膜型基质金属蛋白酶-1上调人乳腺癌细胞血管内皮生长因子的表达并诱导肿瘤血管新生

目的 研究膜型基质金属蛋白酶-1(MT1-MMP)在肿瘤血管新生过程中的作用,探讨其诱导肿瘤血管新生的作用途径.方法 应用基因转染方法 ,将MT1-MMP导入人乳腺癌细胞系MCF-7细胞;应用半定量逆转录聚合酶链反应(RT-PCR)和免疫荧光染色,比较转染前后肿瘤细胞血管内皮生长因子(VEGF)表达的变化;通过裸鼠异种移植瘤模型,检测MT1-MMP对肿瘤生长速度、肿瘤组织微血管密度(MVD)和VEGF表达的影响.结果 在MT1-MMP稳定转染后的MCF-7细胞中,VEGF189、VEGF165和VEGF121 mRNA表达水平显著上调(P＜0.001).免疫荧光检测结果 显示,MT1-MMP组的VEGF蛋白免疫荧光强度为93.8±10.3,明显强于MCF-7组(42.9±5.3)和pcDNA3.1组(41.0±5.4,P＜0.001).裸鼠异种移植瘤模型结果 显示,MTI-MMP能够加快肿瘤生长速度.肿瘤组织MVD检测结果 显示,MT1-MMP能够显著提高肿瘤组织的MVD(P＜0.05).免疫组织化学染色结果 显示,VEGF在MT1-MMP组呈强阳性表达.结论 MT1-MMP能够通过上调肿瘤细胞VEGF表达水平而有效诱导肿瘤血管新生.MT1-MMP的这一作用途径可能为临床抗肿瘤研究及抗肿瘤药物开发提供新思路.     

Significance of Membrane Type 1 Matrix Metalloproteinase Expression in Breast Cancer

Expression of matrix metalloproteinases (MMPs) plays an essential role in tumor metastasis and invasion through the degradation of extracellular matrix (ECM). MT1-MMP (membrane type 1 matrix metalloproteinase), a membrane-type MMP, is responsible for the activation of MMP2. In this study the significance of MT1-MMP expression in human breast tumors was investigated by immunocytochemical assay, and its correlation with clinicobiological features was analyzed. MT1-MMP expression was detected in tumor cells and/or stromal cells, and there was a strong correlation between the expressions of MT1-MMP in the two cell types. Out of 183 primary tumors, 103 (56.2%) showed positive staining of MT1-MMP in tumor cells. MT1-MMP expression showed no significant correlation with any of the clinicobiological parameters examined, including hormone receptor status and angiogenesis. In postoperative survival analysis, MT1-MMP expression itself was not a significant prognostic factor. However, in the particular subgroup with the accumulation of thymidine phosphorylase (TP)-positive stromal cells, which have been activated by various stimuli, such as cytokines and hypoxia, MT1-MMP expression had a significant prognostic value. These data suggested that MT1-MMP might function cooperatively with tumor-associated stromal cells for the progression of breast cancer.     

A notch1 ectodomain construct inhibits endothelial notch signaling, tumor growth, and angiogenesis

Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor-induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy.     

Significance of membrane type 1 matrix metalloproteinase expression in breast cancer.

Expression of matrix metalloproteinases (MMPs) plays an essential role in tumor metastasis and invasion through the degradation of extracellular matrix (ECM). MT1-MMP (membrane type 1 matrix metalloproteinase), a membrane-type MMP, is responsible for the activation of MMP2. In this study the significance of MT1-MMP expression in human breast tumors was investigated by immunocytochemical assay, and its correlation with clinicobiological features was analyzed. MT1-MMP expression was detected in tumor cells and/or stromal cells, and there was a strong correlation between the expressions of MT1-MMP in the two cell types. Out of 183 primary tumors, 103 (56.2%) showed positive staining of MT1-MMP in tumor cells. MT1-MMP expression showed no significant correlation with any of the clinicobiological parameters examined, including hormone receptor status and angiogenesis. In postoperative survival analysis, MT1-MMP expression itself was not a significant prognostic factor. However, in the particular subgroup with the accumulation of thymidine phosphorylase (TP)-positive stromal cells, which have been activated by various stimuli, such as cytokines and hypoxia, MT1-MMP expression had a significant prognostic value. These data suggested that MT1-MMP might function cooperatively with tumor-associated stromal cells for the progression of breast cancer.     

Survivin promotes glioma angiogenesis through vascular endothelial growth factor and basic fibroblast growth factor in vitro and in vivo

Survivin is involved in multiple signaling mechanisms in tumor maintenance, and accumulated studies elucidate that knockdown of survivin in endothelial cells could inhibit angiogenesis; however, the role of survivin in tumor cells to regulate tumor-derived angiogenesis remains largely unclear. In the present study 80 cases of brain glioma were chosen and protein expressions of survivin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in glioma cells were investigated by immunohistochemistry (IHC). Human umbilical vein endothelial cells (HUVEC) were cocultured with human glioma U251 wild-type cells, U251 cells survivin silenced, SHG44 wild-type cells, and SHG44 survivin-overexpressing cells, respectively. The proliferation and migration of HUVEC were evaluated by MTT assay and transwell chamber assay. The microvessels density (MVD) marked by CD31 expression in vascular endothelial cells in glioma xenografts in nude mice was detected by IHC. VEGF, bFGF, and PDGF in the aforementioned cells were detected by quantitive PCR (qPCR), Western blot, ELISA, and IHC in vitro and in vivo. The results showed that VEGF immunoreactivity score (IRS), bFGF IRS, and PDGF IRS were all positively correlated with survivin IRS in gliomas, respectively (P < 0.01). Survivin in human glioma cells could significantly promote the proliferation and migration of HUVEC and increase MVD, which could be contributed to survivin-dependent burst of VEGF and bFGF expression, followed by increase of tumor growth and proliferation. In summary, survivin, through upregulation of VEGF and bFGF, plays an essential role during glioma angiogenesis.     

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.     

Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression could be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influenced vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression was induced in an EMMPRIN- and MMP-dependent fashion, and was further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression was inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulated tumor angiogenesis and growth; both were significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, was regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression.     

Effect of hepatocyte growth factor on invasion of prostate cancer cell lines

Hepatocyte growth factor (HGF) was suggested to play an important role in the regulation of mitogenesis, motogenesis, angiogenesis, migration and invasion for various types of cells, and acts through a specific membrane receptor encoded by c-met proto-oncogene. However, the mechanism of the effect of HGF on tumor invasion of prostate cancer cells remains unclear. We investigated the effect of HGF on the invasion of PC-3 and DU-145 prostate cancer cells through a reconstituted basement membrane (Matrigel), the haptotactic migration to fibronectin substrate, the expression of protein and mRNA for matrix metalloproteinases (MMP)-1 and -9, membrane-type 1-MMP (MT1-MMP), urokinase-type plasminogen activator (u-PA) and its receptor (uPAR). HGF increased both Matrigel invasion and haptotactic migration of prostate cancer cells. Furthermore, HGF also increased the production of MMP-1 and -9, MT1-MMP, u-PA and uPAR of these cells. These results suggested that HGF increased the invasive potential of prostate cancer cells probably through enhancement of cell motility and the production of MMPs and u-PA.     

Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.     

Upregulation of matrix metalloproteinases (MMPs) in breast cancer xenografts: a major induction of stromal MMP-13

In human breast cancer (HBC), as with many carcinoma systems, most matrix metalloproteinases (MMPs) are largely expressed by the stromal cells, whereas the tumour cells are relatively silent in MMP expression. To determine the tissue source of the most relevant MMPs, we xenografted HBC cell lines and HBC tissues into the mammary fat pad (MFP) or bone of immunocompromised mice and measured the expression of human and mouse MMP-2, -9, -11, -13, membrane-type-1 MMP (MT1-MMP), MT2-MMP and MT3-MMP by species-specific real-time quantitative RT-PCR. Our data confirm a stromal origin for most tumour-associated MMPs and indicate marked and consistent upregulation of stromal (mouse) MMP-13 and MT1-MMP in all xenografts studied, irrespective of implantation in the MFP or bone environments. In addition, we show increased expression of both human MMP-13 and human MT1-MMP by the MDA-MB-231 tumour cells grown in the MFP compared to in vitro production. MMP protein and activity data confirm the upregulation of MMP mRNA production and indicate an increase in the activated MMP-2 species as a result of tumour implantation. These data directly demonstrate tumour induction of MMP production by stromal cells in both the MFP and bone environments. These xenografts are a valuable means for examining in vivo production of MMPs and suggest that MMP-13 and MT1-MMP will be relevant targets for inhibiting breast cancer progression.     

MT1-MMP及MMP2在人脑胶质瘤中的表达及意义

目的探讨膜型基质金属蛋白酶-1在人脑胶质瘤中的表达及其与肿瘤生物学行为和预后的关系。方法用免疫组织化学S-P法检测48例人脑胶质瘤组织和10例正常人脑组织中MT1-MMP和MMP2的表达。结果高级别胶质瘤组织(Ⅲ~Ⅳ)中MT1-MMP和MMP2的阳性表达率显著高于低级别胶质瘤组织(Ⅰ~Ⅱ),且两者的表达呈显著正相关性。在单因素生存分析中,MT1-MMP的表达与患者预后不良有关。结论MT1-MMP在恶性胶质瘤组织中高表达,与胶质瘤的进展和侵袭密切相关,可作为胶质瘤恶性表型的有用指标;MT1-MMP的阳性表达与患者的预后不良有关。MT1-MMP可能与MMP2的活化密切相关,直接或间接的促进脑胶质瘤的侵袭。     

The chemokine stromal cell derived factor-1 (CXCL12) promotes glioma invasiveness through MT2-matrix metalloproteinase

Chemokines have been found to alter tumor growth and metastasis. We have described previously that a particular chemokine receptor, CXCR4, was predominantly expressed on various glioma cell lines and in resected glioblastoma specimens. Herein, we have tested the ligand of CXCR4, stromal cell derived factor-1alpha (SDF-1alpha, CXCL12), on the response of human glioma cells. We found that SDF-1alpha increased the expression of membrane type-2 matrix metalloproteinase (MT2-MMP), but not the other MT-MMPs, MMP-2 or MMP-9. The SDF-1alpha enhanced MT2-MMP expression was blocked by a CXCR4 antagonist, AMD3100. Functional invasion assays showed that SDF-1alpha stimulated glioma cells to invade through matrigel-coated chambers and this effect was inhibited in glioma cells by the stable downregulation of MT2-MMP expression using small interfering RNA (siRNA). In vivo and at asymptomatic stages following intracerebral implant of cells, mice harboring MT2-MMP siRNA downregulated clones had smaller and less invasive tumors compared with mice implanted with non-specific siRNA control cells. Analyses at symptomatic stages demonstrate that mice with MT2-MMP siRNA clones survive longer than mice harboring control cells. These results highlight MT2-MMP as an effector of CXCR4 signaling in glioma cells, and they reveal the novel role of MT2-MMP in modulating tumor activity.     

Expression of E1AF, an ets-oncogene transcription factor, highly correlates with malignant phenotype of malignant melanoma through up-regulation of the membrane-type-1 matrix metalloproteinase gene

Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.     

Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.

Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.     

Novel approach to analysis of in vitro tumor angiogenesis with a variable-pressure scanning electron microscope: suppression by matrix metalloproteinase inhibitor SI-27

Degradation of basement membrane by metallo-proteinases (MMP) is a critical step in tumor angiogenesis. To evaluate in vitro angiogenesis, several models have been employed, including bovine cornea, fenestrated rat brain, Matrigel, and others. These models did not provide quantitative analysis of capillary formation. The current study aimed for a novel approach to in vitro assay of angiogenesis with a “wet scanning electron microscope (SEM)” to investigate suppression of tumor angiogenesis by the MMP inhibitor, SI-27. The effects of noncytotoxic concentrations of SI-27 (1–100μM) were determined on nonmitogenic vascular endothelial growth factor (VEGF) (10ng/ml)-mediated cell motility and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). Activities of MMP and tissue inhibitor of metalloproteinase (TIMP) were determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the inhibitory effect of SI-27 was examined on in vitro angiogenesis stimulated by supernatants of human glioma cell lines (U87MG, U251MG, or U373MG). In vitro angiogenesis was quantitatively analyzed with a variable-pressure SEM. Cell motility and in vitro angiogenesis by HUVECs were significantly increased by VEGF along with elevated MMP-1 and-2 activity, whereas SI-27 significantly suppressed VEGF-mediated in vitro angiogenesis and inactivated both MMP-1 and MMP-2, but not inhibited cell motility. The angiogenesis promoted by glioma supernatants showed a significant reduction in the presence of SI-27. SI-27, a novel MMP inhibitor, inhibited tumor angiogenesis in vitro. It can be anticipated to prevent tumor growth through its angiosuppressive effect. Quantitative analysis with a variable-pressure SEM is a novel approach to in vitro angiogenesis assay.     

The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines.

Tumor growth is partially dependent on angiogenesis, a process that relies on angiogenic factors. Tumorigenicity of cancer cells is thought to be associated with the production of various angiogenic factors that stimulate or inhibit the rate of endothelial cell migration and proliferation. However, the relative importance of specific individual factors originally studied in cancer cell lines has yet to be determined in vivo. In this study, we examined seven human glioma cell lines for dynamic changes of two major angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and for doubling time and tumorigenicity in nude mice. Various correlation studies demonstrated that in these glioma cell lines, VEGF expression correlated well with RBC density in tumor sections (r2 = 0.804) and with average tumor weight (r2 = 0.987). In contrast, bFGF expression in the observed glioma cell lines did not correlate with tumorigenicity (r2 = 0.001) or with VEGF expression (r2 = 0.255). Furthermore, there was no correlation between doubling time and tumorigenicity in these cell lines (r2 = 0.160). Taken together, these results suggest that VEGF plays a major role in glioma formation and that down-regulation of VEGF, rather than bFGF, would be a more effective choice for glioma gene therapy.     

Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis

Degradation of extracellular matrix (ECM) is one of the first steps in tumor invasion and metastasis. Matrix metalloproteinases (MMP) have been strongly implicated in this step. Membrane-type MMP-1 (MT1-MMP) was first identified as an activator of proMMP-2 expressed on the surface of tumor cells and later, not only ECM macromolecules but also various biologically important molecules, were shown to serve as substrates for MT1-MMP. Accumulated lines of evidence have demonstrated that MT1-MMP expression level is closely associated with invasiveness and malignancy of tumors, suggesting that MT1-MMP is one of the most critical factors for tumor invasion and metastasis. Despite enthusiasm for MMP inhibitors, phase III trials have not yet demonstrated significance in overall survival and side-effects remain an issue. An understanding of the functions of MT1-MMP could supply clues for developing novel therapeutic strategies targeting MT1-MMP.     

The role of thrombin in the neo-vascularization of malignant gliomas: an intrinsic modulator for the up-regulation of vascular endothelial growth factor

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.     

Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis

The multifunctional growth factor scatter factor/hepatocyte growth factor (SF/HGF) and its receptor tyrosine kinase c-Met have emerged as key determinants of brain tumor growth and angiogenesis. SF/HGF and c-Met are expressed in brain tumors, the expression levels frequently correlating with tumor grade, tumor blood vessel density, and poor prognosis. Overexpression of SF/HGF and/or c-Met in brain tumor cells enhances their tumorigenicity, tumor growth, and tumor-associated angiogenesis. Conversely, inhibition of SF/HGF and c-Met in experimental tumor xenografts leads to inhibition of tumor growth and tumor angiogenesis. SF/HGF is expressed and secreted mainly by tumor cells and acts on c-Met receptors that are expressed in tumor cells and vascular endothelial cells. Activation of c-Met leads to induction of proliferation, migration, and invasion and to inhibition of apoptosis in tumor cells as well as in tumor vascular endothelial cells. Activation of tumor endothelial c-Met also induces extracellular matrix degradation, tubule formation, and angiogenesis in vivo. SF/HGF induces brain tumor angiogenesis directly through only partly known mechanisms and indirectly by regulating other angiogenic pathways such as VEGF. Different approaches to inhibiting SF/HGF and c-Met have been recently developed. These include receptor antagonism with SF/HGF fragments such as NK4, SF/HGF, and c-Met expression inhibition with U1snRNA/ribozymes; competitive ligand binding with soluble Met receptors; neutralizing antibodies to SF/HGF; and small molecular tyrosine kinase inhibitors. Use of these inhibitors in experimental tumor models leads to inhibition of tumor growth and angiogenesis. In this review, we summarize current knowledge of how the SF/HGF:c-Met pathway contributes to brain tumor malignancy with a focus on glioma angiogenesis.     

Glioma-derived versican promotes tumor expansion via glioma-associated microglial/macrophages Toll-like receptor 2 signaling

BackgroundAccumulation and infiltration of microglia/brain macrophages around and into glioma tissue promote tumor invasion and expansion. One tumor-promoting mechanism of microglia/brain macrophages is upregulation of membrane type 1 matrix metalloprotease (MT1-MMP), which promotes the degradation of extracellular matrix. MT1-MMP upregulation is induced by soluble factors released by glioma cells activating microglial Toll-like receptor 2 (TLR2).MethodsVersican identified by proteomics was silenced in glioma cells by short interference RNA and short hairpin RNA approaches and studied in vitro and after injection into mouse brains or organotypic brain slices.ResultsThe splice variants V0/V1 of the endogenous TLR2 ligand versican are highly expressed in mouse and human glioma tissue. Versican-silenced gliomas induced less MT1-MMP expression in microglia both in vitro and in vivo, which resulted in smaller tumors and longer survival rates as compared with controls. Recombinant versican V1 induced significantly higher levels of MT1-MMP in wild-type microglia compared with untreated and treated TLR2 knockout microglial cells. Using glioma-injected organotypic brain slices, we found that the impact of versican signaling on glioma growth depended on the presence of microglia. Moreover, we found that TLR2 expression is upregulated in glioma-associated microglia but not in astrocytes. Additionally, an established TLR2 neutralizing antibody reduced glioma-induced microglial MT1-MMP expression as well as glioma growth ex vivo.ConclusionsOur results show that versican released from glioma promotes tumor expansion through glioma-associated microglial/macrophage TLR2 signaling and subsequent expression of MT1-MMP. This signaling cascade might be a novel target for glioma therapies.