Angiogenesis and cancer: A cross-talk between basic science and clinical trials (the "do ut des" paradigm)

Angiogenesis plays a crucial role in facilitating tumor growth and the metastatic process, and it is the result of a dynamic balance between pro-angiogenic factors, like vascular endothelial growth factor (VEGF) and platelet-derived growth factor, and antiangiogenic factors, like thrombospondin-1 and angiostatin. Many drugs that target human tumors, like bevacizumab and some VEGF-receptor tyrosine-kinase inhibitors (e.g., BAY 43-9006, SU11248 and PTK787/ZK222584) have been studied in clinical trials, with favorable toxicity reports and encouraging results in advanced colorectal cancer, renal cell cancer, breast cancer and non-squamous non-small cell lung cancer, either combined with chemotherapy, or in monotherapy. Another potential approach to inhibiting angiogenesis is through metronomic chemotherapy (low doses of chemotherapy for long periods of time). This review describes the mechanisms of the angiogenic process and evaluates the recent data about antiangiogenic therapies in clinical trials.     

Antiangiogenic strategies, compounds, and early clinical results in breast cancer

Angiogenesis is a multi-step process leading to the formation of new blood vessels from pre-existing vasculature and it is necessary for primary tumor growth, invasiveness and development of metastasis. Experimental and clinical data demonstrated that breast cancer is an angiogenesis-dependent disease and that the vascular endothelial growth factor (VEGF) family plays a key role it being a highly expressed and selective endothelial cell growth factor. Preclinical studies have shown that the angiogenic switch occurs early in the multistage process of breast cancer development. Targeting the molecular pathways involved in tumor progression by biologically-designed treatments is a new therapeutic paradigm aimed to reach cancer growth control. A number of possible therapeutic targets for antiangiogenic agents have been identified. Here we discuss the therapeutic approach based on inhibition of angiogenesis in the context of breast cancer with a focus on the early clinical studies on antiangiogenic agents in advanced disease.     

Antiangiogenic effects of melatonin in endothelial cell cultures

Endothelial cells represent one of the critical cellular elements in tumor microenvironment playing a crucial role in the growth and progression of cancer through controlling angiogenesis. Vascular endothelial growth factor (VEGF) produced from tumor cells is essential for the expansion of breast cancer and may function in both paracrine and autocrine manners to promote proliferation, growth, survival and migration of endothelial cells. Since melatonin regulates tumor microenvironment by decreasing the secretion of VEGF by malignant epithelial cells and also regulates VEGF expression in human breast cancer cells, the aim of the present study was to investigate the anti-angiogenic activity of melatonin against the pro-angiogenic effects of breast cancer cells.In this work, we demonstrate that melatonin strongly inhibited the proliferation as well as invasion/migration of human umbilical vein endothelial cells (HUVECs). Melatonin disrupted tube formation and counteracted the VEGF-stimulated tubular network formation by HUVEC. In addition, conditioned media collected from human breast cancer cells were angiogenically active and stimulated tubule length formation and this effect was significantly counteracted by the addition of anti-VEGF or melatonin. Melatonin also disintegrated preformed capillary network.All these findings demonstrate that melatonin may play a role in the paracrine interactions that take place between malignant epithelial cells and proximal endothelial cells. Melatonin could be important in reducing endothelial cell proliferation, invasion, migration and tube formation, through a downregulatory action on VEGF. Taken together, our findings suggest that melatonin could potentially be beneficial as an antiangiogenic agent in breast cancer with possible future clinical applications.     

乳腺癌患者血清VEGF含量测定及临床意义

血管内皮生长因子 (VEGF)在肿瘤血管的形成中起到非常重要的作用。本研究采用 EL ISA法检测 36例乳腺癌患者术前血清血管内皮生长因子 (VEGF)含量 ,与良性对照组及正常对照组比较 ,并观察 VEGF与临床病理指标的关系。认为乳腺癌组与良性对照组和正常对照组比较有非常显著差异 ,并与临床分期及淋巴结转移有相关性。     

Endogenous stimulators and inhibitors of angiogenesis in gastrointestinal cancers: basic science to clinical application

Progression of cancer is dependent on acquisition of vascular networks within the tumor. Tumor angiogenesis is dependent on up-regulation of angiogenesis stimulators to overcome the endogenous anti-angiogenic barrier. Such disruption of angiogenesis balance to favor neovascularization is a key step for progression of tumor growth and metastasis. In this regard, the vascular basement membrane and the extracellular matrix have been found to be rich sources of angiogenesis stimulators and inhibitors that become bioavailable on proteolysis of the matrix by tumor microenvironment-related enzymes. In this review the subgroup of endogenous angiogenesis stimulators and inhibitors is discussed, and their mechanism of action during tumor angiogenesis is evaluated. The role in regulating tumor growth and the possibility of using them as prognostic markers for human gastrointestinal cancers is discussed. Furthermore, we specifically address the role of vascular endothelial growth factor in human gastrointestinal cancers and discuss the development and use of bevacizumab (Avastin; anti-vascular endothelial growth factor antibody [Genentech, CA]) in the treatment of colorectal and other gastrointestinal cancers.     

Drug insight: antiangiogenic therapies for gastrointestinal cancers--focus on monoclonal antibodies

Tumor angiogenesis is strongly induced by vascular endothelial growth factor (VEGF), which is overexpressed in most human gastrointestinal cancers. VEGF overexpression is known to be associated with poor prognosis and survival in patients with various solid tumors. The humanized monoclonal anti-VEGF antibody bevacizumab (Avastin, Genentech Inc., South San Francisco, CA) is a prototypic antiangiogenic compound, and has proven therapeutic benefit combined with conventional chemotherapy-namely, significantly improved progression-free survival in patients with metastatic colorectal cancer. Bevacizumab is the only anti-VEGF antibody that has been approved by the FDA and the European Medicines Agency for the treatment of metastatic colorectal cancer. Several ongoing clinical studies are evaluating the potential of bevacizumab therapy for other gastrointestinal cancers, in combination with chemotherapy, other targeted therapies and/or radiation. Soluble chimeric receptors, tyrosine kinase inhibitors, and monoclonal antibodies against VEGF and molecular targets in the integrin and Delta-like protein 4-Notch pathways are being developed. As tumors acquire resistance to anti-VEGF therapy, further development of antiangiogenic and vascular targets and therapy is warranted.     

TLR9 agonist acts by different mechanisms synergizing with bevacizumab in sensitive and cetuximab-resistant colon cancer xenografts

Synthetic agonists of Toll-like receptor 9 (TLR9), a class of agents that induce specific immune response, exhibit antitumor activity and are currently being investigated in cancer patients. Intriguingly, their mechanisms of action on tumor growth and angiogenesis are still incompletely understood. We recently discovered that a synthetic agonist of TLR9, immune modulatory oligonucleotide (IMO), acts by impairing epidermal growth factor receptor (EGFR) signaling and potently synergizes with anti-EGFR antibody cetuximab in GEO human colon cancer xenografts, whereas it is ineffective in VEGF-overexpressing cetuximab-resistant GEO cetuximab-resistant (GEO-CR) tumors. VEGF is activated by EGFR, and its overexpression causes resistance to EGFR inhibitors. Therefore, we used IMO and the anti-VEGF antibody bevacizumab as tools to study IMO's role on EGFR and angiogenesis and to explore its therapeutic potential in GEO, LS174T, and GEO-CR cancer xenografts. We found that IMO enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cetuximab, that bevacizumab has no ADCC, and IMO is unable to enhance it. Nevertheless, the IMO-plus-bevacizumab combination synergistically inhibits the growth of GEO and LS174T as well as of GEO-CR tumors, preceded by inhibition of signaling protein expression, microvessel formation, and human, but not murine, VEGF secretion. Moreover, IMO inhibited the growth, adhesion, migration, and capillary formation of VEGF-stimulated endothelial cells. The antitumor activity was irrespective of the TLR9 expression on tumor cells. These studies demonstrate that synthetic agonists of TLR9 interfere with growth and angiogenesis also by EGFR- and ADCC-independent mechanisms affecting endothelial cell functions and provide a strong rationale to combine IMO with bevacizumab and EGFR inhibitory drugs in colon cancer patients.     

The Role of the Hemostatic System in Tumor Growth, Metastasis, and Angiogenesis: Tissue factor is a Bifunctional Molecule Capable of Inducing Both Fibrin Deposition and Angiogenesis in Cancer

Cancer patients are prone to venous thromboembolism (VTE), and this hypercoagulability favors tumor growth and metastasis. After a brief review of the clinical aspects of VTE and cancer, we discuss the pathogenesis of hypercoagulability with an emphasis on the role of tissue factor (TF). The discovery that, in addition to tumor cells, TF is expressed by tumor-associated macrophages and tumor-associated endothelial cells led to studies of the role of TF in the regulation of tumor angiogenesis. In human lung cancer, melanoma, and breast cancer, TF and vascular endothelial growth factor (VEGF) co-localize in tumor cells; a close correlation exists between TF and VEGF synthesis (P = .001) in tumor cell lines and with angiogenesis in vivo in a severe, combined immunodeficient mouse model. Transfection of a TF/VEGF low-producing human tumor cell line with full length TF complementary DNA (cDNA) results in conversion to a high producer of TF and VEGF; transfection of a deletion-mutant TF cDNA lacking cytoplasmic serine residues restores full TF procoagulant activity but not VEGF synthesis to the cells. These results suggest that the cytoplasmic tail of TF is necessary for tumor cell VEGF synthesis. Targeting of TF in tumors and tumor-associated blood vessels is discussed as a strategy for drug delivery and rational anti-cancer and anti-angiogenesis drug design.     

Regulation of vascular endothelial growth factor by melatonin in human breast cancer cells

Melatonin exerts oncostatic effects on breast cancer by interfering with the estrogen‐signaling pathways. Melatonin reduces estrogen biosynthesis in human breast cancer cells, surrounding fibroblasts and peritumoral endothelial cells by regulating cytokines that influence tumor microenvironment. This hormone also exerts antiangiogenic activity in tumoral tissue. In this work, our objective was to study the role of melatonin on the regulation of the vascular endothelial growth factor (VEGF) in breast cancer cells. To accomplish this, we cocultured human breast cancer cells (MCF‐7) with human umbilical vein endothelial cells (HUVECs). VEGF added to the cultures stimulated the proliferation of HUVECs and melatonin (1 mm ) counteracted this effect. Melatonin reduced VEGF production and VEGF mRNA expression in MCF‐7 cells. MCF‐7 cells cocultured with HUVECs stimulated the endothelial cells proliferation and increased VEGF levels in the culture media. Melatonin counteracted both stimulatory effects on HUVECs proliferation and on VEGF protein levels in the coculture media. Conditioned media from MCF‐7 cells increased HUVECs proliferation, and this effect was significantly counteracted by anti‐VEGF and 1 mm melatonin. All these findings suggest that melatonin may play a role in the paracrine interactions between malignant epithelial cells and proximal endothelial cells through a downregulatory action on VEGF expression in human breast cancer cells, which decrease the levels of VEGF around endothelial cells. Lower levels of VEGF could be important in reducing the number of estrogen‐producing cells proximal to malignant cells as well as decreasing tumoral angiogenesis.     

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.     

New antiangiogenetic agents and non-small cell lung cancer

New blood vessel formation, known as angiogenesis is a fundamental event in the process of tumor growth and metastatic dissemination. Due to its central role in tumor angiogenesis, the vascular endothelial growth factor (VEGF) and its receptor have been a major focus of basic research and drug development in the field of oncology, including the treatment of non-small cell lung cancer (NSCLC). Approaches targeting VEGF include monoclonal antibodies and vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs). Bevacizumab (Avastin) is an anti-VEGF recombinant humanized monoclonal antibody. A very recent randomized phase III trial demonstrated a statistically significant advantage in median survival favouring the combination of bevacizumab plus chemotherapy versus chemotherapy alone in the treatment of advanced non-squamous NSCLC. This study represents the first evidence of superior efficacy of targeted therapy combined with chemotherapy over chemotherapy alone in the treatment of NSCLC. ZD6474 is an orally bioavailable inhibitor of VEGFR-2 tyrosine kinase. First evidences of antitumor activity and its excellent toxicity profile make it a promising targeted agent for the treatment of NSCLC. A recent phase I/II study examined the combination of Epidermal Growth Factor Receptor (EGFR)-TKI erlotinib and bevacizumab in patients with non-squamous stage IIIB/IV NSCLC. Data on antitumor activity of this combination have to be considered very promising. Clinical trials of multiple targeted therapy may represent the second generation studies in the treatment of NSCLC.     

Effect of VEGF,P53 and telomerase on angiogenesis of gastric carcinoma tissue

Objective:To investigate the effect of vascular endothelial growth factor(VEGF),P53 and telomerase on angiogenesis in gastric carcinoma tissue.Methods:A total of 95 surgical resection samples of gastric cancer tissue after pathological diagnosis are collected to observe the VEGF,P53 and telomerase expression using immunohistochemical methods.Relationship between their expression and its influence on angiogenesis in gastric carcinoma tissue were analyzed.Results:Microvascular density(MVD)and the expression of VEGF,P53 and telomerase were positively correlated.Expression of VEGF and P53 protein were related to tumor type and lymph metastasis,and also a correlation was observed between P53 and VEGF.The telomerase expression had no correlation with VEGF,and P53.Conclusions:VEGF angiogenesis has a angiogenesis promoting effect on gastric cancer tissue development and plays an important role in tumor generation and metastasis.Mutant P53 promotes the tumor angiogenesis generation by adjusting VEGF.Telomerase has a certain role in promoting activity of angiogenesis through different way rather than P53.     

Anti-angiogenesis therapy in pancreatic carcinoma

Pancreatic adenocarcinoma is a leading cause of cancer death in the United States and represents a challenging chemotherapeutic problem. The treatment of advanced pancreatic cancer with gemcitabine has only modest activity with a small survival benefit, and toxicity continues to be a major obstacle. New therapeutic strategies that notably lack cross resistance with established treatment regimens are much needed in pancreatic cancer. One such approach is the pharmacological control of angiogenesis that represents a novel approach to the management of pancreas cancer, since the pathological development of vascular supply is a critical step for tumor growth and may affect its prognosis. Since pancreatic carcinoma show strong tumor neoangiogenesis, overexpression of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, in pancreatic cancer and consequently are highly vascularized, the role of anti-angiogenic therapies is under exploration at present. Hence, this review covers the summary of the development of anti-angiogenesis as anti-antitumor therapy in pancreatic carcinoma, including matrix-metalloproteinase inhibitors (MMPIs), such as marimastat and BAY 12-9566, anti-VEGF agent, bevacizumab (Avastin, Genentech, South San Francisco, CA, USA), celecoxib (a cyclooxygenase-2 inhibitor), thalidomide and others. Role of markers of angiogenesis in predicting response to therapy is also discussed.     

Sex-steroid regulation of vascular endothelial growth factor in breast cancer

Angiogenesis is a key event, which occurs in both normal and pathological expansion of tissues and provides the nourishment necessary for growth. The role of angiogenic growth factors in breast pathology is rapidly gaining recognition since scientists and clinicians realized that these factors could function as molecular targets event 550 209822 for controlling tumor expansion. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. Although significant advances have been made in understanding the sex-steroid-dependent regulation of this key factor, the role of VEGF in controlling breast tumors is not well understood. In this review, I discuss recent studies describing the role of the female sex steroids estrogens and progestins in the regulation of VEGF in breast cancer cells. Furthermore, I present a summary of recent studies from other biological systems (mainly focused on tumor biology) directed towards providing us with a better understanding of the regulation of classical VEGF and VEGF receptors. I propose that by extending such studies we will gain deeper insights into how we might combat the progression of breast cancer by controlling hormone-dependent angiogenesis within tumor tissue. I believe that information gained from such studies will permit us to target angiogenic growth factors and their initiated signal transduction pathways in a more precise and selective manner, and thereby to control the formation of new blood vessels that fuel the rapid growth of breast tumors. Finally, it is my hope that the concepts discussed here will help elucidate molecular targets in the hormone-dependent angiogenesis pathway that will ultimately allow us to overcome anti-hormone resistance and to provide insights into how we might pursue the concept of chemoprevention by considering 'angioprevention' as the end point.     

Autocrine vascular endothelial growth factor signalling in breast cancer. Evidence from cell lines and primary breast cancer cultures <Emphasis Type="Italic">in vitro</Emphasis>

Inhibition of angiogenesis has become a major target in experimental cancer therapies. Vascular endothelial growth factor (VEGF) and its receptors are essential for breast cancer progression and relevant targets in experimental anti-angiogenesis. VEGF, produced by carcinoma cells, acts in a paracrine fashion on endothelial cells and displays autocrine activity on carcinoma cells. Breast cancer cells express VEGF-A, VEGF-B, VEGF-C and their receptors VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and neuropilin (NP-1/NP-2). VEGF-A triggers cellular signalling, an invasive phenotype of certain breast cancer cell lines and influences cell survival. However, such an autocrine VEGF/VEGFR signalling loop remains to be established. We demonstrate production of VEGF-A in cell lines MDA-MB-468, T47d, MCF-7, HBL-100 and in a primary breast cancer culture. Moreover, these cells showed baseline VEGFR-2 tyrosine-phosphorylation that could be enhanced by VEGF-A stimulation. In addition, VEGF-A leads to increased phosphorylation of ERK1/2 and Akt indicating that VEGF-A stimulation plays a crucial role in the regulation of cell growth, apoptosis, survival and differentiation. Moreover, we have established a novel breast cancer cell culture MW1 that expresses high levels of VEGF-A. We demonstrate that VEGFR-2 on the surface of breast cancer cells is functional and is capable of being stimulated by external VEGF-A. VEGF-A production by and VEGFR-2 activation on the surface of breast cancer cells indicates the presence of a distinct autocrine signalling loop that enables breast cancer cells to promote their own growth and survival by phosphorylation and activation of VEGFR-2. Moreover, this autocrine loop represents an attractive therapeutic target. Correspondence to: Melanie Weigand, Department of Cancer Biology, University of Massachusetts Medical School, LRB-470X, 364 Plantation St, Worcester, MA, USA. Tel: +1-508-856-2579; Fax: +1-508-856-1310; E-Mail: melanie_weigand@hotmail.de     

CD146 is a coreceptor for VEGFR-2 in tumor angiogenesis

CD146 is a novel endothelial biomarker and plays an essential role in angiogenesis; however, its role in the molecular mechanism underlying angiogenesis remains poorly understood. In the present study, we show that CD146 interacts directly with VEGFR-2 on endothelial cells and at the molecular level and identify the structural basis of CD146 binding to VEGFR-2. In addition, we show that CD146 is required in VEGF-induced VEGFR-2 phosphorylation, AKT/p38 MAPKs/NF-κB activation, and thus promotion of endothelial cell migration and microvascular formation. Furthermore, we show that anti-CD146 AA98 or CD146 siRNA abrogates all VEGFR-2 activation induced by VEGF. An in vivo angiogenesis assay showed that VEGF-promoted microvascular formation was impaired in the endothelial conditional knockout of CD146 (CD146(EC-KO)). Our animal experiments demonstrated that anti-CD146 (AA98) and anti-VEGF (bevacizumab) have an additive inhibitory effect on xenografted human pancreatic and melanoma tumors. The results of the present study suggest that CD146 is a new coreceptor for VEGFR-2 and is therefore a promising target for blocking tumor-related angiogenesis.     

VEGF kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases

Tumor growth depends on blood supply, requiring the development of new vessels, and vascular endothelial growth factor (VEGF) plays a central role in neoangiogenic processes. For this reason, VEGF represents a target for the development of new therapeutic antiangiogenic molecules. Clinical trials using anti-VEGF mAbs such as bevacizumab have validated the efficacy of this therapeutic approach but have also revealed adverse effects. Here we report that a VEGF-derived immunogen, consisting of a heterocomplex of a murine (m)VEGF and keyhole limpet hemocyanin, called "mVEGF kinoid," triggered a strong Ab immune response in mice. The anti-VEGF Abs inhibited both the proliferation of human umbilical vein endothelial cells cultured in the presence of mVEGF and the binding of mVEGF to its receptor-2 Flk-1. In mVEGF kinoid-immunized BALB/c mice challenged with syngeneic CT26 colorectal tumor cells, the number and size of lung metastases were significantly decreased. In human (h)VEGF kinoid-immunized BALB/c mice, high levels of serum Abs to hVEGF were present, and purified IgG from these mice decreased by > or =50% the tumor growth of human A673 rhabdomyosarcoma cells and HT29 colon carcinoma xenografted in Swiss nude and NOD/SCID mice, respectively. Tumor cell growth inhibition was similar to that observed in mice receiving therapeutic doses of bevacizumab. These experiments suggest that a therapeutic vaccine containing VEGF kinoid may represent a strategy for safely combating VEGF-dependent neovascularization and metastases occurring in malignant tumors.     

Variability of vascular endothelial growth factor in normal human breast tissue in vivo during the menstrual cycle

Exposure to sex steroids increases the risk of breast cancer, but the mechanisms are poorly understood. Angiogenesis is crucial in tumor development and progression. Very little is known about the regulation of angiogenesis in the normal breast. Vascular endothelial growth factor (VEGF) has a key stimulatory role in angiogenesis. Interferon-inducible protein 10 (IP-10) is a potent inhibitor of angiogenesis in vivo. These factors function in autocrine/paracrine pathways; therefore, direct measurements in the target tissue are needed. I measured VEGF and IP-10 in normal human breast tissue in situ in healthy women, using microdialysis, in the follicular and luteal phase of the menstrual cycle. In breast tissue, VEGF levels increased in the luteal phase, compared with the follicular phase (17.8 +/- 4 pg/ml to 34 +/- 9 pg/ml, P < 0.05). Plasma VEGF did not show a cyclic variation (10.6 +/- 2.8 pg/ml vs. 14.6 +/- 3.5 pg/liter, P = 0.3). IP-10 levels did not vary during the menstrual cycle either in breast tissue (65 +/- 17 pg/ml vs. 75 +/- 21 pg/ml, P = 0.6) or in plasma (64 +/- 7 pg/ml vs. 81 +/- 10 pg/ml, P = 0.06). The data suggests that, in the luteal phase, VEGF and IP-10, in the normal human breast, exhibit a proangiogenic profile. This may be one mechanism by which sex steroids contribute to breast cancer development.