaglandin F2α (PGF2α) has been proposed as a functional luteolysin in primates. However, administration of PGF2α or prostaglandin synthesis inhibitors in vivo both initiate luteolysis. These contradictory findings may reflect changes in PGF2α receptors (PTGFRs) or responsiveness to PGF2α at a critical point during the life span of the corpus luteum. The current study addressed this question using ovarian cells and tissues from female cynomolgus monkeys and luteinizing granulosa cells from healthy women undergoing follicle aspiration. PTGFRs were present in the cytoplasm of monkey granulosa cells, while PTGFRs were localized in the perinuclear region of large, granulosa-derived monkey luteal cells by mid-late luteal phase. A PTGFR agonist decreased progesterone production in luteal cells obtained at mid-late and late luteal phases, but did not decrease progesterone production by granulosa cells or luteal cells from younger corpora lutea. These findings are consistent with a role for perinuclear PTGFRs in functional luteolysis. This concept was explored using human luteinizing granulosa cells maintained in vitro as a model for luteal cell differentiation. In these cells, PTGFRs relocated from the cytoplasm to the perinuclear area in an estrogen- and estrogen receptor-dependent manner. Similar to our findings with monkey luteal cells, human luteinizing granulosa cells with perinuclear PTGFRs responded to a PTGFR agonist with decreased progesterone production. These data support the concept that PTGFR stimulation promotes functional luteolysis only when PTGFRs are located in the perinuclear region. Estrogen receptor-mediated relocation of PTGFRs within luteal cells may be a necessary step in the initiation of luteolysis in primates.

Targeting COX-2 abrogates mammary tumorigenesis: Breaking cancer-associated suppression of immunosurveillance

Three studies addressed the role of cyclooxygenase-2 (COX-2) in mammary tumorigenesis using epithelial and macrophage COX-2 knockout mice. Deletion of COX-2 in either cell restored, at least partially, tumor immunosurveillance either by changing macrophage function to offset pro-tumor effects, or by attracting more cytotoxic T lymphocytes and natural killer cells to the tumor. These studies suggest benefits from targeted COX-2 selective inhibition in combination with immunotherapies.

Abstract A094: mPGES1 deletion increases tumor susceptibility to immune suppression

The pro-tumor contributions of prostaglandin (PG) E2 are established, as is the clinical efficacy of pharmacological inhibition of COX-2, the rate-limiting enzyme in PGE2 synthesis. In addition to the desired suppression of tumor PGE2, collateral loss of endothelial COX-2-derived PGI2 imposes a cardiovascular hazard that limits clinical use of COX-2 inhibitors. mPGES1, the terminal enzyme in the PGE2 synthesis pathway, is an alternative target to interrupt COX-2-driven events in tumors without elevating cardiovascular risk. We engineered mice transgenic for an activated HER2/neu oncogene to lack mPGES1 globally (mPGES1 KOgl) or only in mammary epithelial cells (mPGES1 KOepi). Abdominal mammary glands from wild type (WT), heterozygous (het), mPGES1 KOgl, and mPGES1 KOepi mice were harvested at 22 weeks of age, paraffin embedded, HE 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A094.

Abstract 928: Deletion of mammary epithelial mPGES1 suppresses tumor development in mice: a possible effect of substrate re-diversion

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Microsomal prostaglandin E2 synthase (mPGES1) is the terminal enzyme in cyclooxygenase (COX)-2-mediated prostaglandin (PG) E2 biosynthesis. The pro-tumor contributions of COX-2 and PGE2 are established as is the clinical efficacy of pharmacological COX-2 inhibition. However, in addition to the desired suppression of tumor PGE2, collateral loss of vascular endothelial COX-2-derived PGI2, an endogenous anti-platelet mediator, imposes a cardiovascular hazard that limits clinical use of COX-2 inhibitors. mPGES1 is a promising alternative target to interrupt COX-2-driven events in tumors without elevating cardiovascular risk. We engineered mice transgenic for an activated HER2/neu oncogene to lack mPGES1 in mammary epithelial cells (MEC; mPGES1 KOMEC ). mPGES1 KOMEC mice and their wild type (WT) littermates were sacrificed at 22 weeks and mammary glands harvested. Glands were sectioned, every 10th slide was HE p=0.002). In addition, in a second cohort of mice followed until tumors were palpable, mPGES1 KOMEC mice trended toward a longer tumor free period. Concordant with these observations, shRNA knock down (KD) of mPGES1 in mammary tumor cells dramatically suppressed their growth as orthotopic tumors in syngeneic immune competent WT hosts - 5 out of 6 non-target shRNA control tumors grew successfully while, in all cases, mPGES1 KD cells failed to grow. By immunohistochemistry, no difference was observed in proliferation or apoptosis between WT and mPGES1 KOMEC spontaneous tumors. Interestingly, and in contrast to our previous study of neu-driven mammary tumors in mice lacking MEC COX-2 (COX-2 KOMEC), vascularization was not different between mPEGS-1 KOMEC and WT spontaneous tumors suggesting divergence in the anti-tumorigenic mechanisms in mPEGS-1 KOMEC and COX-2 KOMEC tumors. Multiple studies of mPGES-1 deletion report re-diversion of COX-2-derived substrate to other prostanoid synthetic pathways, in particular PGD2. By Q-PCR, other than reduced mPGES1 mRNA, terminal prostanoid enzymes were not altered in mPGES1 KOMEC tumors. Interestingly, while the expression of PGE2 receptors EP2 and EP4 trended downwards in mPGES1 KOMEC tumors, the DP2 receptor for PGD2 was significantly increased. Taken together, these data establish mPGES1 as a promising therapeutic target in breast cancer. Further, we propose that, in addition to loss of PGE2, re-diversion of COX-2-derived substrate to the PGD2-DP2 pathway may be involved in delayed tumorigenesis. Citation Format: Nune Markosyan, Emer M. Smyth. Deletion of mammary epithelial mPGES1 suppresses tumor development in mice: a possible effect of substrate re-diversion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 928. doi:10.1158/1538-7445.AM2014-928

Abstract A71: HER2/neu oncogene induced tumors in mammary epithelial cell COX-2 deficient mice have disease delaying modifications of tumor microenvironment.

Cyclooxygenase (COX) 2 expression is associated with more than 40% of breast cancers and its inhibition leads to decreased disease risk and recurrence. COX-2 promotes tumor cell proliferation and survival but also contributes to the interplay between tumor and stromal cells that drives disease progression. We examined the specific role of mammary epithelial cell (MEC) COX-2 in a HER2/neu oncogene transgenic mouse model of breast cancer. Deletion of MEC COX-2 (COX-2MECKO) delayed mammary tumor onset compared to wild type (WT) littermates (tumor onset at age 23 and 29 weeks, respectively, n=12-17, p=0.006), and lowered tumor multiplicity (4.1 versus 7.6, respectively, n=14-18, p=0.05). Similarly, in WT mice, growth of HER2/neu expressing mammary cancer cells as orthotopic tumors was suppressed by knock-down of tumor cell COX-2 with shRNA. Compared to WT, COX-2MECKO tumors showed reduced Ki67, a marker of proliferation, and CD31, an endothelial cell marker, indicating decreased tumor growth and vascularization. Reduced gene expression of the angiogenic factor VEGF-A and its receptor VEGFR-2, as well as VEGFR-3, the receptor for the lymphangiogenic factor VEGF-C, were all lower in COX-2MECKO tumors, consistent with the reported pro-angiogenic function of COX-2 in tumors. Flow cytometric analysis of COX-2MECKO tumors indicated higher percentages of tumor-associated CD3+CD8+ cytotoxic T lymphocytes, CD3-CD8+ natural killer cells, as well as CD3+CD4+ T helper (Th) lymphocytes, compared to WT. Leukocytes were isolated from COX-2MECKO and WT tumors, using microbeads coated with antibodies against the pan-leukocyte marker CD45, and analyzed for phenotypic marker gene expression. A higher ratio of Tbet (Th1 marker)/GATA3 (Th2 marker), and reduced Retnla (a M2 macrophage marker), was evident in leukocytes from COX-2MECKO tumors compared to WT tumors, indicating prevalence of pro-immune Th1 over immune suppressive Th2 lymphocytes, as well as reduced polarization of macrophages to the pro-tumorigenic M2 phenotype. Taken together, these data reveal a less tolerant immune microenvironment in COX-2MECKO tumors, with enhanced cytotoxic and pro-immune type 1, and suppressed pro-tumorigenic and immune suppressive type 2 tumor-associated macrophages and lymphocytes. This restoration of immune surveillance in COX-2MECKO tumors may be explained, at least in part, by decreased gene expression of the T lymphocyte co-inhibitory receptors CTLA4 and PD1, as well as the PD1 ligand PD-L1. Indeed, PD-L1 protein, measured by flow cytometry, was decreased in IFNγ-treated COX-2 knock-down mammary cancer cells in vitro, and was restored by treatment with prostaglandin (PG) E2, the dominant product of COX-2 in tumors. The data strongly support that, in addition to its pro-angiogenic role, tumor cell COX-2 is paracrine mediator that promotes a permissive tumor immune microenvironment and contributes to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy. Citation Format: Nune Markosyan, Chen Edward, Victoire Ndong, Emer Smyth. HER2/neu oncogene induced tumors in mammary epithelial cell COX-2 deficient mice have disease delaying modifications of tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A71.