Mayumi Ono

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.

Induction of Vascular Endothelial Growth Factor by Tumor Necrosis Factor α in Human Glioma Cells POSSIBLE ROLES OF SP-1

The expression of vascular endothelial growth factor (VEGF) has been implicated in brain tumor angiogenesis, and the promoter region for the VEGF gene contains several SP-1 and AP-1 (c-Fos and c-Jun) binding motifs. Among eight human glioma cell lines, cellular mRNA levels of transcription factors SP-1 and AP-1 (c-Fos and c-Jun) were found to be closely correlated with those of VEGF. VEGF expression appears to be highly susceptible to hypoxia or exogenous cytokines and growth factors. Of various cytokines and growth factors, basic fibroblast growth factor (bFGF), tumor necrosis factor α (TNF-α), and interleukin 1 most potently enhanced VEGF mRNA levels of a glioma cell line, U251. Incubation of the glioma cells with bFGF or TNF-α increased both VEGF and SP-1 mRNA at 30 min and c-Fos mRNA at 1-3 h, over 5-fold. Nuclear run-on assays showed an apparent increase of the transcription of the VEGF gene as well as the SP-1 gene by bFGF or TNF-α. Gel mobility shift assays demonstrated that only SP-1 binding activity was increased 1 h after exposure to bFGF or TNF-α, and also that AP-1, but not SP-1, activity was significantly activated by hypoxia. Mithramycin, an inhibitor of SP-1, at 1-10 nM inhibited activation of the VEGF gene by bFGF or TNF-α but not that by hypoxia. Western blot analysis also demonstrated an increase in cellular amounts of VEGF by TNF-α and a decrease by co-administration with mithramycin. The promoter activity of the VEGF gene, which contains five SP-1 binding sites and one AP-1 binding site but not hypoxia regulatory elements, was enhanced by bFGF or TNF-α but not by hypoxia. The chloramphenicol acetyltransferase assay with VEGF promoter deletion constructs demonstrated that four clusterized SP-1 binding sites in the proximal promoter were essential for the basal transcription and the TNF-α-dependent activation. These data indicated that the expression of the VEGF gene enhanced by bFGF or TNF-α appeared to be mediated in part through the transcription factor SP-1, suggesting a different mechanism from that for hypoxia-induced activation of the VEGF gene.

Activating Mutations in the Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Are Associated with Improved Survival in Gefitinib-Treated Chemorefractory Lung Adenocarcinomas

Purpose: Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. Experimental Design: We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non–small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non–small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Results: Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). Conclusions: The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: Possible involvement of TNFα and IL‐1α

We examined whether macrophage infiltration is associated with angiogenesis in cutaneous melanoma. The numbers of macrophages and microvessels increased significantly with increasing depth of tumor and with tumor angiogenesis. Macrophage infiltration thus appeared to provide a useful diagnostic marker for the progression of cutaneous melanoma. We further examined whether human melanoma cells produce angiogenic factors in response to macrophage‐derived cytokines, tumor necrosis factor alpha (TNFα) and interleukin‐1 alpha (IL‐1α). Treatment of melanoma cells with TNFα and IL‐1α in vitro enhanced the production of interleukin‐8 (IL‐8) and vascular endothelial growth factor (VEGF), and of basic fibroblast growth factor (bFGF) to a lesser degree, in human melanoma cells. Lipopolysaccharide (LPS)‐activated human monocytes enhanced production of IL‐8, VEGF, TNF α, as well as IL‐1α, but not bFGF. Co‐culture of human monocytes and human melanoma cells was also found to significantly enhance production of IL‐8 and VEGF in the absence and presence of LPS, compared with either monocytes or melanoma cells alone. The production of IL‐8 and VEGF from co‐cultured melanoma cells and LPS‐activated monocytes was blocked when anti‐TNF‐α antibody or anti‐IL‐1α antibody was co‐administrated. This is direct evidence that production of the potent angiogenic factors IL‐8 and VEGF from melanoma cells is up‐regulated through TNFα and/or IL‐1α secreted by activated monocytes/macrophages, influencing both tumor growth and angiogenesis in melanomas. Int. J. Cancer 85:182–188, 2000. ©2000 Wiley‐Liss, Inc.

EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLC) to tyrosine kinase inhibitors (TKIs) has been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression and their dysregulation has been implicated in tumorigenesis. To understand the role of microRNAs in TKI-resistant NSCLC, we examined TK receptor-mediated microRNA changes. Here we report that miR-30b/c and miR-221/222, modulated by both EGF and MET receptors, and miR-103, -203, controlled only by MET, play important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition (EMT) of NSCLC cells, in vitro and in vivo, by inhibiting the expression of Bim, APAF-1, PKC-ε and SRC genes. The finding suggests that modulation of specific microRNAs may provide a therapeutic approach for future treatment of NSCLC.

Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined.

A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis

The genetic alteration of p53 is associated with neovascularization during progression of glioma to its more malignant form, glioblastoma. Hence, one or more of the genes transactivated by p53 is likely to function as an angiogenesis inhibitors. We isolated a novel p53-inducible gene that encodes a 1584-amino-acid product containing five thrombospondin type 1 (TSP-type 1) repeats and is specifically expressed in the brain. A recombinant protein corresponding to the TSP-type 1 repeats of this gene product inhibited in vivo neovascularization induced by bFGF in the rat cornea. The expression of this gene, designated BAI1 (brain-specific angiogenesis inhibitor 1) was absent or significantly reduced in eight of nine glioblastoma cell lines, suggesting BAI1 plays a significant role in angiogenesis inhibition, as a mediator of p53.

Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non–small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non–small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.

Molecular links between tumor angiogenesis and inflammation: inflammatory stimuli of macrophages and cancer cells as targets for therapeutic strategy.

Both inflammation and angiogenesis are exacerbated by increased production of chemokines/cytokines, growth factors, proteolytic enzymes, proteoglycans, lipid mediators and prostaglandins. It has been reported that approximately 15–20% of all malignancies are initiated or exacerbated by inflammation. Initiation and progression of cancer are also closely linked to angiogenesis. Infiltration of macrophages is a dramatic and common feature of inflammation, angiogenesis and cancer, and has been recently highlighted in an attempt to develop novel strategies for treating cancer. The recruitment and infiltration of macrophages in the tumor microenvironment activates them to support the malignant progression of cancer cells, and these macrophages are called tumor‐associated macrophages. In a model of experimental angiogenesis using mouse corneas, macrophages infiltrated tissue in response to inflammatory cytokines and produced chemokines and angiogenesis‐promoting factors, such as vascular endothelial growth factor‐A, interleukin‐8, matrix metalloproteinases, prostanoids and reactive oxygen species. Moreover, in a cancer xenograft model, inflammatory stimuli by a representative inflammatory cytokine, interleukin‐1β, enhanced tumor growth and angiogenesis with infiltration and activation of macrophages. Co‐culture of cancer cells with macrophages synergistically stimulated production of various angiogenesis‐related factors when stimulated by the inflammatory cytokine. This inflammatory angiogenesis in both mouse cornea and a tumor model was mediated, in part, by activation of nuclear factor κB and activator protein 1 (Jun/Fos). Administration of either nuclear factor κB‐targeting drugs or cyclooxygenase 2 inhibitors or depletion of macrophages could block both inflammatory angiogenesis and tumor angiogenesis. Thus, both inflammatory and angiogenic responses in tumor stroma could be targets for development of anticancer therapeutic drugs. (Cancer Sci 2008; 99: 1501–1506)

Infiltration of COX-2–expressing macrophages is a prerequisite for IL-1β–induced neovascularization and tumor growth

Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1beta promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1beta-induced angiogenesis and cell inflammation. IL-1beta induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti-Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1beta- or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1-deficient (MCP-1(-/-)) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1beta-induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1beta-induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1beta (LLC/IL-1beta) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1(-/-) mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1beta. Thus, macrophage involvement might be a prerequisite for IL-1beta-induced neovascularization and tumor progression.