Amy M. Fulton

Increased cyclooxygenase-2 (cox-2) expression and activity in a murine model of metastatic breast cancer.

Elevated prostaglandin E2 (PGE2) production is a common feature of human malignancies. This activity has often been attributed to increased metabolic activity of the cyclooxygenase enzymes, although a direct comparison of these 2 parameters i.e., prostaglandin production and cox protein expression, is rarely performed in the same malignant tissue. Using a murine model of metastatic breast cancer, we show that PGE2 levels are positively correlated with increased tumorigenic and metastatic potential. Because prostaglandin synthesis is a product of 2 isoforms of the cyclooxygenase enzyme, we examined the expression and activity of both isoforms. All tumor cell lines examined, regardless of phenotype, express both cox‐1 and cox‐2 proteins in vitro. In contrast to the uniform cox‐2 expression in vitro, only tumors resulting from the transplantation of metastatic cell lines express cox‐2 in vivo. Cox‐1 is detected in both metastatic and nonmetastatic tumors. Thus, this is the first evidence that, in the tumor milieu, cox‐2 expression can be regulated differently in metastatic vs. nonmetastatic lesions. Examination of PGE2 synthesis in vitro reveals that nearly complete inhibition of prostaglandin synthesis occurs in the presence of either indomethacin, which inhibits both isoforms, or NS398, which is selective for the cox‐2 isoform. Thus, even though cell lines express both isoforms, the majority of the prostaglandin synthesis stems from the activity of the inducible, cox‐2 isoform. Likewise, cell growth is inhibited by both indomethacin and NS398 in a dose‐dependent manner, albeit at higher drug concentrations than required to ablate PGE2 synthesis. Despite the inhibition of prostaglandin synthesis, the cox‐2 enzyme levels (protein and mRNA) were increased by either indomethacin or NS398.

Restoring Immune Function of Tumor-Specific CD4+ T Cells during Recurrence of Melanoma

Recurrent solid malignancies are often refractory to standard therapies. Although adoptive T cell transfer may benefit select individuals, the majority of patients succumb to their disease. To address this important clinical dilemma, we developed a mouse melanoma model in which initial regression of advanced disease was followed by tumor recurrence. During recurrence, Foxp3+ tumor-specific CD4+ T cells became PD-1+ and represented >60% of the tumor-specific CD4+ T cells in the host. Concomitantly, tumor-specific CD4+ T effector cells showed traits of chronic exhaustion, as evidenced by their high expression of the PD-1, TIM-3, 2B4, TIGIT, and LAG-3 inhibitory molecules. Although blockade of the PD-1/PD-L1 pathway with anti–PD-L1 Abs or depletion of tumor-specific regulatory T cells (Tregs) alone failed to reverse tumor recurrence, the combination of PD-L1 blockade with tumor-specific Treg depletion effectively mediated disease regression. Furthermore, blockade with a combination of anti–PD-L1 and anti–LAG-3 Abs overcame the requirement to deplete tumor-specific Tregs. In contrast, successful treatment of primary melanoma with adoptive cell therapy required only Treg depletion or Ab therapy, underscoring the differences in the characteristics of treatment between primary and relapsing cancer. These data highlight the need for preclinical development of combined immunotherapy approaches specifically targeting recurrent disease.

Sublethal oxidative stress inhibits tumor cell adhesion and enhances experimental metastasis of murine mammary carcinoma

We have postulated that murine mammary tumor progression is fueled, in part, by tumor-associated macrophages that deliver sub-lethal oxidative stress to tumor cells. In the present study, we determined whether oxidative stress would affect murine mammary tumor cell attachment to laminin and fibronectin, critical functions in the metastatic process. Sublethal oxidative stress generated by exposure of cells to hydrogen peroxide (H2O2, 1–1000 μM/L) inhibited tumor cell attachment to immobilized laminin or fibronectin. This oxidant effect was blocked in the presence of catalase which removes H2O2. The inhibitory effect on attachment was rapid, with significant inhibition occurring at 5 min; total inhibition was achieved at 60 min with 1 mM H2O2. The oxidative stress effect was partially reversible at 20 h post-treatment and occurred at concentrations of H2O2 that do not adversely affect cell viability or growth. Pretreatment of tumor cells with H2O2 or hypoxanthanine and xanthine oxidase (to generate superoxide radical and H2O2) prior to intravenous injection, enhanced experimental lung tumor colony formation. The enhancement of experimental metastatic potential with enzyme-generated oxidative stress was completely reversed by catalase; the H2O2-mediated enhancement was only partially reversed with catalase. Thus, treatments that inhibit tumor cell attachment to extracellular matrix proteins in vitro enhance experimental metastasis in vivo.

Short-term dietary administration of celecoxib enhances the efficacy of tumor lysate-pulsed dendritic cell vaccines in treating murine breast cancer.

Cyclooxygenase‐2 (COX‐2) is a rate‐limiting enzyme in the synthesis of prostaglandins. It is over‐expressed in multiple cancers and has been associated with diminished tumor immunity. Dendritic cells (DCs) are considered candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC‐based vaccines have exhibited minimal effectiveness against established tumors. In this study, we evaluated the effect of short‐term administration of the selective COX‐2 inhibitor celecoxib on the efficacy of DC‐based vaccines in preventing and treating established 4T1 murine mammary tumors. We show that dietary celecoxib alone significantly suppresses the growth of primary tumors and the incidence of lung metastases in the prophylactic setting but is less effective against pre‐established tumors. However, we demonstrate that celecoxib administered after primary tumor establishment synergizes with tumor lysate‐pulsed DC and the adjuvant, GM‐CSF, to improve the antitumor immune response by suppressing primary tumor growth and markedly reducing the occurrence of lung metastases. This triple combination therapy elicits a tumor‐specific immune response evidenced by elevated IFN‐γ and IL‐4 secretion by CD4+ T cells and results in increased infiltration of CD4+ and CD8+ T cells to the tumor site. In addition, dietary celecoxib inhibits angiogenesis evidenced by decreased vascular proliferation within the tumor and serum vascular endothelial growth factor levels. These studies suggest that short‐term celecoxib therapy in combination with DC vaccines may be safely used for treating metastatic breast cancer.

A prostaglandin E (PGE) receptor EP4 antagonist protects natural killer cells from PGE2-mediated immunosuppression and inhibits breast cancer metastasis

Cyclooxygenase-2 is frequently upregulated in epithelial tumors and contributes to poor outcomes in multiple malignancies. The COX-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1–4). Using a novel small molecule EP4 antagonist (RQ-15986) and a syngeneic murine model of metastatic breast cancer, we determined the effect of EP4 blockade on innate immunity and tumor biology. Natural killer (NK)-cell functions are markedly depressed in mice bearing murine mammary tumor 66.1 or 410.4 cells owing to the actions of PGE2 on NK cell EP4 receptors. The EP4 agonist PGE1-OH inhibits NK functions in vitro, and this negative regulation is blocked by RQ-15986. Likewise, the treatment of tumor-bearing mice with RQ-15986 completely protected NK cells from the immunosuppressive effects of the tumor microenvironment in vivo. RQ-15986 also has direct effects on EP4 expressed by tumor cells, inhibiting the PGE2-mediated activation of adenylate cyclase and blocking PGE2-induced tumor cell migration. The pretreatment of tumor cells with a non-cytotoxic concentration of RQ-15986 inhibited lung colonization, a beneficial effect that was lost in mice depleted of NK cells. The oral administration of RQ-15986 inhibited the growth of tumor cells implanted into mammary glands and their spontaneous metastatic colonization to the lungs, resulting in improved survival. Our findings reveal that EP4 antagonism prevents tumor-mediated NK-cell immunosuppression and demonstrates the anti-metastatic activity of a novel EP4 antagonist. These observations support the investigation of EP4 antagonists in clinical trials.

Antagonism of the prostaglandin E receptor EP4 inhibits metastasis and enhances NK function

Cyclooxygenase-2 (COX-2) is associated with aggressive breast cancers. The COX-2 product prostaglandin E2 (PGE2) acts through four G-protein-coupled receptors designated EP1–4. Malignant and immortalized normal mammary epithelial cell lines express all four EP. The EP4 antagonist AH23848 reduced the ability of tumor cells to colonize the lungs or to spontaneously metastasize from the mammary gland. EP4 gene silencing by shRNA also reduced the ability of mammary tumor cells to metastasize. Metastasis inhibition was lost in mice lacking either functional Natural Killer (NK) cells or interferon-γ. EP4 antagonism inhibited MHC class I expression resulting in enhanced ability of NK cells to lyse mammary tumor target cells. These studies support the hypothesis that EP4 receptor antagonists reduce metastatic potential by facilitating NK-mediated tumor cell killing and that therapeutic targeting of EP4 may be an alternative approach to the use of COX inhibitors to limit metastatic disease.

Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5 microM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumor cell lines.

Promoter methylation regulates cyclooxygenase expression in breast cancer.

IntroductionOverexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract.MethodsUsing bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential.ResultsWe showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis).ConclusionsThese results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype.

Prospects of Controlling Breast Cancer Metastasis by Immune Intervention

Different immune effectors control distinct steps in tumor metastasis; T cells inhibit the growth of primary and metastatic tumors, while NK control tumor cells in transit. Vaccination with DNA encoding Her-2 which is expressed on primary and metastasized breast cancer cells induced both humoral and cellular immunity to inhibit tumor growth. Vaccination efficacy can be amplified by depleting CD4;+CD25;+Foxp3;+ regulatory T cells (Treg), but the risk of inducing autoimmunity warrants new strategies to selectively amplify anti-tumor immunity when modulating Treg. In the tumors, the major cyclooxygenase (COX)-2 product is prostaglandin E2(PGE2) which suppresses T and NK cells while amplifying Treg. These cellular responses to PGE2 are mediated through four E prostanoid (EP) receptors. Cox inhibitors and EP antagonists enhance NK activity to inhibit tumor metastasis; but they may down regulate MHC class I expression. Since T and NK cells have opposite requirements for MHC class I expression, their relative contribution to cancer control may vary with the stage of the disease. To enhance tumor infiltration by immune effectors, the role of CXCL9 is discussed. The complex nature of tumor metastasis necessitates a comprehensive approach to achieve successful immune intervention.

Multiple drug resistance-associated protein 4 (MRP4), prostaglandin transporter (PGT), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) as determinants of PGE2 levels in cancer.

The cyclooxygenase-2 (COX-2) enzyme and major lipid product, prostaglandin E2 (PGE2) are elevated in many solid tumors including those of the breast and are associated with a poor prognosis. Targeting this enzyme is somewhat effective in preventing tumor progression, but is associated with cardiotoxic secondary effects when used chronically. PGE2 functions by signaling through four EP receptors (EP1-4), resulting in several different cellular responses, many of which are pro-tumorigenic, and there is growing interest in the therapeutic potential of targeting EP4 and EP2. Other members in this signaling pathway are gaining more attention. PGE2 is transported out of and into cells by two unique transport proteins. Multiple Drug Resistance-Associated Protein 4 (MRP4) and Prostaglandin Transporter (PGT) modulate PGE2 signaling by increasing or decreasing the levels of PGE2 available to cells. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) metabolizes PGE2 and silences the pathway in this manner. The purpose of this review is to summarize the extensive data supporting the importance of the COX-2 pathway in tumor biology with a focus on more recently described pathway members and their role in modulating PGE2 signaling. This review describes evidence supporting roles for MRP4, PGT and 15-PGDH in several tumor types with an emphasis on the roles of these proteins in breast cancer. Defining the importance of these latter pathway members will be key to developing new therapeutic approaches that exploit the tumor-promoting COX-2 pathway.