Masato Nakatsuji

COX-2 inhibition alters the phenotype of tumor-associated macrophages from M2 to M1 in ApcMin/+ mouse polyps

Macrophages are a major component of tumor stroma. Tumor-associated macrophages (TAMs) show anti- (M1) or protumor (M2) functions depending on the cytokine milieu of the tumor microenvironment. Cyclooxygenase-2 (COX-2) is constitutively expressed in a variety of tumors including colorectal cancer. TAMs are known to be a major source of COX-2 in human and mice intestinal tumors. COX-2 inhibitor reduces the number and size of intestinal adenomas in familial adenomatous polyposis patients and Apc(Min/+) mice. Although COX-2 inhibitor is thought to regulate cancer-related inflammation, its effect on TAM phenotype remains unknown. Here, we examined the effects of COX-2 inhibition on TAM phenotype and cytokine expression both in vivo and in vitro. Firstly, the selective COX-2 inhibitor celecoxib changed the TAM phenotype from M2 to M1, in proportion to the reduction in number of Apc(Min/+) mouse polyps. Concomitantly, the expression of M1-related cytokine interfron (IFN)-γ was significantly upregulated by celecoxib, although the M2-related cytokines interleukin (IL)-4, IL-13 and IL-10 were not significantly altered. Secondly, IFN-γ treatment attenuated M2 phenotype of mouse peritoneal macrophages and oriented them to M1 even in the presence of M2-polarizing cytokines such as IL-4, IL-13 and IL-10. Thus, our results suggest that COX-2 inhibition alters TAM phenotype in an IFN-γ-dependent manner and subsequently may reduce intestinal tumor progression.

Nardilysin and ADAM proteases promote gastric cancer cell growth by activating intrinsic cytokine signalling via enhanced ectodomain shedding of TNF‐α

Nardilysin (NRDc), a metalloendopeptidase of the M16 family, promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of ADAM proteins. Here, we show the growth‐promoting role of NRDc in gastric cancer cells. Analyses of clinical samples demonstrated that NRDc protein expression was frequently elevated both in the serum and cancer epithelium of gastric cancer patients. After NRDc knockdown, tumour cell growth was suppressed both in vitro and in xenograft experiments. In gastric cancer cells, NRDc promotes shedding of pro‐tumour necrosis factor‐alpha (pro‐TNF‐α), which stimulates expression of NF‐κB‐regulated multiple cytokines such as interleukin (IL)‐6. In turn, IL‐6 activates STAT3, leading to transcriptional upregulation of downstream growth‐related genes. Gene silencing of ADAM17 or ADAM10, representative ADAM proteases, phenocopied the changes in cytokine expression and cell growth induced by NRDc knockdown. Our results demonstrate that gastric cancer cell growth is maintained by autonomous TNF‐α–NF‐κB and IL‐6–STAT3 signalling, and that NRDc and ADAM proteases turn on these signalling cascades by stimulating ectodomain shedding of TNF‐α.

Inhibitory role of Gas6 in intestinal tumorigenesis

Growth arrest-specific gene (Gas) 6 is a γ-carboxyglutamic acid domain-containing protein, which shares 43% amino acid identity with protein S. Gas6 has been shown to enhance cancer cell proliferation in vitro. On the other hand, recent studies have demonstrated that Gas6 inhibits toll-like receptor-mediated immune reactions. Immune reactions are known to affect intestinal tumorigenesis. In this study, we investigated how Gas6 contributes to tumorigenesis in the intestine. Administration of recombinant Gas6 weakly, but significantly, enhanced proliferation of intestinal cancer cells (SW480 and HT29), whereas it suppressed the inflammatory responses of Lipopolysaccharide (LPS)-stimulated monocytes (THP-1). Compared with Gas6(+/+) mice, Gas6(-/-) mice exhibited enhanced azoxymethane/dextran sulfate sodium (DSS)-induced tumorigenesis and had a shorter survival. Gas6(-/-) mice also exhibited more severe DSS-induced colitis. DSS-treated Gas6(-/-) mice showed attenuated Socs1/3 messenger RNA expression and enhanced nuclear factor-kappaB activation in the colonic stroma, suggesting that the target of Gas6 is stromal cells. Bone marrow transplantation experiments indicated that both epithelial cells and bone marrow-derived cells are Gas6 sources. Furthermore, the number of intestinal tumors in Apc(Min) Gas6(-/-) mice was higher than that in Apc(Min) Gas6(+/+) mice, resulting in shorter survival. In a group of 62 patients with advanced colorectal cancer, Gas6 immunoreactivity in cancer tissues was positively correlated with prognosis. Thus, we revealed a unique in vivo inhibitory role of Gas6 during the progression of intestinal tumors associated with suppression of stromal immune reactions. These results suggest a novel therapeutic approach for colorectal cancer patients by regulation of stromal immune responses.

EP4 Receptor–Associated Protein in Macrophages Ameliorates Colitis and Colitis-Associated Tumorigenesis

Prostaglandin E2 plays important roles in the maintenance of colonic homeostasis. The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.

EP4 Receptor–Associated Protein in Macrophages Protects against Bleomycin-Induced Pulmonary Inflammation in Mice

Excessive activation of inflammatory macrophages drives the pathogenesis of many chronic diseases. EP4 receptor–associated protein (EPRAP) has been identified as a novel, anti-inflammatory molecule in macrophages. In this study, we investigated the role of EPRAP using a murine model of bleomycin (BLM)-induced pulmonary inflammation. When compared with wild-type mice, EPRAP-deficient mice exhibited significantly higher mortality, and increased accumulation of macrophages and proinflammatory molecules in the lung 7 d post-BLM administration. Accordingly, the levels of phosphorylated p105, MEK1/2, and ERK1/2 were elevated in EPRAP-deficient alveolar macrophages following BLM administration. In contrast, macrophage-specific EPRAP overexpression decreased the production of proinflammatory cytokines and chemokines, suggesting that EPRAP in macrophages plays a key role in attenuating BLM-induced pulmonary inflammation. As EPRAP is phosphorylated after translation, we examined the role of posttranslational modifications in cellular inflammatory activation using mouse embryo fibroblasts (MEFs) expressing mutant EPRAP proteins. Expression of mutant EPRAP, in which serine–108 and serine–608 were replaced with alanine (EPRAP S108A/S608A), markedly suppressed TNF-α production in LPS-treated MEFs. Conversely, the serine phosphatase 2A (PP2A) inhibitor, cantharidic acid, increased LPS-induced TNF-α production in MEFs expressing wild-type EPRAP, but not in MEFs expressing EPRAP S108A/S608A. Immunoprecipitation analyses demonstrated that EPRAP associated with PP2A in both MEFs and alveolar macrophages from BLM-treated mice. Our data suggest that PP2A dephosphorylates EPRAP, which may be a crucial step in exertion of its anti-inflammatory properties. For these reasons, we believe the EPRAP–PP2A axis in macrophages holds the key to treating chronic inflammatory disorders.

Abstract 4708: Anti-tumor phenotype of tumor-associated macrophages are induced by COX-2 inhibition via the up-regulation of IFN-γ.

BACKGROUNDS AND AIMS Macrophages are a major constituent in tumor stroma, and macrophages infiltrating tumors are called tumor-associated macrophages (TAMs). Macrophages have a functional plasticity depending on the stimuli from microenvironment. M1 macrophages are induced by Th1 cytokines, and exerted anti-tumor function. On the contrary, M2 macrophages are induced by Th2 cytokines, and showed pro-tumor phenotype. TAMs are considered to shift from M1 to M2 during tumor progression. However, the mechanism for this M1/M2 switch is poorly understood. Cyclooxygenase-2 (COX-2)/PGE 2 pathway is up-regulated in a variety of cancers including colorectal cancers, and promotes the cancer progression. COX-2 inhibition shows anti-tumor effect in familial adenomatous polyposis patients and in Apc knockout mice. Although COX-2 inhibition affects cytokine expression profiles and tumor microenvironments, the relationship between COX-2/PGE 2 pathway and TAM phenotypes remains unclear. The aim of this study is to uncover the effects of COX-2 inhibition on TAM phenotypes and cytokine expression. METHODS Cytokine expression and the phenotypes of TAMs infiltrating the stroma of Apc Min/+ mouse polyps were examined by qRT-PCR and immunohistochemistry in terms of M1 and M2 dichotomy. Selective COX-2 inhibitor was administered to Apc Min/+ mice, and the effects of COX-2 inhibition on cytokine expression and TAM phenotypes were evaluated. Furthermore, by using mouse peritoneal macrophages, the direct effects of COX-2 inhibitor on Th1- or Th2-related cytokines on macrophage phenotypes were examined. RESULTS Th2 cytokines were predominant in Apc Min/+ mouse polyps, and TAMs were polarized to M2. Selective COX-2 inhibitor skewed TAM phenotypes from M2 to M1, accompanied by tumor regression in Apc Min/+ mice. Concomitantly, the expression of M1-related cytokine IFN-γ was significantly up-regulated by COX-2 inhibition, although that of M2-related cytokines IL-4, IL-13, and IL-10 was not significantly altered. Administration of COX-2 inhibitor alone was insufficient to alter the phenotypes of mouse peritoneal macrophages from M2 to M1. However, IFN-γ treatment polarized the macrophage phenotypes from M2 to M1 even in the presence of M2-related cytokines IL-4, IL-13, and IL10. CONCLUSIONS Our results suggest that COX-2 inhibition induced anti-tumor (M1) phenotype of TAMs via the up-regulation of IFN-γ, and subsequently, may suppress intestinal tumor progression in Apc Min/+ mouse polyps. Citation Format: Yuki Nakanishi, Masato Nakatsuji, Hiroshi Seno, Tsutomu Chiba. Anti-tumor phenotype of tumor-associated macrophages are induced by COX-2 inhibition via the up-regulation of IFN-γ. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2013-4708