[Cell Adhesion & Migration 3:2, 230-235; April/May/June 2009]; ©2009 Landes Bioscience
Brain tumors exhibit marked and aberrant blood vesl forma-tion indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vesls are ud for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players. The current angiogenesis inhibitors ud in clinical trials mostly target single angiogenic proteins and so far show limited effects on tumor growth. Besides the conventional angiogenesis inhibitors, RNA-bad inhibitors such as small-interfering RNAs (siRNAs) are being analyzed for their capacity to silence the message of proteins involved in neovascularization. More recently, a new family of non-coding RNAs, named angiomirs [microRNAs (miRNAs) involved in angiogenesis] has emerged. The small RNAs have the advantage over siRNAs in that they have the potential of silencing multiple messages at the same time and therefore they might become therapeutically relevant in a “one-hit multiple-target” context against brain tumor angiogenesis. In this review we will discuss the emerging technologies in anti-angiogenesis empha-sizing on RNA-bad therapeutics.
Brain Tumor Angiogenesis
Malignant gliomas are characterized by a marked increa in blood vesl formation (angiogenesis), which is crucial for tumor growth and colonization in the brain.1 Glioma blood vesls show endothelial cell proliferation which is a key feature of high grade gliomas in the WHO grading system.2,3 Increasing evidence supporting the critical
role of angiogenesis in the biological behavior of the tumors, and patients prognosis led to a variety of studies on basic mechanisms of tumor angiogenesis.4 Several factors are involved in this process which results in the recruitment, proliferation and alignment of the endothelial blood vesl cells through a complex interaction between the cells and tumor cells.1 Glioma cells are known to alter the mRNA profile of endothelial cells as shown by microarray analysis in co-cultures of human umbilical vein endothelial cells and glioma cells.5 Further, differences in levels of specific proteins have been obrved between normal and tumor endothelial cells, which contribute in part to the distinct angiogenic blood vesl morphology in tumors.6,7
Glioma cells clearly need the vasculature for the delivery of nutrients and oxygen for tumor growth. In addition, many reports demonstrated that high grade glioma cells may also u the vascu-lature for migration to different parts of the brain.8,9 High grade gliomas show two types of infiltration into the normal brain. The first is a diffu infiltrative migration of single cells into the brain parenchyma.
The cond type is a clear perivascular migration along the microvasculature. Brain tumor infiltration has been shown to be one of the main reasons for tumor recurrence.8,9 The infiltrated cells cannot be rected by surgery and are known to be resistant to current chemo- and radiotherapies.10,11 Increasing awareness of the importance of the tumor vasculature for glioma growth and migra-tion has led the effort to focus on developing novel anti-angiogenic drugs which are summarized in Figure 1A.12-16 T wo major pathways are undertaken for this type of therapy: (1) cutting the oxygen and nutrients supplies; and (2) limiting possible migration routes for infil-trating glioma cells. Brain tumor angiogenesis is regulated through a complex network of molecules which will be partly discusd here, offering veral potential points of intervention.
Conventional Angiogenesis Inhibitors
Glioma cells crete many pro-angiogenic factors.13,17 Interestingly, the cells also crete substances that could inhibit angiogenesis.17,18
*Correspondence to: Thomas Würdinger/ Bakhos A. Tannous; Molecular Neurogenetics Unit; Massachutts General Hospital-East; Building 149; 13th Street; Charlestown, MA 02129 USA; Tel.: 617.726.6026; Fax: 617.724.1537; Email: twurdinger@mgh.harvard.edu/ Tel.: 617.726.6026; Fax: 617.724.1537; Email: btannous@hms.harvard.edu
Submitted: 11/18/08; Accepted: 01/22/09
Previously published online as a Cell Adhesion & Migration E-publication: /journals/celladhesion/article/7910Special Focus: Angeogenesis in the Central Nervous System
Glioma angiogenesis
Towards novel RNA therapeutics
Thomas Würdinger 1,2,4,5 and Bakhos A. Tannous 1-4
1Molecular Neurogenetics Unit; Department of Neurology; 2Center for Molecular Imaging Rearch; Department of Radiology; and 3Department of Pathology; Massachutts
General Hospital; and 4Program in Neuroscience; Harvard Medical School; Boston, MA USA; 5Neuro-oncology Rearch Group; Cancer Center Amsterdam; Department of Neurosurgery; VU University Medical Center; Amsterdam, Netherlands
Abbreviations: GBM, glioblastoma; CNV , choroidal neovascularization; MRI, magnetic resonance im
aging; siRNAs, small-interfering RNAs; miRNAs, microRNAs; uPA, urokina-type plasminogen activator; uPAR, urokina-type plasminogen activator receptor; MMP-9, matrix metalloproteina-9; LNA, locked nucleic acid; HBMVECs, human brain microvascular endothelial cells; VEGF , vascular endothelial growth factor; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; shRNAs, short hairpin RNAs; TLR3, toll-like receptor 3; NF κB, nuclear factor κB; IL, interleukin
Key words: glioma, angiogenesis, anti-angiogenesis therapy, siRNA, miRNA, endothelial cells, blood vesls
Following rection and radiation combined with temozo-lomide treatment, the median time for glioma recurrence or progression is six months.11,19 The area of the recurrent tumor, defined as progression of the contrast enhanced lesion on a magnetic resonance imaging (MRI) scan, is in the majority of cas localized at the rection cavity of the primary tumor, where infiltrating tumor cells with a low proliferation rate reside. The phenomenon of fast relap may be explained by the hypothesis that a large primary tumor cretes not only stimulators of its own angiogenesis, but also produces angiogenesis inhibitors that inhibit neovas-cularization in more distal parts—and thus inhibit further
growth—of the infiltrated tumor cells.9,18,20 The u of angiogenesis inhibitors could therefore have a beneficial effect on glioma recur-rence.
A number of endogenous inhibitors of angiogenesis have been discovered. The most commonly studied is angiostatin, a polypep-tide of approximately 200 amino acids.21Angiostatin is produced by the cleavage of plasminogen, a plasma protein that is important for dissolving blood clots. Another commonly ud anti-angiogenic inhibitor is endostatin, a polypeptide of 184 amino acids.21 Endostatin is compod of the globular domain found at the C-terminal of Type XVIII collagen (a collagen found in blood vesls), which is cut off from the parent molecule. Understanding the function of collagen and collagen receptors can help in revealing the role of the extracel-lular matrix in glioma angiogenesis. Glioma cells express integrins, which are collagen receptors, by which they anchor the extracellular matrix to the outside of the cell membrane.22,23 It has been shown that the new blood vesls in tumors express a vascular integrin, designated alphavbeta3, which is not found on blood vesls of normal tissues.23 Endostatin significantly inhibited angiogenesis and tumor growth in veral orthotopic human brain tumor models, including the U87 mou model and the BT4C rat model.24,25 Unfortunately, clinical trials with endostatin showed that, although safe to u, it was not effective in 40 patients when given as a single agent in the treatment of advanced neuroen
docrine tumors.26,27 Monoclonal antibodies and small molecule drugs directed against integrins have shown promising results in mice and in clinical trials in humans.22,28,29 One of the promising integrin inhibitors is cilengitide, a potent αvβ3 and αvβ5 integrin inhibitor.22 Preclinical studies and clinical trials evaluating cilengitide showed that the inte-grin inhibitor is active and synergizes with radiotherapy in preclinical glioblastoma (GBM) models. In clinical trials for recurrent GBM, single-agent cilengitide showed antitumor benefits and minimal toxicity. Among newly diagnod patients with GBM, cilengitide combined with standard radiotherapy and temozolomide showed promising therapeutic respon with no incread toxicity. The results lead to a planned randomized Pha III trial.22 Further testing is required in order to analyze the final effectiveness of the newly developed antibodies and small molecules.
Cancer cells are also known to produce excess quantities of growth factors.1,13,17 The factors have autocrine stimulating capacities to induce tumor growth and endothelial neovascularization. Angiogenic growth factors acting on specific receptors are currently being studied extensively for anti-cancer drug development.16,30 Anti-angiogenesis drugs targeting growth factors like vascular endothelial growth factor (VEGF), or growth factor receptors like VEGFR, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), are being tested in clinical tri
resulting
als to determine their effect in reducing tumor blood vesl formation, or to normalize the aber-rant glioma blood vesl system, either alone or in combination with standard or experimental therapeutics. Recently, patients with newly diagnod WHO grade 3 malignant glioma were treated in a Pha II clinical trial with bevacizumab, a monoclonal antibody to VEGF, in combination with irinotecan.31 Evaluation with physical examina-tion and MRI showed that twenty patients (61%) had at least partial respon with limited adver events. This study concludes that the combination of bevacizumab and irinotecan is an active regimen with acceptable toxicity for patients with recurrent WHO grade 3 malignant gliomas and that anti-VEGF treatment is therapeutically relevant. Although the treatment was shown to be sub-optimal, further studies showing long term effects are warranted.
RNA-Bad Angiogenesis Inhibitors
Besides small molecules and antibodies, an additional class of RNA-bad inhibitors is arising. Currently, RNA interfer-ence (RNAi) is widely ud as a mechanism to silence individual genes.32,33There are marketed small-interfering RNA (siRNA) products being analyzed by ophthalmologists to prevent age-related macular degeneration, caud by aberrant neovascularization in the eye, and resulting in completely blinding of some of the older patients.34,35 Further, veral groups are focusing on using siRNAs to knock-down specific genes i
nvolved in glioma angiogenesis. A recent study by Gondi et al.36,37showed that intraperitoneal injection of plasmids encoding short hairpin RNAs (shRNAs) directed against urokina-type plasminogen activator (uPA) and its receptor (uPAR), as well as proteina matrix metalloproteina-9 (MMP-9) resulted in significant reduction of glioma angiogenesis and tumor growth in preclinical mou models. The results also suggest that the intrap-eritoneal route of administration can be ud for successful delivery of RNA-bad drugs. Kargiotis et al.38 showed that the delivery of siRNA against MMP-2 using adenovirus vectors resulted in impaired invasion, as well tumor-induced angiogenesis leading to inhibition of tumor growth. Niola et al.39 showed that siRNAs against VEGF reduced GBM angiogenesis in xenograft mou model. Clinical trials are also ongoing in which siRNAs are being delivered into target cells in order to prevent neovascularization. For example, siRNAs designed to target and to abrogate the expression of VEGF or its receptor are currently being tested. Clinical trials were initiated to analyze intraocular injection of siRNAs in patients with chor-oidal neovascularization (CNV), a late stage of age-related macular degeneration. Preclinical CNV mou models were ud to show
Figure 1. Different class of angiogenesis inhibitors.
the same time may have veral advantages, mainly bad on the u of miRNAs to regulate genes
in a more natural phenomenon shaped by evolution. Several methods have been developed to alter single miRNA expression levels, thereby changing the expression pattern of multiple genes. The action of overexpresd intra-cellular miRNAs can be inhibited by the delivery of anti-n single-strand RNA-bad oligonucleotides. Inhibition of specific endogenous miRNAs has been achieved by the administration of synthetic anti-n oligonucleotides that are complementary to the mature endogenous miRNAs.62-68 Improved miRNA inhibitors are now available containing various chemical modifications. The modifica-tions were shown to have different effects on specificity and efficacy.69 In addition, efficient locked nucleic acid (LNA) anti-n oligonu-cleotides have been designed and tested for their silencing capacity in vitro and vivo.70-73 More recently, antagomirs, miRNA inhibitors conjugated to cholesterol groups, have been developed. Antagomirs have been described to efficiently inhibit miRNA activity in various organs as well as in tumor vasculature, when injected into mice, and may have therapeutic potential.74-76
Various laboratories are currently working on improving the targeting of miRNA modulators into the cells of interest. Since tumors take up small molecules more efficiently than normal cells, they form a logical target for the delivery of miRNA mimics and miRNA inhibitors. Nevertheless, the delivery of inhibitors of the small RNAs to cancer cells in order to block tumor growth remains challenging, m
ore likely due to the heterogeneity of cancer cells. On the other hand, tumor blood vesls offer an attractive target for the delivery of miRNA inhibitors or precursor/mimics in order to reduce tumor growth since the cells are not transformed and conquently less heterogeneous.
Recently, a new group of miRNAs related to angiogenesis was identified and named angiomirs.76,77 The miRNAs were shown to play a significant role in neovascularization. Specifically, miR-296 controls the overexpression of growth factor receptors on tumor endothelial cells. Our group showed that inhibition of miR-296 resulted in reduced tumor angiogenesis in vitro and in vivo.76 We showed that glioma cells and angiogenic growth factors (including VEGF), induce miR-296 in primary human brain microvascular endothelial cells (HBMVECs) in culture, as well as in primary tumor endothelial cells isolated from human brain tumors. Exposing endothelial cells to glioma cells or a cocktail compod of angiogenic growth factors resulted in incread tubule formation in an in vitro matrigel angiogenesis assay (Fig. 2A). miR-296 knockdown resulted in inhibition of both glioma-induced tubule formation as well as endothelial cell migration (Fig. 2B and C). On the other hand, overexpression of miR-296 using pre-miR-296 molecules resulted in an incread tubule formation in a similar manner to endothelial cells expod to glioma cells or growth factors (Fig. 2D). Further, inhibition of miR-296 inhibited tumor vascularization in mice.76 Angiomirs,
such as miR-296, can regulate a broad range of ‘natural’ mRNA targets and therefore they might prove more efficient for fine-tuning cellular switch programs than siRNAs.78 Wang et al.,61 Fish et al.56 and Kuhnert et al.58 showed that miR-126 plays an important role in developmental angiogenesis and vascular integrity. Knock-out experiments in zebrafish and mice demonstrated aber-rant vasculature attributed to the abnce of miR-126 molecules.
that injections with naked VEGF siRNA (Bevasiranib) or VEGFR1 siRNA (AGN211745; siRNA-027) suppresd lar-injury-induced CNV .40-43
Intriguingly, it was recently found that siRNAs in general can cau toll-like receptor 3 (TLR3) activation, irrespective of their quence.44 TLR3 is a member of the toll-like receptor family of pattern recognition receptors of the innate immune system and recognizes double-stranded RNA, resulting in activation of the transcription factor nuclear factor-kappaB (NF κB) thereby inducing interferons as an immune defen mechanism.45 Kleinman et al.44 showed that endothelial TLR3 activation by siRNAs had a direct effect on neovascularization. This work reveals an unexpected aspect of RNAi for angiogenesis which demonstrates that any 21-nucleotide or longer double-strand RNA could affect angiogenesis and that the two siRNAs tested in clinical trials could owe their anti-angiogenic effect not to target knock-down only, but also becau of TLR3 activation.44,46 Since ma
ny different proteins are involved in angio-genesis, it is unlikely that single-target strategies are sufficient to treat complex process such as brain tumor angiogenesis inhibition. Although the activation of TLR3 by siRNAs can have anti-angio-genic effect, it probably will need complementation from other anti-angiogenic molecules in order to inhibit brain tumor angiogen-esis. One possibility may be to u a cocktail of multiple siRNAs to silence multiple genes, besides activating TLR3. A recent study in a mou model for collagen-induced arthritis showed that siRNA directed against different targets can act synergistically. Khoury et al.47 showed that weekly injections of siRNAs against either inter-leukin 1 (IL-1), IL-6 or IL-18 reduced the collagen-induced arthritis, however, the most striking therapeutic effect was obrved when a combination of all three siRNAs were delivered. The disadvantage of such a complex multi-component silencing cocktail is that it might result in numerous off-target effects (the unexpected silencing of ‘good’ genes) and therefore ems currently not yet practically feasible.48
The discovery of microRNAs (miRNAs) and their role in the complex control of gene expression may offer new strategies to change gene expression profiles. miRNAs are part of a highly conrved group of small non-coding RNAs that can block mRNA translation.49-51 One important principle of miRNA function is that the complementarity between their nucleotides and their targets is not comple
te and therefore a single miRNA can regulate multiple protein species by interacting with up to hundreds of different target mRNAs.52,53 miRNAs are usually about 22 nucleotides long, which is relatively short for a genetic transcript. The human genome is estimated to contain thousands of them. miRNAs can efficiently ba pair with their protein-coding mRNA targets in order to block their expression. This biological mechanism is described as a “one-hit multiple target” mechanism, which enables a single miRNA to control whole ts of protein-coding genes.52,53 All the accumu-lating evidence indicates that regulation of miRNA expression is very important for proper development and differentiation of many cell types and tissues and that deregulated miRNA expression is a common feature of angiogenesis.52-61
Angiogenesis modulation in a “one-hit multiple target” fashion may be a more attractive strategy then single-target anti-angiogenesis therapy. The u of our own miRNAs to regulate many genes at
cells. Currently, studies are focusing on elucidating tumor-induced changes in miRNA expression in endothelial blood vesl cells. Our group found that 5% of the human miRNAs are deregulated upon exposure of endothelial cells to glioma cells.76 The results support the hypothesis that glioma cells stimulate endothelial blood vesl formation at least in part by altering the expression levels of specific endothelial miRNAs. The discovery of the small non-coding RNAs has incread our kno
wledge regarding the complex control of gene expression involved in regulating tumor growth and is an attractive strategy for anti-angiogenesis therapy, especially in view of a “one-hit multiple target” approach.
Acknowledgements
We would like to acknowledge the Steve Kaplan Fellowship from the American Brain T umor Association and we would like to thank Dr. David P . Noske for critical reading of the manuscript, Mrs. Suzanne McDavitt and Mrs. Lee-Ann Tjon for skilled editorial assistance.
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