The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma

Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen-activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors.     

Knockdown of VEGF receptor-1 (VEGFR-1) impairs macrophage infiltration, angiogenesis and growth of clear cell renal cell carcinoma (CRCC)

Angiogenesis is essential for tumor growth and metastasis. VEGF has been shown to be a central player in this process. The biological activity of VEGF is mainly mediated by two tyrosine kinase receptors, VEGFR-1 and VEGFR-2. While increasing evidence suggests that VEGF/VEGFR-1 signaling is crucial for tumor angiogenesis, its molecular mechanism is not well understood. Here we show that VEGFR-1 knockdown dramatically inhibits tumor growth. This inhibition is associated with significant decrease of tumor VEGF levels and tumor angiogenesis as well as an increased tumor necrosis. Moreover, we demonstrate that VEGF in CRCC tumors is mainly produced by tumor stromal cells instead of the tumor cells themselves. It has been shown that macrophages constitute a significant part of tumor stromal cells and produce a large amount of VEGF. We therefore examined the macrophage infiltration in the xenograft tumors. Remarkably, VEGFR-1 knockdown attenuates the tumor macrophages infiltration. To understand the mechanism, we investigated the impact of VEGFR-1 knockdown on the expression of monocyte chemoattractant protein-1 (MCP-1), one of the main chemoattractants for macrophages. Significantly, VEGFR-1 knockdown inhibits MCP-1 expression of CRCC cells. Taken together, these data indicate that VEGF/VEGFR-1 signaling plays an essential role in initiating tumor angiogenesis by regulating MCP-1 expression, which in turn, attracts macrophages infiltration and VEGF production. Thus, these studies suggest that blockade of VEGFR-1 function may provide a tumor-specific, VEGF-based therapeutic strategy for treatment of CRCC.     

Vascular endothelial growth factor receptor-2: its unique signaling and specific ligand,VEGF-E

Vascular endothelial growth factor receptor-2 (VEGFR-2/KDR/Flk-1) is a high-affinity receptor for vascular endothelial growth factor-A (VEGF-A), and mediates most of the endothelial growth and survival signals from VEGF-A. VEGFR-2 has a typical tyrosine kinase receptor structure with seven immunoglobulin (Ig)-like domains in the extracellular region, as well as a long kinase insert in the tyrosine kinase domain. It utilizes a unique signaling system for DNA synthesis in vascular endothelial cells, i.e. a phospholipase Cγ-protein kinaseC-Raf-MAP kinase pathway. Although VEGF-A binds two receptors, VEGFR-1 and -2, a newly isolated ligand VEGF-E (Orf-virus-derived VEGF) binds and activates only VEGFR-2. Transgenic mice expressing VEGF-E_NZ-7 showed a dramatic increase in angiogenesis with very few side effects (such as edema and hemorrhagic spots), suggesting strong angiogenic signaling and a potential clinical utility of VEGF-E. VEGF family members bear three loops produced via three intramolecular disulfide bonds, and cooperation between loop-1 and loop-3 is necessary for the specific binding and activation of VEGFR-2 for angiogenesis. As it directly upregulates tumor angiogenesis, VEGFR-2 is an appropriate target for suppression of solid tumor growth using exogenous antibodies, small inhibitory molecules and in vivo stimulation of the immune system.     

Mechanisms of resistance to vascular endothelial growth factor blockade

Angiogenesis is essential for the growth of primary tumors and for their metastasis. This process is induced by factors, such as vascular endothelial growth factors (VEGFs), that bind to transmembrane VEGF receptors (VEGFRs). VEGF-A is the primary factor involved with angiogenesis; it binds to both VEGFR-1 and VEGFR-2. The inhibition of angiogenesis by obstructing VEGF-A signaling has been investigated as a method to treat solid tumors, but the development of resistance to this blockade has complicated treatment. The major mechanisms of this resistance to VEGF-A blockade include signaling by redundant receptors, such as the fibroblast growth factors, angiopoietin-1, ephrins, and other forms of VEGF. Other major mechanisms of resistance are increased metastasis of hypoxia-resistant tumor cells, recruitment of cell types capable of promoting VEGF-independent angiogenesis, and increased circulation of nontumor proangiogenic factors. Additional mechanisms of resistance to VEGF-A blockade include heterogeneity of responsiveness among tumor cells, use of anti-VEGF-A agents at insufficient doses or for insufficient duration, altered sensitivity to anti-VEGF-A agents by mutations in endothelial cells or vascular remodeling, maintenance of vascular sleeves that allow for easy regrowth of tumor vasculature upon discontinuation of therapy, vascular cooption, and intussusceptive angiogenesis. An understanding of these mechanisms may lead to the development of targeted therapies that overcome this resistance. Some of these approaches include the combined inhibition of redundant angiogenic pathways, proper patient selection for various therapies based on gene expression profiles, blockade of cellular migration by inhibition of colony-stimulating factor, or the use of agents to disrupt vascular architecture.     

Targeting Angiogenesis in Cancer Therapy: Moving Beyond Vascular Endothelial Growth Factor

Angiogenesis, or the formation of new capillary blood vessels, occurs primarily during human development and reproduction; however, aberrant regulation of angiogenesis is also a fundamental process found in several pathologic conditions, including cancer. As a process required for invasion and metastasis, tumor angiogenesis constitutes an important point of control of cancer progression. Although not yet completely understood, the complex process of tumor angiogenesis involves highly regulated orchestration of multiple signaling pathways. The proangiogenic signaling molecule vascular endothelial growth factor (VEGF) and its cognate receptor (VEGF receptor 2 [VEGFR-2]) play a central role in angiogenesis and often are highly expressed in human cancers, and initial clinical efforts to develop antiangiogenic treatments focused largely on inhibiting VEGF/VEGFR signaling. Such approaches, however, often lead to transient responses and further disease progression because angiogenesis is regulated by multiple pathways that are able to compensate for each other when single pathways are inhibited. The platelet-derived growth factor (PDGF) and PDGF receptor (PDGFR) and fibroblast growth factor (FGF) and FGF receptor (FGFR) pathways, for example, provide potential escape mechanisms from anti-VEGF/VEGFR therapy that could facilitate resumption of tumor growth. Accordingly, more recent treatments have focused on inhibiting multiple signaling pathways simultaneously. This comprehensive review discusses the limitations of inhibiting VEGF signaling alone as an antiangiogenic strategy, the importance of other angiogenic pathways including PDGF/PDGFR and FGF/FGFR, and the novel current and emerging agents that target multiple angiogenic pathways for the treatment of advanced solid tumors.

Implications for Practice:

Significant advances in cancer treatment have been achieved with the development of antiangiogenic agents, the majority of which have focused on inhibition of the vascular endothelial growth factor (VEGF) pathway. VEGF targeting alone, however, has not proven to be as efficacious as originally hoped, and it is increasingly clear that there are many interconnected and compensatory pathways that can overcome VEGF-targeted inhibition of angiogenesis. Maximizing the potential of antiangiogenic therapy is likely to require a broader therapeutic approach using a new generation of multitargeted antiangiogenic agents.     

Vascular endothelial growth factor (VEGF)-C differentially affects tumor vascular function and leukocyte recruitment: role of VEGF-receptor 2 and host VEGF-A

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.     

Different cascades in the signaling pathway of two vascular endothelial growth factor (VEGF) receptors for the VEGF-mediated murine hepatocellular carcinoma development

It has been shown that the interaction between the potent angiogenic factor; the vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2), plays a pivotal role in tumor development, including hepatocellular carcinoma (HCC). However, the properties of the respective VEGF receptor in the signaling transduction pathway of VEGF-mediated effects in HCC have not been elucidated yet. The aim of this study was to examine the respective signaling pathway of two VEGFRs in the VEGF-mediated murine HCC development and angiogenesis. We examined the signaling cascades of VEGFR-1 and VEGFR-2 in the VEGF-mediated HCC development in combination with a retroviral tetracycline (tet)-regulated (Retro-Tet) gene expression system, which can manipulate the gene expression in vivo by providing tet in the drinking water, as well as VEGFR-1 and VEGFR-2 specific neutralizing monoclonal antibodies (R-1mAb and R-2mAb, respectively). Both R-1mAb and R-2mAb significantly suppressed the VEGF-mediated tumor growth associated with reduction of the tumoral neovascularization, and the combination treatment with both mAbs almost completely attenuated the tumor development and angiogenesis. The protein kinase-C (PKC) and MEK1/2 activities in the tumor were markedly attenuated by treatment with R-2mAb, whereas R-1mAb did not alter these activities. These results suggested that both VEGFR-1 and VEGFR-2 play important roles, and lie in the different signaling cascades by which VEGF augments HCC development and angiogenesis.     

Expression and regulation of neuropilin-1 in human astrocytomas

Vascular endothelial growth factor (VEGF), through activation of its endothelial receptors VEGFR-1 and VEGFR-2, is an important positive modulator of tumor angiogenesis and edema in solid tumors such as malignant astrocytomas. Neuropilin-1 (Npn-1) is a transmembrane receptor expressed by both endothelial and non-endothelial cells, including tumor cells. Npn-1 has been postulated to function as a co-factor in activation of the biologically relevant VEGFR-2, by the most abundant VEGF165 isoform. However, the function of Npn-1 in normal and pathological angiogenesis, its expression pattern in relation to VEGF in tumors such as astrocytomas and whether it is similarly or differentially regulated compared to VEGF remain unknown. In our study, the expression pattern of Npn-1 and VEGF by human astrocytoma cell lines and specimens was closely correlated and associated with malignant astrocytomas. Mitogens, such as epidermal growth factor and activation of p21-Ras, previously demonstrated to be relevant in astrocytoma proliferation and induction of VEGF, also induce Npn-1 expression. Hypoxia, the main physiological inducer of VEGF expression, decreased Npn-1 expression. Increased Npn-1 expression was also demonstrated in a transgenic mouse astrocytoma model. Astrocytomas are an ideal system for furthering our understanding of the functional relevance, if any, of Npn-1 in tumor angiogenesis.     

Ang-2、VEGF及VEGFR3在喉鳞癌中的表达及其临床意义

 目的 研究喉鳞癌组织中促血管生成素-2(Angiopoietin-2,Ang-2)、血管内皮生长因子（vascular endothelial growth factor,VEGF）及受体-3（VEGFR-3）的表达及其与临床病理学特征间的关系。 方法 采用免疫组织化学方法检测喉鳞癌患者癌组织及癌旁正常组织中促血管生成素-2（Ang 2）、血管内皮生长因子（VEGF）及血管内皮生长因子受体-3（VEGFR-3）的表达。 结果 Ang-2、VEGF及VEGFR-3在喉鳞癌组织中表达显著高于癌旁正常组织（P均<0.01）;Ang-2表达在肿瘤病理分化程度及有无淋巴结转移上差异无统计学意义（P>0.05）, VEGF与VEGFR 3的表达在病理分化程度上的比较差异无统计学意义（P>0.05）,而有无淋巴结转移上的比较差异有统计学意义（P<0.05）; Pearson积差相关性分析：喉鳞癌组织中Ang-2与VEGF的表达呈正相关（r=0.8193,P<0.01）;VEGF与VEGFR-3的表达也呈正相关（r=0.7365,P<0.01）。 结论 Ang-2、VEGF及VEGFR-3与肿瘤的血管生成和成熟密切相关,它们在喉鳞癌组织中的过表达可能在喉鳞癌的发展过程中起重要作用。     

The histone deacetylase inhibitor NVP-LAQ824 inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-alpha and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.     

AAV2-mediated gene transfer of VEGF-Trap with potent suppression of primary breast tumor growth and spontaneous pulmonary metastases by long-term expression

Vascular endothelial growth factor (VEGF) is an important signaling protein and a predominant mediator of angiogenesis in tumor growth and metastasis. Therefore, antagonism of the VEGF pathway results in inhibition of abnormal angiogenesis, then suppression of tumor growth and metastasis. VEGF-Trap, a high-affinity soluble decoy receptor, is currently in phase II clinical trails, and has demonstrated more efficacy in different types of solid tumors by intravenous injection every two weeks. In our study, we used recombinant AAV2 as a delivery vehicle to achieve long-lasting expression of VEGF Trap protein in a mouse model for the first time. We report that AAV2-VEGF-Trap can be safely administered and sustained expression in?vivo via a single intravenously administration, simultaneously suppressing primary tumor growth and preventing the pulmonary metastases of 4T1 tumors. Decreased microvessel density and increased tumor cell apoptosis were observed in the treatment group. AAV2-VEGF-Trap can obviously decrease not only the concentration of VEGF in sera, but also the concentration of other angiogenic factors, such as aFGF, bFGF, angiopoietin-1 and others. These studies suggest that AAV-mediated long-term expression of VEGF-Trap is a useful and safe tool to block tumor progression and inhibit spontaneous pulmonary metastases.     

Modulation of the angiogenesis response through Ha-ras control,placenta growth factor,and angiopoietin expression in mouse skin carcinogenesis

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.     

A receptor for vascular endothelial growth factor that stimulates endothelial apoptosis.

Vascular endothelial growth factor (VEGF) is a dimeric angiogenic factor that is overexpressed by many tumors and stimulates tumor angiogenesis. VEGF initiates signaling by dimerizing the receptors VEGFR-1 and VEGFR-2. The Fas receptor stimulates apoptosis, and artificial dimerization of the Fas cytoplasmic domain has been shown to induce apoptosis. We constructed a chimeric receptor (VEGFR2Fas) combining the extracellular and transmembrane domains of VEGFR-2 with the cytoplasmic domain of Fas receptor. When VEGFR2Fas was stably expressed in endothelial cells in vitro, treatment with VEGF rapidly induced cell death with features characteristic of Fas-mediated apoptosis. These findings demonstrate that VEGFR2Fas functions as a VEGF-triggered death receptor and raise the possibility that introduction of VEGFR2Fas into tumor endothelium or tumor cells in vivo may convert tumor-derived VEGF from an angiogenic factor into an antiangiogenesis agent.     

Expression of VEGF and its receptor genes in intracranial schwannomas

Vascular endothelial growth factor (VEGF) is considered to be a major regulator of angiogenesis in various brain tumors. In this study, we determined the expression levels of VEGF, and vascular endothelial growth factor receptor (VEGFR)-1 and -2 mRNA in 46 intracranial schwannomas by quantitative real-time PCR, and correlated these with various clinical factors or other molecular markers. We found that these tumors expressed significant amounts of VEGF mRNA in comparison with other brain tumors, including malignant gliomas and meningiomas. In addition, we performed immunohistochemical studies for VEGF and VEGFR-1, and confirmed that these tumors prominently express these proteins. The expression levels of VEGF and VEGFR-1 mRNA in recurrent tumors were higher than those in primary tumors. When we divided patients into two groups according to VEGF mRNA expression in the tumor, there was no significant difference in patient age, gender, or cranial nerves of origin between groups; however, the tumor volume tended to be larger in the high VEGF group than in the low VEGF group. The levels of VEGFR-1 mRNA and neurofibromatosis-2 mRNA in the high VEGF group were significantly greater than those in the low VEGF group. Levels of VEGFR-2 mRNA and DNA topoisomerase IIα mRNA, and the MIB-1 labeling index in the high VEGF group were slightly higher than those in the low VEGF group; however, the difference was not statistically significant. Based on these observations, the significance of VEGF and its receptor genes in intracranial schwannomas is discussed.     

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.     

Vascular endothelial growth factor-mediated decrease in plasma soluble vascular endothelial growth factor receptor-2 levels as a surrogate biomarker for tumor growth

Vascular endothelial growth factor (VEGF) is a potent proangiogenic protein that activates VEGF receptor (VEGFR) tyrosine kinases expressed by vascular endothelial cells. We previously showed that one of these receptors, VEGFR-2, has a truncated soluble form (sVEGFR-2) that can be detected in mouse and human plasma. Because activation of VEGFR-2 plays an important role in tumor angiogenesis, clinical interest in monitoring plasma sVEGFR-2 levels in cancer patients has focused on its potential exploitation as a surrogate biomarker for disease progression as well as assessing efficacy/activity of antiangiogenic drugs, particularly those that target VEGF or VEGFR-2. However, no preclinical studies have been done to study sVEGFR-2 during tumor growth or the mechanisms involved in its modulation. Using spontaneously growing tumors and both localized and metastatic human tumor xenografts, we evaluated the relationship between sVEGFR-2 and tumor burden as well as underlying factors governing protein level modulation in vivo. Our results show an inverse relationship between the levels of sVEGFR-2 and tumor size. Furthermore, using various methods of VEGF overexpression in vivo, including cell transfection and adenoviral delivery, we found plasma sVEGFR-2 decreases to be mediated largely by tumor-derived VEGF. Finally, in vitro studies indicate VEGF-mediated sVEGFR-2 modulation is the result of ligand-induced down-regulation of the VEGFR-2 from the cell surface. Taken together, these findings may be pertinent to further clinical exploitation of plasma sVEGFR-2 levels as a surrogate biomarker of VEGF-dependent tumor growth as well as an activity indicator of antiangiogenic drugs that target the VEGFR system.