Search

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling., Kosuke Watari et al. show that N-myc downstream-regulated gene 1 (NDRG1) stimulates new blood vessel formation that is induced by VEGF-A, using Ndrg1 knockout mice. They find that PLCγ1/ERK signaling mediates this regulation, providing mechanistic insights into how vascular endothelial cells form new vessels.

[Background] Vascular endothelial growth factor (VEGF)-A is a key regulator of tumor angiogenesis that is essential for tumor growth and progression. The unraveling of the precise mechanisms behind VEGF-A-induced tumor angiogenic process will further contribute to development of novel and potent anti-cancer therapeutics. N-myc downstream regulated gene 1 (NDRG1) has been shown to play essential roles in multiple biological processes including embryogenesis, tissue development, cell growth, differentiation, and tumorigenesis. We previously reported that NDRG1 expression levels in cancer cells are closely correlated with tumor angiogenesis and growth (Hosoi et al., Cancer Res., 2009; Murakami et al., J Biol Chem., 2013), and also that NDRG1 promotes tumor angiogenesis through enhanced VEGF-A production by tumor-associated macrophages (Watari et al., Sci Rep., 2016). However, it remains unclear whether NDRG1 expression in vascular endothelial cells (ECs) plays any crucial role in VEGF-A-induced tumor angiogenesis. In our present study, we further ask whether and how NDRG1 in ECs could specifically regulate tumor angiogenesis. We also present our finding of intrinsic importance that NDRG1 functions as an essential factor for VEGF-A-induced angiogenesis. [Methods] NDRG1 deficient mice: The NDRG1 deficient mice on C57BL6 background were purchased from Laboratory Animal Resource Bank, National Institutes of Biomedical Innovation, Health and Nutrition (Osaka, Japan). Isolation of mouse endothelial cells: CD31+ endothelial cells were isolated from mouse lung by magnetic sorting using CD31 MicroBeads. Aortic ring assay: 1 mm mouse aortic rings were embedded in 3-dimensional growth factor reduced Matrigel, treated with or without FGF-2 (50 ng/mL) or VEGF (25 ng/ml), and incubated at 37°C. Vascular length and branching point were measured at day 7. [Results] [1] Analysis of TCGA datasets revealed that NDRG1 expression was positively correlated with VEGF-A expression in patients with various cancer types. [2] In breast cancer patients in Kurume University hospital, NDRG1 was expressed in both cancer cells and ECs. In NDRG1 deficient mice, we observed following experimental results. [3] Both tumor growth and angiogenesis were all suppressed in syngeneic tumors. [4] VEGF-A-induced angiogenesis was specifically impaired in corneal micropocket assay and aortic ring assay, whereas FGF-2 could induce angiogenesis in both assays. [5] NDRG1 formed a complex with PLC-γ, and this complex formation was requisite for the VEGF-A-induced PLCγ/ERK activation in ECs. [Conclusion] We first present an indispensable role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ/ERK activation in ECs. The NDRG1 could be a novel candidate target for development of therapeutics for VEGF-A-induced tumor angiogenesis and other vascular diseases. Citation Format: Kosuke Watari, Tomohiro Shibata, Ai Shinoda, Hideyuki Abe, Akihiko Kawahara, Yuichi Murakami, Eiji Oki, Jun Akiba, Yoshihiko Maehara, Michihiko Kuwano, Mayumi Ono. N-myc downstream regulated gene 1 (NDRG1) is indispensable for VEGF-A-induced tumor angiogenesis through PLCγ/ERK signaling activation in vascular endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 183.

N-myc downstream regulated gene 1 (NDRG1) is a responsible gene for a hereditary motor and sensory neuropathy-Lom (Charcot–Marie–Tooth disease type 4D). This is the first study aiming to assess the contribution of NDRG1 to differentiation of macrophage lineage cells, which has important implications for bone remodeling and inflammatory angiogenesis. Ndrg1 knockout (KO) mice exhibited abnormal curvature of the spine, high trabecular bone mass and reduced number of osteoclasts. We observed that serum levels of macrophage colony-stimulating factor (M-CSF) and macrophage-related cytokines were markedly decreased in KO mice. Differentiation of bone marrow (BM) cells into osteoclasts, M1/M2-type macrophages and dendritic cells was all impaired. Furthermore, KO mice also showed reduced tumor growth and angiogenesis by cancer cells, accompanied by decreased infiltration of tumor-associated macrophages. The transfer of BM-derived macrophages from KO mice into BM-eradicated wild type (WT) mice induced much less tumor angiogenesis than observed in WT mice. Angiogenesis in corneas in response to inflammatory stimuli was also suppressed with decreased infiltration of macrophages. Taken together, these results indicate that NDRG1 deficiency attenuates the differentiation of macrophage lineage cells, suppressing bone remodeling and inflammatory angiogenesis. This study strongly suggests the crucial role of NDRG1 in differentiation process for macrophages.

[Background] Angiogenesis is essential for malignant progression of cancer. Over the last couple of decades, it was highlighted that the progenitor cells derived from bone marrow (BM) differentiated, became part of the tumor stroma, and involved in tumor angiogenesis. In particular, macrophages, which differentiated into tumor-associated macrophages in tumor microenvironment, are known to play crucial roles in tumor angiogenesis. However, the specific molecules and markers of these “angiogenesis-supporting macrophages” have not been fully defined (Qian and Pollard, Cell. 2010). N-myc downstream regulated gene 1 (NDRG1), a gene responsible for a hereditary motor and sensory neuropathy (Lon-Charcot-Marie disease), plays pleiotropic roles in cell proliferation, development, differentiation, and tumorigenesis. We have previously reported that NDRG1 expression levels in cancer cells were closely correlated with tumor angiogenesis (Maruyama et al., Cancer Res. 2006; Hosoi et al., Cancer Res. 2009; Ureshino et al., PLoS One. 2012; Murakami et al., J Biol Chem. 2013). However, how NDRG1 could affect tumor angiogenesis remains unclear. In this study, we asked how NDRG1 could specifically modulate tumor angiogenesis through its differentiation control of macrophage lineage cells by using NDRG1 knock out (KO) mice. [Material and methods] NDRG1 KO mice: The NDRG1 KO mice on C57BL6 background have been kindly donated by Dr. Toshiyuki Miyata (National Cerebral and Cardiovascular Center) (Okuda et al., Mol Cell Biol., 2004). Preparation of BM derived macrophages (BMDMs): BMDMs were obtained by flushing mouse femurs from mice. Cells were seeded in dishes and washed twice 3 hours later. Adherent cells were incubated in DMEM supplemented with 10% FBS and 20ng/ml M-CSF for 7 days. Matrigel plug assay in BM suppression mice: wild type (WT) recipient mice were exposed to 3-Gy sublethal whole-body irradiation to suppress BM and temporarily deplete leukocytes. Seven days after the irradiation, the mice were injected subcutaneously with growth factor-reduced Matrigel containing B16/BL6 cells, with or without BMDMs from WT or NDRG1 KO mice. [Results] In NDRG1 KO mice as compared to their WT counterparts, [1] serum levels of M-CSF and macrophage-related cytokines were all decreased, and BM cells showed much slower growth rates in response to M-CSF in vitro; [2] trabecular bone mass and number of osteoclasts were decreased in vivo; [3] tumor growth and angiogenesis were markedly suppressed, accompanying by decreased infiltration of tumor-associated macrophages; [4] the transfer of BMDMs from KO mice into BM-eradicated WT mice induced much less tumor angiogenesis than observed in WT mice; [5] VEGF-A expression in BMDMs from KO mice were much less than those in cells from WT mice when stimulated by LPS or IL-1β. [Conclusion] We discovered the central role of NDRG1 in differentiation of macrophage lineage cells, which affected bone remodeling and tumor angiogenesis. We will discuss which molecules are specifically regulated by NDRG1 during differentiation processes of monocytes/macrophages into angiogenesis-supporting macrophages, and whether these molecules in macrophages could have potentially important target for anti-angiogenesis cancer therapeutics. Citation Format: Kosuke Watari, Tomohiro Shibata, Hiroshi Nabeshima, Ai Shinoda, Yuichi Fukunaga, Akihiko Kawahara, Kazuyuki Karasuyama, Jun-ichi Fukushi, Yuichi Murakami, Michihiko Kuwano, Mayumi Ono. Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B199.

Widespread metastases including lymph node have often led to the failure of cancer patient treatments. Lymph node metastasis is closely associated with lymphangiogenesis and poor prognosis in lung cancer. Identifying the molecules involved in these processes could help to advance therapeutic strategies for lung cancer patients. We have asked how cancer cells acquire malignant characteristics including lymph node metastasis and lymphangiogenesis, and also how such highly malignant tumor could be therapeutically overcome. Human malignancies are often initiated and promoted by inflammation, in close association with angiogenesis and lymphangiogenesis, while the recruitment of macrophages and neutrophils to the tumor microenvironment activates cells to support cancer progression. Interleukin (IL-1), a representative inflammatory cytokine, is often expressed in human malignancies including lung cancers with poor prognosis. In this study we examined how IL-1 promotes lymph node metastasis and lymphangiogenesis by lung cancer cell lines expressing higher IL-lα. In our present study, we observed following experimental results. [1] Tumor growth, lymph node metastasis, lymphangiogenesis and enhanced infiltration of neutrophils and macrophage were all markedly augmented by the highly metastatic cancer cells. [2] In the highly metastatic tumors, we observed higher expression of IL-1α, IL-1β and CXC chemokines by cancer cells, and also that of VEGF-A and VEGF-C by tumor-associated macrophages, respectively. These tumor-associated macrophages were found to be mainly of the M2 type. [3] Treatment with an IL-1R antagonist (IL-1Ra) significantly blocked expression of CXC chemokines by cancer cells, and also expression of VEGFs by macrophages when co-cultured with cancer cells in vitro. [4] Administration of IL-1Ra significantly suppressed tumor growth, lymph node metastasis and lymphangiogenesis, accompanying by significantly decreased infiltration of M2-type macrophage in the highly metastatic tumors. Together, targeting IL-1/IL-1R signaling pathway and/or M2-type macrophages could be expected to provide useful therapeutic strategy against lymph node metastasis by inflammatory malignant cancer cells. Citation Format: Kosuke Watari, Tomohiro Shibata, Akihiko Kawahara, Yuichi Murakami, Hiroshi Nabeshima, Ai Shinoda, Koichi Azuma, Hiroto Izumi, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Tumor-derived interleukin-1 promotes lymphangiogenesis and lymph node metastasis through activation of M2-type macrophages by lung cancer cells. [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 3662. doi:10.1158/1538-7445.AM2014-3662

Tumors formed by a highly metastatic human lung cancer cell line are characterized by activated signaling via vascular endothelial growth factor (VEGF)-C through its receptor (VEGFR-3) and aggressive lymph node metastasis. In this study, we examined how these highly metastatic cancers acquired aggressive lymph node metastasis. Compared with their lower metastatic counterparts, the highly metastatic tumors formed by this cell line expressed higher amounts of interleukin (IL)-1α, with similarly augmented expression of IL-1α and IL-1β by tumor stromal cells and of VEGF-A and VEGF-C by tumor-associated macrophages. These tumor-associated macrophages were mainly of the M2 type. Administration of a macrophage-targeting drug suppressed the production of these potent angiogenic and lymphangiogenic factors, resulting in decreased tumor growth, angiogenesis, lymphangiogenesis, and lymph node metastasis. In Matrigel plug assays, the highly metastatic cells formed tumors that were extensively infiltrated by M2-type macrophages and exhibited enhanced angiogenesis and lymphangiogenesis. All of these responses were suppressed by the IL-1 receptor (IL-1R) antagonist anakinra. Thus, the IL-1α-driven inflammatory activation of angiogenesis and lymphangiogenesis seems to provide a highly metastatic tumor microenvironment favorable for lymph node metastasis through cross-talk with macrophages. Accordingly, the IL-1R/M2-type macrophage axis may be a good therapeutic target for patients with this form of lung cancer.