Pathways for aberrant angiogenesis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although the specific mechanisms that dictate its biological aggressiveness are not clearly established, it is characterized by a variety of molecular alterations as well as by the overexpression of mitogenic and angiogenic growth factors and their receptors. PDACs also express high levels of vascular endothelial growth factor (VEGF). Recent studies indicate that suppression of VEGF expression attenuates pancreatic cancer cell tumorigenicity in a nude mouse model, and that VEGF can exert direct mitogenic effects on some pancreatic cancer cells. These findings suggest that cancer cell derived VEGF promotes pancreatic cancer growth in vivo via a paracrine angiogenic pathway and an autocrine mitogenic pathway, and provide novel opportunities for therapeutic intervention in this deadly disease.     

Inhibition of K562 leukemia angiogenesis and growth by expression of antisense vascular endothelial growth factor (VEGF) sequence

Vascular endothelial growth factor (VEGF), a major angiogenic factor, plays a key role in the growth of solid tumor. Recently, expression of VEGF and its receptors has been found on leukemic cells as well as on endothelial cells. VEGF may fulfill a fundamental role in promoting tumor angiogenesis and proliferation by stimulating both endothelial cells and leukemic cells. To investigate the role of VEGF in the angiogenesis and growth of leukemic cell, we used an antisense strategy to downregulate VEGF expression in K562 cells, a human erythroleukemia cell line. Expression of antisense-VEGF in K562 cells reduced the secretion of VEGF protein and inhibited cell survival. The proliferation and migration of human umbilical vein endothelial cells were decreased in response to the conditioned medium (CM) from K562 cells expressed antisense-VEGF, compared to CM from K562 cells transfected with vector control. Moreover, subcutaneous injection of nude mice with antisense-VEGF K562 cells inhibited tumor growth with a reduction of the density of microvessels and an increased apoptosis in those tumors, compared to vector control K562 cells. These results suggest that the efficient downregulation of the VEGF production in leukemic cells using antisense-VEGF may constitute a novel strategy of treatment in leukemia.     

Vascular endothelial growth factor-trap suppresses tumorigenicity of multiple pancreatic cancer cell lines.

PURPOSE: Vascular endothelial growth factor A (VEGF-A) is a potent angiogenic agent that binds to two high affinity VEGF receptors (VEGFRs), a process facilitated by the low affinity neuropilin receptors. Although VEGF-A is overexpressed in pancreatic ductal adenocarcinoma, it is not known whether the in vivo growth of multiple pancreatic cancer cells can be efficiently blocked by VEGF-A sequestration. EXPERIMENTAL DESIGN: Four human pancreatic cancer cell lines were grown s.c. in athymic nude mice. One cell line also was used to generate an orthotopic model of metastatic pancreatic cancer. The consequences of VEGF-A sequestration on tumor growth and metastasis were examined by injecting the mice with a soluble VEGFR chimer (VEGF-Trap) that binds VEGF-A with high affinity. RESULTS: VEGF-Trap, initiated 2 days after tumor cell inoculation, suppressed the s.c. growth of four pancreatic cancer cell lines and markedly decreased tumor microvessel density. Analysis of RNA from tumors generated with T3M4 cells revealed that VEGF-Trap decreased the expression of VEGFR-1 and neuropilin-1 and -2. VEGF-Trap, initiated 3 weeks after tumor implantation, also attenuated intrapancreatic tumor growth and metastasis in an orthotopic model using PANC-1 cells. CONCLUSIONS: VEGF-Trap is a potent suppressor of pancreatic tumor growth and metastasis and also may act to attenuate neuropilin-1 and -2 and VEGFR-1 expression. Therefore, VEGF-Trap may represent an exceedingly useful therapeutic modality for pancreatic ductal adenocarcinoma.     

A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing's sarcoma growth in a xenograft mouse model.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.     

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth,metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.     

Antiangiogenicity of docosahexaenoic acid and its role in the suppression of breast cancer cell growth in nude mice.

The addition of the omega-3 fatty acid (n-3 FA) docosahexaenoic acid (DHA), 4%, to a 20% (wt/wt) fat diet containing 4% linoleic acid (LA, n-6 FA) partially suppressed the growth of the MDA-MB-231 human breast cancer cell line as solid tumors in athymic nude mice. This reduced tumor growth was associated with significant inhibition of cell proliferation, as indicated by diminished Ki-67 nuclear proliferation marker expression, and an increase in TUNEL positive (apoptotic) cells (both p<0.001). The microvessel counts were also reduced in tumors from the DHA-supplemented dietary group of mice (p<0.001), and this suppression of angiogenesis was positively correlated with loss of Ki-67 expression. Tumor vascular endothelial cell growth factor (VEGF) concentrations were not reduced in the DHA-fed mice. It is postulated that the antiangiogenicity of DHA in this breast cancer model is related to our demonstrated inhibition of LA-derived prostaglandin E2, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE synthesis, reducing the paracrine stimulation by these known angiogenic eicosanoids on microvessel endothelial cells.     

Molecular basis of the synergistic antiangiogenic activity of bevacizumab and mithramycin A

The impact of antiangiogenic therapy on the Sp1/vascular endothelial growth factor (VEGF) pathway and that of alteration of Sp1 signaling on the efficacy of antiangiogenic therapy is unclear, yet understanding their interactions has significant clinical implications. Treatment with bevacizumab, a neutralizing antibody against VEGF, suppressed human pancreatic cancer growth in nude mice. Gene expression analyses revealed that this treatment substantially up-regulated the expression of Sp1 and its downstream target genes, including VEGF and epidermal growth factor receptor, in tumor tissues, whereas it did not have this effect on pancreatic cancer cells in culture. Treatment with mithramycin A, an Sp1 inhibitor, suppressed the expression of Sp1 and its downstream target genes in both cell culture and tumors growing in nude mice. Combined treatment with bevacizumab and mithramycin A produced synergistic tumor suppression, which was consistent with suppression of the expression of Sp1 and its downstream target genes. Thus, treatment with bevacizumab may block VEGF function but activate the pathway of its expression via positive feedback. Given the fact that Sp1 is an important regulator of the expression of multiple angiogenic factors, bevacizumab-initiated up-regulation of Sp1 and subsequent overexpression of its downstream target genes may profoundly affect the potential angiogenic phenotype and effectiveness of antiangiogenic strategies for human pancreatic cancer. Therefore, this study is the first to show the significance and clinical implications of alteration of Sp1 signaling in antiangiogenic therapy for pancreatic cancer and other cancers.     

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.     

Inhibition of fibroblast growth factor receptor 2 attenuates proliferation and invasion of pancreatic cancer

The alternative splicing of the extracellular domain of fibroblast growth factor receptor (FGFR)‐2 generates the IIIb and IIIc isoforms. Expression of FGFR‐2 IIIb correlates with vascular endothelial growth factor‐A (VEGF‐A) expression and venous invasion of pancreatic ductal adenocarcinoma (PDAC). By contrast, FGFR‐2 IIIc expression correlates with faster development of liver metastasis after surgery, and increased proliferation rates and invasion of the cancer. In this study, we analyzed the expression and roles of total FGFR‐2 (both isoforms) to determine the effectiveness of FGFR‐2‐targeting therapy for PDAC. Immunohistochemically, FGFR‐2 was highly expressed in 25/48 (52.1%) PDAC cases, and correlated with advanced stage cancer. In FISH analysis, FGFR2 was amplified in 3/7 PDAC cell lines. We stably transfected an FGFR‐2 shRNA targeting the IIIb and IIIc isoforms into FGFR2‐amplified PDAC cells. The proliferation rates, migration, and invasion of FGFR‐2‐shRNA‐transfected cells were lower than those of control cells in vitro. In response to FGF‐2, FGFR‐2‐shRNA‐transfected cells showed decreased phosphorylation of ERK compared with control cells. The FGFR‐2‐shRNA‐transfected cells also expressed lower levels of vascular endothelial growth factor‐A than control cells, and formed smaller s.c. tumors in nude mice. These findings suggest that FGFR‐2 is a therapeutic target for inhibition in PDAC.     

Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia: an in vitro and in vivo study

Angiogenesis in pancreatic ductal adenocarcinomas depends on the presence of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and is thought to be stimulated by hypoxia. We tested the angiogenic potential of 9 cell lines of pancreatic ductal carcinoma origin by screening mRNA and protein expression of VEGF and bFGF and the release of VEGF into culture medium under normoxic and hypoxic (5% or 0.2% O2) conditions. Angiogenic activity was determined using 2- and 3-D endothelial cell assays. Furthermore, VEGF expression and tumor vascularization were studied in human pancreatic carcinoma tissues from orthotopic xenografts and resection specimens. All cell lines expressed (mRNA, protein) and secreted VEGF, whereas bFGF was only found in 3 cell lines and was secreted into the medium in low concentrations. In addition to the dominant isoforms VEGF121,VEGF165 and VEGF189, 2 isoforms described recently, VEGF145 and VEGF183, were detected. Severe hypoxia (0.2% O2), but not moderate hypoxia (5% O2) raised VEGF mRNA expression and protein secretion in 7/9 and 5/9 cell lines, respectively. Conditioned media from 7/9, 6/9, 8/9 and 7/9 cell lines stimulated endothelial cell proliferation under normoxic (24 and 48 hr) or hypoxic (24 hr, 0.2% and 48 hr 5% O2) conditions, respectively. Conditioned media from 4/9 cell lines also induced capillary-like sprouting under normoxic conditions and from 6/9 under hypoxic (0.2% O2) conditions. In xenografted carcinoma tissues microvessel density was found not to be increased around areas of ischemic necrosis. In resected ductal carcinomas showing tumor necrosis VEGF expression and microvessel density were only increased in 3/12 and 2/13 cases, respectively. In conclusion, in vitro most pancreatic ductal carcinomas show a distinct VEGF related angiogenic potential, as demonstrated by 2- and 3-D endothelial cell proliferation, which may be promoted by severe hypoxia. Surprisingly, perinecrotic tumor areas, which are supposed to be hypoxic, only rarely showed the expected increase in microvessel density and VEGF expression.     

Inhibiting Colorectal Carcinoma Growth and Metastasis By Blocking the Expression of VEGF Using RNA Interference

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis using RNA interference (RNAi) technology. Four small interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into human colorectal carcinoma (CRC) SW620 cells. Stable transfection of these plasmids decreased VEGF protein expression, leading to the potent suppression of tumor cell proliferation, migration, invasion, and angiogenesis in vitro. Furthermore, in subcutaneous and intrasplenic/portal injection models involving athymic nude mice, the tumor growth and metastasis of SW620 cells expressing VEGF siRNA were significantly inhibited compared with untransfected cells or cells transfected with control vector alone. Immunohistochemical analyses of tumor sections revealed a decreased vessel density and decreased VEGF expression in the animals where siRNA against VEGF were expressed. These results indicate that RNAi of VEGF can be an effective antiangiogenic strategy for CRC.     

Aberrant expression of neuropilin-1 and -2 in human pancreatic cancer cells.

PURPOSE: Neuropilin (Np)-1 and -2 are coreceptors for vascular endothelial growth factor (VEGF). This study was designed to assess their role in pancreatic ductal adenocarcinoma (PDAC). EXPERIMENTAL DESIGN: We assessed Np-1 and Np-2 expression by real-time quantitative PCR in relation to the expression of VEGF ligands and receptors in pancreatic cancer cell lines and tissues. RESULTS: ASPC-1, CAPAN-1, and PANC-1 pancreatic cancer cells and tumor-derived, laser-captured pancreatic cancer cells exhibited higher Np-1 and Np-2 mRNA levels than VEGF receptor-1, -2, or -3 mRNA levels. Transfection of Np-1 and Np-2 cDNAs in COS-7 cells, and treatment with tunicamycin revealed that both proteins were glycosylated. Both proteins were expressed in pancreatic cancer cell lines, in the PDAC samples, and in acinar cells adjacent to the cancer cells. The normal pancreas was devoid of Np-1 immunoreactivity, whereas Np-2 immunoreactivity was present in the endocrine islets and in some acinar cells, but not in ductal cells. CONCLUSIONS: The aberrant localization of Np-1 and Np-2 in the cancer cells in PDAC suggests that in addition to exerting proangiogenic effects, these coreceptors may contribute to novel autocrine-paracrine interactions in this malignancy.     

Kallikrein-binding protein suppresses growth of hepatocellular carcinoma by anti-angiogenic activity

Effect of kallikrein-binding protein (KBP), an endogenous angiogenic inhibitor, on the growth of hepatocellular carcinoma and the possible mechanism were investigated. KBP inhibited proliferation and induced apoptosis of endothelial cells, but had no effect on the proliferation and apoptosis of hepatocarcinoma cell line HepG2. Intraperitoneal injection of KBP significantly suppressed the tumor growth and inhibited intratumoral neovascularization both in grafted hepatocarcinoma mice and xenografted hepatocarcinoma athymic mice. Moreover, KBP reduced expression of VEGF and HIF-1alpha nuclear translocation in HepG2 cells and xenografts. Down-regulation of VEGF in tumor cells through inhibiting HIF-1alpha may represent a novel mechanism for the anti-angiogenic and anti-tumor activity of KBP.     

Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.