Tumor Angiogenesis Factor Research Articles

The Changes of HIF-1α and VEGF Expression After TACE in Patients With Hepatocellular Carcinoma.

As a common malignant tumor, hepatocellular carcinoma (HCC) has a high prevalence and is a serious threat to human health. The surgical resection rate of HCC is low, and the prognosis is poor. Although transarterial chemoembolization (TACE) is the main treatment for HCC patients who are not candidates for surgical resection, it is not considered a curative procedure. For HCC, poor TACE efficacy or TACE failure may be related to tumor angiogenesis of the residual disease. Among the many regulatory factors in tumor angiogenesis, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play vital roles in this process. In this paper, we conducted a review of the dynamic change and relevance of HIF-1α and VEGF levels after TACE of HCC patients.

Anti-hepatoma activity of the stiff branched β-d-glucan and effects of molecular weight.

The water soluble β-(1 → 3)-d-glucan with short branches (AF1) isolated from Auricularia auricula-judae was successfully fractionated by ultrasonication into three fractions with different weight-average molecular weights (Mws). The results of static and dynamic laser light scattering, viscometry and atomic force microscopy confirmed that the AF1 samples adopted a stiff chain conformation in water, and the coexistence of individuals and aggregates occurred gradually with increasing concentration. The AF1 sample with the highest Mw easily self-entangled, and exhibited a strong shear rate-dependence of viscosity in water. The glucans displayed anti-hepatoma activity and significantly inhibited H22 tumour growth without cytotoxicity towards normal tissues. They displayed both molecular weight- and dosage-dependencies of anti-tumour activity, and the sample with an Mw of 7.7 × 105 at the dosage of 5 mg kg-1 exhibited the highest inhibition ratio of ∼77% against H22 tumour, even significantly higher than the positive control of cytoxan. The immunohistochemical and western blot analyses revealed that the AF1 glucans triggered cell apoptosis, indicated by the activation of caspase 3/9 and down-regulated tumour angiogenesis factors of VEGF and CD31. The underlying antitumor mechanism was suggested to induce tumour cell apoptosis and to inhibit angiogenesis in tumour tissues via enhancement of the immune-response. Taken together, the AF1 β-glucan was a potent natural drug candidate with high anti-cancer activities and less cytotoxicity, and the AF1 sample with a moderate molecular weight existed in aqueous solution as a more extended chain conformation, which plays an important role in activating immune responses.

Influence Of The Number Of Platelets And Hemoglobin Concentrations In Predicting The Development Of Proteinuria Induced By The Administration Of Bevacizumab

Abstract Bevacizumab neutralizes a key stimulation factor in tumour angiogenesis - vascular endothelial growth factor (VEGF). The origin of VEGF is related to platelets, and the basic stimulus for its production is hypoxia. By neutralizing VEGF, changes occur in the blood that are manifested at the platelet and haemoglobin levels. Study results indicate that proteinuria is a side effect of bevacizumab treatment. The aim of the present study was to examine the percentages of proteinuria in patients with metastatic colorectal cancer after the administration of bevacizumab. The correlation between the degree of proteinuria and changes in the number of platelets and concentration of haemoglobin in patients, as well as in relation to the age (< 60 i ≥ 60) and gender of the patients, was examined. The study group of respondents included 20 patients with metastatic colorectal cancer (9 men and 11 women) ranging in age from 43 to 73 years. Proteinuria was tested using “Laboquick” test strips. The levels of platelets and haemoglobin were measured using an autoanalyser, the type Cell Dyn 3700 – Abbott, according to the protocol prescribed by The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Statistically significant differences were recorded for the platelet measurements before and after therapy in the overall group of respondents (p= 0.025). Statistically significant decreases in platelet counts and increases in haemoglobin concentrations were also noted in women (Plt: p=0.0036; Hb: p=0.0286) and in patients < 60 years old (Plt: p=0.0270; Hb: p=0.0553). No significant differences were identified in men or in patients ≥ 60 years of age. Proteinuria was detected in one case in which demonstrating an increase in only the recorded number of platelets and a decrease in the level of haemoglobin. Platelets and haemoglobin may serve as prognostic biomarkers of the risk of proteinuria. Based on the initial values of these parameters, we can predict the target group, in terms of gender and age, that may obtain the most benefit from bevacizumab therapy.

5-Fluoruracil blocked giant cell tumor progression by suppressing osteoclastogenesis through NF-kappaB signals and blocking angiogenesis

Giant cell tumor of bone (GCTB) is a bone destroying tumor comprised of spindle-like stromal cells and monocytes of myeloid lineage that are differentiated into osteoclast-like multinucleated giant cells. Nuclear factor-Kappa B (NF-κB) has been identified to be essential for GCT progression. Herein, we found that 5-Fluorouracil (5-FU), a widely used chemotherapeutics, is a promising anticancer agent for GCT both targeting spindle-like stromal cells and osteoclast giant cells through NF-κB pathway. In this study, in vitro 5-FU not only directly blocked both stromal cell- and RANKL-induced osteoclastogenesis through NF-κB pathway, but also indirectly inhibited osteoclast formation and angiogenesis by suppressing the expression of osteoclast-activating factors including IL-1β, MCP-1 and tumor angiogenesis factor VEGF in stromal cells. In vivo, we found that 5-FU blocked GCT progression through NF-κB pathway by utilizing our chick embryo chorioallantoic membrane (CAM) model. Taken together, our results suggest that 5-FU can inhibit GCT development by suppressing osteoclast formation through NF-κB pathway and blocking angiogenesis, and may serve as a novel agent in the treatment of GCT.

The novel tumor angiogenic factor, adrenomedullin-2, predicts survival in pancreatic adenocarcinoma

BackgroundTumor angiogenesis has been demonstrated to have an important role in the development, progression, and metastasis of pancreas cancer. Adrenomedullin-2 (ADM2) is a calcitonin gene-related peptide that has recently been shown to be a novel tumor angiogenesis factor, acting via mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/Akt, and vascular endothelial growth factor/vascular endothelial growth factor-2 signaling pathways. Through the use of tissue microarray (TMA) technology, we hypothesize that ADM2 is an important tumor angiogenesis factor in pancreatic cancer. MethodsMultiple TMAs were created using tissue from pancreatic cancer patients resected between January 1996 and December 2006. Core tissue samples of formalin-fixed, paraffin-embedded blocks of pancreatic cancer tissue were collected through an institutional review board-approved protocol and linked to available clinicopathologic data. Two TMAs consisting of 112 and 60 patients with pancreatic adenocarcinoma were studied for ADM2 protein expression using a quantitative, automated immunofluorescent microscopy system, a technology that removes potential observer bias in TMA analysis. The results were analyzed using independent Student t-test, chi-square, log-rank regression, and Kaplan–Meier methods. ResultsOne hundred sixteen patients were identified for complete analysis, and 56 patients had complete survival data. Median follow-up for survivors was 14.5 mo. Total cellular levels of ADM2 were found to be a predictor of survival in pancreatic cancer. Low ADM2 levels were associated with a higher 5-y survival compared with high ADM2 levels (18% versus 6%, P = 0.05). Median survival was also worse in high ADM2 expressers (18.7 versus 8.6 mo). In accordance with prior-published pancreatic cancer data, a worse histologic grade (P = 0.001), tumor (T) stage (P = 0.009), and overall disease stage (P = 0.004), all portended a worse survival. ConclusionsFor the first time, we have demonstrated that high levels of ADM2 expression predict a poorer survival in patients with pancreatic adenocarcinoma. This suggests a possible role of ADM2 in pancreas cancer and as a novel biomarker that predicts poorer survival. Additional study of ADM2 in pancreatic cancer will help reveal its true angiogenic role in pancreas cancer and its potential role as a novel therapeutic target.

Dll4 blockade potentiates the anti-tumor effects of VEGF inhibition in renal cell carcinoma patient-derived xenografts.

BackgroundThe Notch ligand Delta-like 4 (Dll4) is highly expressed in vascular endothelium and has been shown to play a pivotal role in regulating tumor angiogenesis. Blockade of the Dll4-Notch pathway in preclinical cancer models has been associated with non-productive angiogenesis and reduced tumor growth. Given the cross-talk between the vascular endothelial growth factor (VEGF) and Delta-Notch pathways in tumor angiogenesis, we examined the activity of a function-blocking Dll4 antibody, REGN1035, alone and in combination with anti-VEGF therapy in renal cell carcinoma (RCC).Methods and ResultsSevere combined immunodeficiency (SCID) mice bearing patient-derived clear cell RCC xenografts were treated with REGN1035 and in combination with the multi-targeted tyrosine kinase inhibitor sunitinib or the VEGF blocker ziv-aflibercept. Immunohistochemical and immunofluorescent analyses were carried out, as well as magnetic resonance imaging (MRI) examinations pre and 24 hours and 2 weeks post treatment. Single agent treatment with REGN1035 resulted in significant tumor growth inhibition (36–62%) that was equivalent to or exceeded the single agent anti-tumor activity of the VEGF pathway inhibitors sunitinib (38–54%) and ziv-aflibercept (46%). Importantly, combination treatments with REGN1035 plus VEGF inhibitors resulted in enhanced anti-tumor effects (72–80% growth inhibition), including some tumor regression. Magnetic resonance imaging showed a marked decrease in tumor perfusion in all treatment groups. Interestingly, anti-tumor efficacy of the combination of REGN1035 and ziv-aflibercept was also observed in a sunitinib resistant ccRCC model.ConclusionsOverall, these findings demonstrate the potent anti-tumor activity of Dll4 blockade in RCC patient-derived tumors and a combination benefit for the simultaneous targeting of the Dll4 and VEGF signaling pathways, highlighting the therapeutic potential of this treatment modality in RCC.

Abstract 5427: Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors

Abstract Vascular endothelial growth factor (VEGF) is a key tumor angiogenesis factor that is overexpressed by many tumors; the Fas/CD95 death receptor induces apoptosis when activated by its ligand FasL. Agents that inhibit VEGF such as bevacizumab, or activate Fas receptor have been intensively developed as cancer therapeutics. Bevacizumab only modestly improves overall survival or has no effect on overall survival in most cancer patients. More effective VEGF-targeted agents are needed. We conceived of a new strategy that goes beyond VEGF inhibition and converts VEGF to act as a tumor cell death factor. Our aim is to co-opt overexpressed VEGF within the tumor microenvironment to initiate apoptosis via Fas death receptors expressed on tumor cells and/or stromal cells. We created a recombinant homotrimeric fusion protein designated R1FasL that combines the VEGF-binding domain of human VEGFR1 with the trimerization and death receptor-binding domains of human FasL ligand. R1FasL was produced in CHO cells and purified by FLAG antibody affinity chromatography to yield a 135 kD homotrimer. In vitro studies confirmed that VEGF binds to and crosslinks the R1FasL fusion protein to form higher-order complexes that efficiently aggregate and activate Fas death receptors to induce apoptosis. Jurkat cells were killed by R1FasL only when VEGF was added. R1FasL induced apoptosis in diverse human cancer cell lines in vitro, including renal cell, glioblastoma and breast cancers. When VEGF was sequestered by a VEGFR1-Fc trap protein, R1FasL did not induce apoptosis, demonstrating that R1FasL used the VEGF secreted by the cancer cells to kill them. We previously demonstrated that a single intravenous dose of R1FasL induced widespread tumor apoptosis in a xenograft model of human renal cell carcinoma. We have now tested R1FasL in an intracranial xenograft model of human glioblastoma. Nude mice bearing U87 intracranial xenografts were treated with a single intratumoral injection of R1FasL (0.025 to 0.9 mg/kg in 10 microliters). Twenty-four hours later tumors were collected and analyzed for evidence of apoptosis by caspase-3 immunohistochemistry. Mice treated with a single intratumoral dose of R1FasL at 0.1 mg/kg demonstrated significant apoptosis within the tumor but not in surrounding brain. In a safety study, intracranial injection of R1FasL (0.1 or 0.8 mg/kg) into normal brains of nude mice did not cause increased weight loss or death versus PBS injection. In a subsequent survival study, mice treated with a single intratumoral dose of R1FasL (0.8 mg/kg) survived significantly longer than PBS-treated mice (39.5 versus 30 days, p=0.018). Non-invasive imaging demonstrated a median 59% reduction in tumor size 48 hours after R1FasL injection. By converting VEGF to act as a Fas death receptor ligand, the R1FasL fusion protein may be an effective agent to simultaneously target VEGF and death receptors to selectively induce apoptosis in the tumor microenvironment. Citation Format: Timothy Quinn, YJ Lu, Raquel Santos, Tomoko Ozawa, David James, Mary C. Nakamura. Inducing tumor apoptosis by redirecting VEGF to activate cell death receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5427. doi:10.1158/1538-7445.AM2014-5427

GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids.

Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell-cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane-localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane-localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.

The Novel Tumor Angiogenic Factor, Adrenomedullin-2 (ADM2) Predicts Survival in Pancreatic Adenocarcinoma

Tumor angiogenesis has been demonstrated to have an important role in the development, progression, and metastasis of pancreas cancer. Adrenomedullin-2 (ADM2) is a calcitonin gene-related peptide (CGRP) that has recently been shown to be a novel tumor angiogenesis factor, acting via ERK, Akt, and VEGF/VEGF-2 signaling pathways. Through the use of tissue microarray (TMA) technology, we hypothesize that ADM2 is an important tumor angiogenesis factor in pancreatic cancer.

GLOBAL EXISTENCE AND ASYMPTOTIC BEHAVIOR OF A TUMOR ANGIOGENESIS MODEL WITH CHEMOTAXIS AND HAPTOTAXIS

We consider a system of differential equations modeling tumor angiogenesis. The system consists of three equations: two parabolic equations with chemotactic terms to model endothelial cells and tumor angiogenesis factors coupled to an ordinary differential equation which describes the evolution of the fibronectin concentration. We study global existence of solutions and, under extra assumption on the initial data of the fibronectin concentration we obtain that the homogeneous steady state is asymptotically stable.

Research of hypoxia-inducible factor in tumor angiogenesis

Hypoxia-inducible factor (HIF) is an important transcriptional factor,which plays crucial role in response to hypoxia.HIF is highly expressed in many human cancers,and it is closely related to tumor angiogenesis.HIF promotes tumor growth,invasion and metastasis via up-regulating vascular endothelial growth factor gene and starting associated angiogenesis.With the in-depth study of HIF regulation of tumor angiogenesis mechanism,HIF is expected to become an important target for anti-angiogenesis therapy. Key words: Hypoxia-inducible factor; Vascular endothelial growth factors; Neovascularization,pathologic

Abstract 2176: R1FasL: a recombinant fusion protein that converts VEGF to act as a cell death factor.

Abstract Vascular endothelial growth factor (VEGF) is a key tumor angiogenesis factor that is overexpressed by many tumors; the Fas/CD95 death receptor induces apoptosis when activated by its ligand FasL. Agents that inhibit VEGF such as bevacizumab, or activate Fas receptor have been intensively developed as cancer therapeutics. Bevacizumab only modestly improves overall survival or has no effect on overall survival in most cancer patients. More effective VEGF-targeted agents are needed. We conceived of a new strategy that goes beyond VEGF inhibition and converts VEGF to act as a tumor cell death factor. Our aim is to co-opt overexpressed VEGF within the tumor microenvironment to initiate apoptosis via Fas death receptors expressed on tumor cells and/or stromal cells. We created a recombinant homotrimeric fusion protein designated R1FasL that combines the VEGF-binding domain of human VEGFR1 with the trimerization and death receptor-binding domains of human FasL ligand. R1FasL was produced in CHO cells and purified by FLAG antibody affinity chromatography to yield a 135 kD homotrimer. In vitro studies confirmed that VEGF binds to and crosslinks the R1FasL fusion protein to form higher-order complexes that efficiently aggregate and activate Fas death receptors to induce apoptosis. Jurkat cells were killed by R1FasL only when VEGF was added. R1FasL induced apoptosis in diverse human cancer cell lines in vitro, including renal cell, glioblastoma and breast cancers. When VEGF was sequestered by a VEGFR1-Fc trap protein, R1FasL did not induce apoptosis, demonstrating that R1FasL used the VEGF secreted by the cancer cells to kill them. R1FasL-induced apoptosis was inhibited by ZVAD caspase inhibitor and potentiated when combined with chemotherapeutic agents. R1FasL was tested in vivo in a xenograft model of human renal cell carcinoma. Nude mice bearing A498 human renal carcinoma xenografts (∼300 mm3)were treated with a single intravenous dose of R1FasL ranging from 0.08 mg/kg to 10 mg/kg. Twenty-four hours later tumors were collected and analyzed for evidence of apoptosis by histology and caspase-3 immunohistochemistry. Mice treated with a single dose of R1FasL at 2 mg/kg or 10 mg/kg demonstrated widespread tumor apoptosis 24 hours later. R1FasL therefore differs from numerous agents that target the VEGF/VEGFR system, such as bevacizumab, aflibercept and VEGFR kinase inhibitors, none of which induce acute tumor apoptosis. Serum ALT was mildly elevated at the 2 mg/kg dose, but in mice without tumors R1FasL at 4 mg/kg did not cause ALT elevation. These results suggest that R1FasL may not have significant toxicity. By converting VEGF to act as a Fas death receptor ligand, the R1FasL fusion protein may be an effective agent to simultaneously target VEGF and death receptors to selectively induce apoptosis in the tumor microenvironment. Citation Format: Timothy Quinn, Erene Niemi, Mary Nakamura. R1FasL: a recombinant fusion protein that converts VEGF to act as a cell death factor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2176. doi:10.1158/1538-7445.AM2013-2176

Vascular Endothelial Growth Factor Receptor-1 Expression in Breast Cancer and Its Correlation to Vascular Endothelial Growth Factor A

VEGF-A is the most potent angiogenic factor in tumour angiogenesis. Its effects are mediated via two receptors VEGFR-1 and VEGFR-2. Primary aim of our study was to examine the expression of VEGFR-1 in breast cancer and its correlation to VEGF expression, lymph node status, tumour size, histological grade, and hormone receptor status. To examine the VEGFR-1 and VEGF expressions in tumour and surrounding tissue of 51 breast cancer patients, and in healthy breast tissue of 30 benign breast diseases patients, we used three-step immunohistochemical staining. VEGFR-1 and VEGF expressions were significantly increased in breast cancer tumour in relation to surrounding tissue (P < 0.01), and the VEGF expression was significantly increased in lymph node positive breast cancer patients (P < 0.01). VEGFR-1 and VEGF expressions were significantly higher in breast cancer tumour compared with healthy breast tissue (P < 0.01). Significant correlation between VEGF and VEGFR-1 expressions was found (P < 0.05). No significant correlations between VEGF and VEGFR-1 expressions and tumour size, histological grade, and hormone receptor status were found. Increased expression of VEGFR-1 and VEGF in breast cancer tumour and significant correlation between these proteins suggest the possible role of VEGF/VEGFR-1 signalization in breast cancer development, although VEGFR-1 potential prognostic value was not confirmed.

Antiangiogenic Therapy for Cancer: An Update

The idea of antiangiogenic therapy was the brainchild of Dr. Judah Folkman in the early 1970s. He proposed that by cutting off the blood supply, cancer cells would be deprived of nutrients and, hence, treated. His efforts paid off when bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor, was approved as antiangiogenic therapy in 2004 for the treatment of colon cancer. Since then, an array of antiangiogenic inhibitors, either as monotherapy or in combination with other cytotoxic and chemotherapy drugs, have been developed, used in clinical trials, and approved for the treatment of cancer. Despite this important breakthrough, antiangiogenic therapy for cancer met with a number of hurdles on its way to becoming an option for cancer therapy. In this article, we summarize the most current information on the mechanisms of tumor angiogenesis, proangiogenic and antiangiogenic factors, potential targets and their mechanisms of action, and experimental evidences, as well as the most recent clinical trial data on antiangiogenic agents for cancer therapy.

Serum Values of Osteopontin and Vascular Endothelial Growth Factor in Association with Clinical Parameters in Patients with Multiple Myeloma

AbstractAbstract 4993 Background:the role of osteopontin (OPN), glyco-phosphoprotein produced by variety of tissues, has been widely investigated in the progression of many solid tumors, but on the other side, there are not many studies performed in multiple myeloma (MM). The role of Vascular Endothelial Growth Factor (VEGF) in tumor angiogenesis, as well as in MM, has been extensively analysed, but the significance of its serum level is still not well recognize. The aim of the present study was to determine the serum levels of both above mentioned cytokines and compare their values with clinical parameters of myeloma patients. Methods:serum level of OPN and VEGF was determinated by ELISA in 44 newly-diagnosed, reviously untreated myeloma patients (21 men, 22 women; median age of 69, range 44 – 86) and 24 age-matched healthy persons who consisted control group. The obtained values were correlated with main clinical parameters such as anaemia (hemoglobine value 20 g/L below the lower limit of normal), renal dysfunction (serum creatinine level above the uper limit of normal) and bone disease (any of lytic lesions or severe osteopenia with compressive fractures on standard radiographs of the bones). The level of cytokines were also correlated with serum beta-2 microglobulin as a prognostic factor related to biological behaviour of MM, and Durie-Salmon Staging System as a measure of tumor mass. Results:serum value of OPN was significantly higher in myeloma patients (median 6. 5, range 0. 3 – 21. 7) in compare to control group (medain 2. 4, range 0. 2 – 8. 9) (p<0. 0001), while these difference could not be observed with VEGF (52. 5 versus 60. 5) (p=0. 6673). Serum level of beta2-microglobuline was positively associated with value of OPN (p=0. 0461) but not with VEGF (p=0. 79). Patients with lower Durie-Salmon Stage had significantly lower both- OPN and VEGF serum levels (p=0. 0456, p=0, 0371 respectively). Higher OPN serum value was found in MM patients with evident bone lesions (p=0. 0268), while this difference was not observed with VEGF (p= 0. 7962). There was no correlation between serum levels of OPN and anaemia (p=0. 2991) or increased serum creatinine (p=0. 1338). VEGF serum level, also, did not correlated with anemia (p=0. 4309) and renal dysfunction (p=0. 5119). Conclusion:our preliminary results support the serum level of OPN as a dual marker of tumor aggressiveness and bone destrucion. On the contrary, present study did not confirm similar clinical value of VEGF serum level which represent a critical angiogenic factor in tumor micro-eco system. However, in our investigation both cytokines positively correlated with tumor burden. Further analysis with more included patient sholud provide definitive information about possible role of OPN as a useful clinical biomarker for monitoring of bone disease and tumor mass, as well as prognostic factor during course of disease. Disclosures:No relevant conflicts of interest to declare.

Abstract 2558: VEGF-dependent suppression of prostaglandin transporter (PGT) expression in Kras mutant human bronchial epithelial cells

Abstract Prostaglandin transporters are expressed in cyclooxygenase-positive cells and provide the dominant mechanisms for transport of prostaglandins (PG) across the cell membrane. Specifically, the prostaglandin transporter PGT, expressed in many cell types including lung epithelia, is responsible for the uptake of prostaglandin E2 (PGE2) for intracellular metabolism and degradation by 15-prostaglandin dehydrogenase (15-PGDH). The regulation of prostaglandin transporters has not been fully explored in lung cancer. We hypothesize that loss of PGT expression is important in lung carcinogenesis and its loss may be enhanced in the presence of common mutations in premalignant bronchial epithelial cells and non-small cell lung cancer (NSCLC). Our preliminary studies show that PGT expression (protein and mRNA) and 15-PGDH are significantly downregulated in Kras mutant human bronchial epithelial cells (HBEC) and in NSCLC. It is well known that Kras mutant cancers are associated with increased production of VEGF, an important factor in tumor angiogenesis. In HBEC, we found significantly higher VEGF levels in Kras mutants compared to vector controls (2242 versus 628 pg/mg, p = 0.04). We proposed that VEGF modulates PGT expression and tested our hypothesis by treating HBEC and NSCLC with human recombinant VEGF. PGT expression is downregulated with exogenous VEGF treatment and neutralizing VEGF production (neutralizing VEGF antibody) prevented the loss of PGT expression. The MEK1/2 inhibitor PD98059 upregulated PGT expression in HBEC Kras. Our results suggest Kras mutation is associated with loss of PGT expression and may be dependent on VEGF production. Modulation of PGT expression may be important in lung carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2558. doi:1538-7445.AM2012-2558

Anti-VEGF/VEGFR Therapy for Cancer: Reassessing the Target

Judah Folkman recognized that new blood vessel formation is important for tumor growth and proposed antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as the primary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or its receptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidly growing mouse tumors. However, results in human cancer have been less impressive. A number of reasons have been offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumor vasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vessel types that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated in mouse tissues by an adenoviral vector expressing VEGF-A(164). Once formed, 4 of the 6 types lose their VEGF-A dependency, and so their responsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have a greater impact on human cancer, targets other than VEGF and its receptors will need to be identified on these resistant tumor vessels.

Effect of siRNA-mediated downregulation of VEGF in Tca8113 cells on the activity of monocyte-derived dendritic cells

Vascular endothelial growth factor (VEGF) is a tumor angiogenesis factor that is important in immune regulation. In our previous study, we found that VEGF expression in the peripheral blood and neoplasm nest from patients with oral squamous cell carcinoma (OSCC) was positively correlated with the course of disease, while an inverse correlation between VEGF expression and dendritic cells (DCs) was identified in the peripheral blood. Therefore, in the present study, we investigated whether inhibition of human VEGF in the human tongue carcinoma cell line Tca8113 had effects on the activity of monocyte-derived DCs. We knocked down the expression of human VEGF in Tca8113 cells using the small interfering RNA (siRNA) technique. Tca8113 cells pre-transfected with siRNA targeting VEGF were co-cultured with monocyte‑derived immature and mature DCs. Cell proliferation was evaluated by a WST-8 assay. Cell apoptosis, cell cycle and cell phenotypes were determined by flow cytometry. The data revealed that downregulation of the human VEGF significantly inhibited the proliferation of Tca8113 cells and increased apoptosis. Inhibition of human VEGF arrested the cell cycle of Tca8113 cells at the G0/G1 phase. Our results showed that the co-culture of DCs with Tca8113 cells markedly inhibited the expression of the mature markers of DCs including HLA-DR, CD80, CD86, CD40 and CD1a, as well as the immature marker CD83, while inhibition of human VEGF in Tca8113 cells significantly reversed these effects. Therefore, human VEGF in Tca8113 cells may not only regulate the cell proliferation and apoptosis of oral squamous cell carcinoma cells, but may also inhibit DC maturation.

I111n-Labeled Cystine-Knot Peptides Based on the Agouti-Related Protein for Targeting Tumor Angiogenesis

Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops. The cell adhesion receptor integrin αvβ3 is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors. AgRP mutants have been engineered to bind to integrin αvβ3 with high affinity and specificity using directed evolution. Here, AgRP mutants 7C and 6E were radiolabeled with 111In and evaluated for in vivo targeting of tumor integrin αvβ3 receptors. AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N′, N″, N‴-tetraacetic acid (DOTA) and radiolabeled with 111In. The stability of the radiopeptides 111In-DOTA-AgRP-7C and 111In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively. Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines. Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors. Both AgRP peptides were easily labeled with 111In in high yield and radiochemical purity (>99%). The two probes exhibited high stability in phosphate-buffered saline and mouse serum. Compared with 111In-DOTA-AgRP-6E, 111In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74 ± 1.60 and 1.29 ± 0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake. Moreover, the tumor uptake of 111In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK). Thus, 111In-DOTA-AgRP-7C is a promising probe for targeting integrin αvβ3 positive tumors in living subjects.

Pleiotrophin is a potential colorectal cancer prognostic factor that promotes VEGF expression and induces angiogenesis in colorectal cancer

Pleiotrophin (PTN) is an important developmental secretory cytokine expressed in many types of cancer and involved in angiogenesis and tumor growth; however, the significance of PTN expression in colorectal cancer (CRC) has not been established. Immunohistochemistry, western blot, and enzyme-linked immunosorbent assay were used to detect PTN expression in CRC patients. The relationship between PTN expression and clinicopathological characteristics and survival time was statistically analyzed, and the relationship between PTN and vascular endothelial growth factor (VEGF) in tumor angiogenesis was further analyzed. Of CRC tissues, 74.70% (62/83) stained positive, with a strong positive ratio of 60.24% (50/83). The expression of PTN in CRC tissues was much higher than in normal colorectal tissues. PTN serum levels in CRC patients (mean = 254.59 ± 261.76 pg/ml) were significantly higher than those of normal volunteers (mean = 115.23 ± 79.53 pg/ml; p < 0.001). PTN expression was related to CRC differentiation and TNM staging. High level of PTN is a predictor of a poor prognosis and high expression of PTN is accompanied by high expression of VEGF in CRC patients. Investigation of the relationship between PTN and VEGF revealed that PTN, through the PTN/RPTPβ/ζ signaling pathway, increased tyrosine phosphorylation of β-catenin, leading to an increase in VEGF. Our study identifies PTN as an essential growth factor for CRC. PTN promotes VEGF expression and cooperates with VEGF in promoting CRC angiogenesis. PTN could serve as a prognostic factor for this cancer. Considering that PTN shows very limited expression in normal tissue, it may represent an attractive new target for CRC therapy.