Kosuke Watari

Inflammatory stimuli from macrophages and cancer cells synergistically promote tumor growth and angiogenesis.

The focus of the present study was whether and how infiltrating macrophages play a role in angiogenesis and the growth of cancer cells in response to the inflammatory cytokine interleukin (IL)‐1β. Lewis lung carcinoma cells overexpressing IL‐1β grew faster and induced greater neovascularization than a low IL‐1β‐expressing counterpart in vivo. When macrophages were depleted using clodronate liposomes, both neovascularization and tumor growth were reduced in the IL‐1β‐expressing tumors. Co‐cultivation of IL‐1β‐expressing cancer cells with macrophages synergistically augmented neovascularization and the migration of vascular endothelial cells. In these co‐cultures, production of the angiogenic factors vascular endothelial growth factor‐A and IL‐8, monocyte chemoattractant protein‐1, and matrix metalloproteinase‐9 were increased markedly. The production of these factors, induced by IL‐1β‐stimulated lung cancer cells, was blocked by a nuclear factor (NF)‐κB inhibitor, and also by the knockdown of p65 (NF‐κB) and c‐Jun using small interference RNA, suggesting involvement of the transcription factors NF‐κB and AP‐1. These results demonstrated that macrophages recruited into tumors by monocyte chemoattractant protein‐1 and other chemokines could play a critical role in promoting tumor growth and angiogenesis, through interactions with cancer cells mediated by inflammatory stimuli. (Cancer Sci 2007; 98: 2009–2018)

Role of macrophages in inflammatory lymphangiogenesis: Enhanced production of vascular endothelial growth factor C and D through NF-κB activation ☆

The close association of inflammation, angiogenesis and cancer progression is now highlighted, and in this study we especially focused on a close association of inflammation and lymphangiogenesis. We found that proinflammatory cytokine, interleukin-1beta (IL-1beta), could induce lymphangiogenesis in mouse cornea through enhanced production of potent lymphangiogenic factors, VEGF-A, VEGF-C and VEGF-D. IL-1beta-induced lymphangiogenesis, but not angiogenesis, was inhibited by administration of a selective anti-VEGF receptor-3 (VEGFR-3) neutralizing antibody. And in mouse cornea we observed recruitment of monocyte/macrophages and neutrophils by IL-1beta implanted cornea. Depletion of macrophages by a bisphosphonate encapsulated in liposomes inhibited this IL-1beta-induced lymphangiogenesis and also up-regulation of VEGF-A, VEGF-C, and VEGF-D. Furthermore, IL-1beta-induced lymphangiogenesis and angiogenesis were suppressed by NF-kappaB inhibition with marked suppression of VEGF-A, VEGF-C, and VEGF-D expression.

Erlotinib Resistance in Lung Cancer Cells Mediated by Integrin β1/Src/Akt-Driven Bypass Signaling

EGF receptor (EGFR) kinase inhibitors, including gefitinib and erlotinib, exert potent therapeutic efficacy in non-small cell lung cancers harboring EGFR-activating mutations. However, most patients ultimately develop resistance to these drugs. Here, we report a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors and the reversal of which could improve clinical outcomes. In erlotinib-resistant lung cancer cells harboring activating EGFR mutations that we established, there was increased expression of Src, integrin β1, α2, and α5 along with enhanced cell adhesion activity. Interestingly, RNAi-mediated silencing of integrin β1 restored erlotinib sensitivity and reduced activation of Src and Akt after erlotinib treatment. Furthermore, Src silencing inhibited Akt phosphorylation and cell growth, with this inhibitory effect further augmented by erlotinib treatment. Increased expression of integrin β1, α5, and/or α2 was also observed in refractory tumor samples from patients with lung cancer treated with erlotinib and/or gefitinib. Together, our findings identify the integrin β1/Src/Akt signaling pathway as a key mediator of acquired resistance to EGFR-targeted anticancer drugs.

Identification of sites subjected to serine/threonine phosphorylation by SGK1 affecting N-myc downstream-regulated gene 1 (NDRG1)/Cap43-dependent suppression of angiogenic CXC chemokine expression in human pancreatic cancer cells

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Y-box binding protein-1 (YB-1) promotes cell cycle progression through CDC6-dependent pathway in human cancer cells

Y-box binding protein-1 (YB-1) plays pivotal roles in acquisition of global drug resistance and cell growth promotion through transcriptional activation of genes for both drug resistance and growth factor receptors. In this study, we investigated whether YB-1 is involved in regulation of the cell cycle and cell proliferation of human cancer cells. Treatment with YB-1 siRNA caused a marked suppression of cell proliferation and expression of a cell cycle related gene, CDC6 by cancer cells. Of cell cycle of cancer cells, S phase content was specifically reduced by knockdown of YB-1. The overexpression of CDC6 abrogated this inhibition of both cell proliferation and S phase entry. ChIP assay demonstrated that YB-1 binds to a Y-box located in the promoter region of the CDC6 gene. Expression of cyclin D1, CDK1 and CDK2 was also reduced with increased expression of p21(Cip1) and p16(INK4A) when treated with YB-1 siRNA. Furthermore, the nuclear YB-1 expression was significantly associated with the level of CDC6 nuclear expression in patients with breast cancer. In conclusion, YB-1 plays an important role in cell cycle progression at G1/S of human cancer cells. YB-1 thus could be a potent biomarker for tumour growth and cell cycle in its close association with CDC6.

Infiltration of thymidine phosphorylase-positive macrophages is closely associated with tumor angiogenesis and survival in intestinal type gastric cancer

Thymidine phosphorylase (TP), an enzyme catalyzing the reversible phospholysis of thymidine, deoxyuridine and their analogs at their respective bases and 2-deoxyribose-1-phosphate, thus promoting angiogenesis, is often expressed in macrophages present in tumor stroma. In this study, we investigated whether infiltration of TP-positive macrophages as well as tumor-associated macrophages affected tumor angiogenesis. TP was expressed in human macrophage-like cells, but not in gastric cancer cells in culture. The expression level of TP, the number of infiltrating CD68+ and CD163+ macrophages, and microvessel density (MVD) in the tumor were further analyzed by immunohistochemistry in 111 patients with gastric cancer. Biostatistical analysis of digitized data obtained by image analysis showed that TP expression was significantly correlated with the number of infiltrating macrophages and MVD in intestinal type gastric cancer (p<0.05). The number of infiltrating macrophages was also correlated with MVD in both the intestinal and diffuse types (p<0.05). An increased number of CD68+ macrophages was significantly associated with poor outcome in patients with intestinal type (p<0.001), but not diffuse type cancer. TP could be a specific marker enzyme that is expressed in tumor-infiltrating macrophages, being associated with tumor angiogenesis and poor prognosis in patients with intestinal-type gastric cancer.

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 (NDRG1) Promotes Metastasis of Human Scirrhous Gastric Cancer Cells through Epithelial Mesenchymal Transition

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition.

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.

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids.