Tomohiro Shibata

Tumor-Derived Interleukin-1 Promotes Lymphangiogenesis and Lymph Node Metastasis through M2-Type Macrophages

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.

N-myc downstream-regulated gene 1 promotes tumor inflammatory angiogenesis through JNK activation and autocrine loop of interleukin-1α by human gastric cancer cells.

Background: Expression of N-myc downstream-regulated gene 1 (NDRG1) is significantly correlated with tumor angiogenesis by human gastric cancer. Results: NDRG1 overexpression induced JNK activation and expression of IL-1α and angiogenic CXC chemokines accompanied by tumor angiogenesis. Conclusion: NDRG1 promoted IL-1α-induced tumor angiogenesis through JNK/AP-1 activation. Significance: NDRG1/JNK/IL-1 axis could be a useful target for development of a novel anti-angiogenic strategy in gastric cancer. The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

NDRG1/Cap43/Drg-1 may predict tumor angiogenesis and poor outcome in patients with lung cancer

Objective: Expression of N-myc downstream-regulated gene 1 (NDRG1)/Cap43 is a prognostic indicator of human malignancies according to the tumor type in which it occurs. We investigated how NDRG1/Cap43 could affect tumor growth and angiogenesis in non–small-cell lung cancer (NSCLC) in vivo using an animal experimental model, and also how it could affect tumor angiogenesis and prognosis in NSCLC patients. Methods and Results: Knockdown of NDRG1/Cap43 in lung cancer cells using a specific small interfering RNA resulted in growth rates in culture that were similar to those of counterpart control cells, but decreased tumor growth rates in vivo markedly. Stable NDRG1/Cap43 knockdown did not induce consistent changes in the expression of Epidermal growth factor receptor (EGFR) family proteins and c-Met in two human lung cancer cell lines in vitro. However, cell lines with NDRG1/Cap43 knockdown showed markedly decreased production of the potent angiogenic factors vascular endothelial growth factor-A and interleukin-8. Cells with knockdown of NDRG1/Cap43 showed marked reduction of tumor-induced angiogenesis. Using immunohistochemistry, we examined 182 surgically resected specimens of NSCLC for expression of NDRG1/Cap43 and tumor angiogenesis. High microvessel density in the tumor was significantly associated with nuclear positivity for NDRG1/Cap43 in both adenocarcinoma (p = 0.003) and squamous cell carcinoma (p=0.041). For both adenocarcinoma (p = 0.031) and squamous cell carcinoma (p=0.034), the survival curve of patients negative for nuclear NDRG1/Cap43 expression differed significantly from that of patients who were positive. Conclusion: Therefore, the expression of NDRG1/Cap43 may be predictive of tumor angiogenesis and poor prognosis in NSCLC.

Y-box binding protein-1 contributes to both HER2/ErbB2 expression and lapatinib sensitivity in human gastric cancer cells

Gene amplification of HER2/ErbB2 occurs in gastric cancer and the therapeutic efficacy of the HER2-targeted antibody, trastuzumab, has recently been improved against HER2-positive advanced stomach cancer. Here, we examined whether Y-box-binding protein-1 (YB-1) could selectively control HER2 gene expression and cellular sensitivity to EGF receptor (EGFR) family protein-targeted drugs in human gastric cancer cells. HER2 expression was specifically downregulated by YB-1 silencing using its cognate siRNA, whereas there was less change in the expression of EGFR and HER3. A chromatin immunoprecipitation assay revealed the specific binding of YB-1 to its consensus sequence on the 5′-regulatory region of HER2. YB-1 knockdown induced drug resistance to lapatinib, a dual EGFR and HER2 kinase inhibitor, and also to erlotinib, an EGFR kinase inhibitor. Moreover, phosphorylation of protein kinase B (Akt) was not markedly affected by lapatinib or erlotinib when YB-1 was silenced. Nuclear YB-1 expression was significantly (P = 0.026) associated with HER2 expression, but not with EGFR or HER3, in patients with gastric cancer (n = 111). The YB-1-HER2 axis may therefore be useful for the further development of personalized therapeutics against gastric cancer by HER2-targeted drugs. Mol Cancer Ther; 12(5); 737–46. ©2013 AACR.

The antitumor effect of a novel angiogenesis inhibitor (an octahydronaphthalene derivative) targeting both VEGF receptor and NF-κB pathway

Development of a novel type of angiogenesis inhibitor will be essential for further improvement of therapeutics against cancer patients. We examined whether an octahydronaphthalene derivative, AMF‐26, which was screened as an inhibitor of intercellular adhesion molecule‐1 (ICAM‐1) production stimulated by inflammatory stimuli in vascular endothelial cells, could block angiogenesis in response to vascular endothelial growth factor (VEGF) and/or inflammatory cytokines. Low dose AMF‐26 effectively inhibited the tumor necrosis factor‐α (TNF‐α)‐ or the interleukin‐1β (IL‐1β)‐induced production of ICAM‐1 in human umbilical vascular endothelial cells (HUVECs). We found that the TNF‐α‐induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor, alpha (IκBα) and nuclear translocation of p65 were impaired by AMF‐26 in both endothelial cells and cancer cells. AMF‐26 was found to inhibit the phosphorylation of VEGF receptor 1 (VEGFR1), VEGFR2 and the downstream signaling molecules Akt, extracellular signal‐regulated kinase (ERK)1/2 stimulated by VEGF in HUVECs. Therefore, the VEGF‐induced proliferation, migration and tube formation of vascular endothelial cells was highly susceptible to inhibition by AMF‐26. Oral administration of AMF‐26 significantly blocked VEGF‐ or IL‐1β‐induced angiogenesis in the mouse cornea, and also tumor angiogenesis and growth. Together, our results indicate that AMF‐26 inhibits angiogenesis through suppression of both VEGFR1/2 and nuclear factor‐κB (NF‐κB) signaling pathways when stimulated by VEGF or inflammatory cytokines. AMF‐26 could be a promising novel candidate drug for cancer treatments.

Breast Cancer Resistance to Antiestrogens Is Enhanced by Increased ER Degradation and ERBB2 Expression

Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545-56. ©2016 AACR.

Impaired differentiation of macrophage lineage cells attenuates bone remodeling and inflammatory angiogenesis in Ndrg1 deficient mice

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.

Phosphorylation of mTOR Ser2481 is a key target limiting the efficacy of rapalogs for treating hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

Abstract C13: Direct binding of ERα to YB-1 suppresses HER2 expression in human breast cancer

[Background] ERα and HER2 are two major biomarkers of therapeutic efficacy in breast cancer patients. Y-box binding protein YB-1 is an oncoprotein involved in breast cancer and a transcription factor for HER2/ErbB2. However, regulatory mechanisms of ERα and HER2 expression by YB-1 are largely unknown. In our present study, we examined how nuclear YB-1 regulates expression of HER2 and ERα in breast cancer cells. [Materials and methods] We established YB-1/Tet-On system in which nuclear YB-1 expression was induced by doxycycline, and examined how YB-1 could modulate expression of HER2 and ERα in breast cancer cells. Furthermore, we established the ERα/Tet-On system, in which ERα expression was markedly induced after treatment with doxycycline. To assess whether the YB-1-HER2-ERα correlation was affected by menopause, we analyzed biopsy samples of 116 premenopausal and 114 postmenopausal patients, who had not received any therapeutic drugs, by IHC. [Results] We first examined whether YB-1 regulates expression of ERα and HER2 by using various human breast cancer cell lines, and observed following findings. (1) Nuclear YB-1 overexpression increased HER2 expression and decreases ERα expression, while HER2 expression was suppressed by estradiol and enhanced by anti-estrogen drugs. (2) ERα binding to YB-1 suppressed YB-1 binding to the HER2 promoter region. Furthermore, binding of YB-1 to ERα was enhanced by estradiol and suppressed by anti-estrogen drugs. (3) YB-1 induced proteasomal degradation of ERα when both interact directly. We have next examined whether expression of HER2, nuclear YB-1, and ERα are significantly associated by IHC analysis of biopsy samples of breast cancers. In breast cancers of postmenopausal, but not premenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. [Conclusions] In our study, we presented a new finding that YB-1 promoted proteasomal degradation of ERα by direct interaction and that YB-1-induced HER2 expression was suppressed by ERα. Furthermore, in breast tumors of postmenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. Therefore, this study could contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Kosuke Watari, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Yuichi Murakami, Ryuji Takahashi, Uhi Toh, Ken-ichi Ito, Maki Tanaka, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Direct binding of ERα to YB-1 suppresses HER2 expression in human breast cancer. [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 C13.

Abstract B199: Novel anti-angiogenic cancer therapeutic strategy by targeting differentiated macrophage lineage cells through N-myc downstream regulated gene 1 (NDRG1)

[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.