William B. Coleman and Gregory J. Tsongalis (eds.)The Molecular Basis of Human Cancer10.1007/978-1-59745-458-2_11

11. Novel Cancer Therapies Targeting Angiogenesis

Monte S. Willis¹ and Nancy Klauber-DeMore²

(1)

Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina School of Medicine, Chapel Hill, NC, USA

(2)

Department of Surgery, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425, USA

Nancy Klauber-DeMore

Email: demore@musc.educ

Keywords

Novel cancer therapiesAngiogenesisCancer progressionGrowth factorsReceptor tyrosine kinases

11.1 Tumors Overcome Growth Limitations by Directing the Formation of New Blood Supply

There are four general strategies by which cancers can enhance their blood supply. They can (1) stimulate angiogenesis, (2) utilize existing vessels directly, (3) induce vasculogenesis, and/or (4) form vasculogenic networks without vascular cells. The secretion of proangiogenic factors and/or inhibition of antiangiogenic factors induce vascular sprouting from preexisting capillaries and venules constitutes the process of angiogenesis. This is the most common way cancers gain access to the vasculature. Cancers may also utilize existing vessels directly by growing along beside them, as is the case in astrocytomas [1]. The strategy of vasculogenesis involves the formation of blood vessels from bone marrow precursors. Vasculogenesis differs distinctly from angiogenesis in that the source of the cells that make up the vessels are from the bone marrow and not from preexisting vessels. However, many of the soluble mediators that initiate this process, notably VEGF, parallel those found in angiogenesis [2]. Lastly, tumors themselves can form lumens which can be used to transport blood, lacking endothelial cells or other vascular components. The contribution of these types of networks to cancer progression has been debated [3, 4]. Multiple strategies may be in play in a particular tumor, depending on their stage and malignancy. We focus here on the major pathways of angiogenesis as its contribution is most common as applied to therapeutic intervention.

11.2 Evidence that Angiogenesis is Necessary for Cancer Progression

The initial evidence that angiogenesis is necessary for the growth of cancer came from studies transplanting cancer cells into the avascular corneas of rabbits [5]. In these studies, tumors did not grow in rabbit corneas before sprouting vessels were able to grow to connect to the tumor. Moreover, inhibiting vessel formation would prevent cancer growth beyond 0.4 mm [5]. Other investigators similarly found that tumors placed in chicken embryo chorioallantoic membranes shrank during the first three days after placement [6]. However, new vessel formation could be seen to form from existing vessels after the cancers were placed. When these new vessels connected to the tumor, cancer growth continued. These studies not only identified a significant role of vessel formation in cancer progression but also determined that cancers elicit the growth of vessels from existing vessels. This suggests that cancer release diffusible factors that initiate angiogenesis (vessel formation from existing vasculature) to continue and maintain their growth.

The ability of cancer cells to induce angiogenesis is not a constitutive trait. In mice, the ability to induce angiogenesis appears to be initiated by tumor progression in models of pancreatic cancer [7, 8] and dermal fibrosarcomas [9–11]. The initiation of a program to induce angiogenesis also occurs in the development of human tumors. Evidence for this comes from comparing vasculature in precancerous and cancerous lesions of the breast, where the microvessel density has been reported to be relatively increased [12]. Similarly, increased capillary density has been reported to increase in cervical squamous cell carcinoma as the stage progresses [13, 14], suggesting that as a cancer becomes more aggressive, its ability to stimulate angiogenesis increases. It is believed that the switch that cancer undergo to induce angiogenesis not only come from the malignant tissue itself, but also from the surrounding tissue and infiltrating immune cells [15]. The targets of these soluble factors are endothelial cells as well as other vascular cells in the microenvironment of the tumor [15]. The interplay between the soluble proangiogenic and antiangiogenic factors and their cognate receptors on the targets is the basis of tumor-induced angiogenesis necessary for tumors to continue their unrestrained growth. Each step in the angiogenic process presents an opportunity for targeted inhibition of angiogenesis. This review focuses on the main steps of angiogenesis for which targeted therapy is being developed.

11.3 Growth Factors/Receptor Tyrosine Kinases

11.3.1 Vascular Endothelial Growth Factor Family

An essential mediator of angiogenesis is the vascular endothelial growth factor (VEGF) family, which consists of five family members of secreted proteins (VEGFA, VEGFB, VEGFC, VEGFD, VEGFE), and platelet-derived growth factor (PDGF) [16], that bind and activate three receptor tyrosine kinases (VEGFR-1, VEGFR-2, and VEGFR-3). VEGFA stimulates endothelial cell proliferation, migration, tube formation, and vascular permeability. VEGFB was identified as an endothelial cell growth factor expressed in heart and skeletal muscle [17]; however, its function as an angiogenesis factor is not clearly defined. VEGFC and VEGFD play a critical role in lymphangiogenesis and expression has been correlated with the development of lymph node metastases [18]. Placental growth factor (PGF) promotes the survival of endothelial cells and modulates the activity of VEGF signaling [19].

Regulation of VEGF family genes expression is under the control of hypoxia, along with other forms of stress such as acidity and hypoglycemia, which stimulate transcription and increase mRNA stability, resulting in increased protein expression. Under normoxic conditions, prolyl residues in hypoxia-inducible factors (HIF) proteins are hydroxylated by prolyl hydroxylase in a reaction that uses molecular oxygen. Hydroxylation of HIF proteins targets them for ubiquitin-mediated proteolysis. Reduction in the concentration of oxygen decreases the efficiency of this process: HIF proteins are stabilized and therefore become available to bind to hypoxia-response elements in the promoters of target genes, thereby activating transcription [20].

VEGF proteins bind to receptor tyrosine kinases (VEGFR-1, VEGFR-2, and VEGFR-3) [21], which then mediate cell signaling resulting in the biologic effects of VEGF. VEGFR-1 (Flt-1) binds three of the VEGF family ligands, VEGF-A, VEGF-B, and PGF. Activation of VEGFR-1 results in embryonic vessel development, hematopoiesis, macrophage chemotaxis and recruitment of endothelial progenitor cells to tumor blood vessels from the bone marrow [22]. VEGFR-2 (Flk-1/KDR) is the key mediator of VEGF-stimulated tumor angiogenesis and is critical in embryonic vascular development. When VEGF ligands VEGFR-2, the receptor is phosphorylated and activates downstream signaling molecules including phospholipase C, protein kinase C, Raf, MAP kinase, PI3K, and FAK pathways, resulting in endothelial cell proliferation, migration, and tube formation, and anti-apoptosis [23]. VEGFR-3 binds VEGF-C and -D and is implicated in the formation of lymphatics in normal tissue and tumors [24].

In 2004 the humanized version of a monoclonal antibody to VEGFA, bevacizumab (Avastin; Genentech), became the first Food and Drug Administration (FDA)-approved antiangiogenic drug in the United States [18]. It was approved as a first-line treatment agent for metastatic colorectal cancer, in combination with 5-fluorouracil [25] and was subsequently approved for treatment metastatic non-squamous-cell lung cancer, breast cancer, and glioblastoma multiforme [26]. Ranibizumab (Lucentis; Genentech), another monoclonal antibody recognizing VEGFA, and pegaptanib (Macugen; Pfizer, New York, NY), a single-stranded nucleic acid aptamer that binds specifically to the heparin-binding domain of VEGFA165, are FDA-approved antiangiogenesis inhibitors in use for treating the wet type of age-related macular degeneration [27].

Additional FDA-approved drugs that block VEGF signaling are sorafenib and sunitinib, both receptor tyrosine kinase inhibitors, which are both administered orally. Sorafenib, in addition to blocking VEGFR signaling, also blocks FLT3, PDGFRB, and KIT signaling [28]. Similarly, sunitinib blocks signaling from VEGFR1-3, FLT3, RET, PDGFRA, and PDGFRB [28]. Sorafenib has been approved for unresectable hepatocellular carcinoma and advanced renal cell carcinoma, whereas sunitinib has been approved for gastrointestinal stromal tumors and metastatic renal cell carcinoma [18] and neuroendocrine tumors [29].

11.3.1.1 Fibroblast Growth Factor

Fibroblast growth factors (FGFs ) are involved in maintaining endothelial cell function. FGF1 and FGF2 promote endothelial cell migration and proliferation and stimulate angiogenesis [30]. FGFs produce their biological effects by binding to transmembrane tyrosine kinase receptors, FGFR1 through FGFR4 [31]. FGFR can activate PLC-γ, thereby stimulating the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). This, in turn, releases intracellular calcium and activates Ca²⁺-dependent PKCs. The activation of the PI3K-Akt cell survival pathway is one of the important biological responses induced by FGF2 in endothelial cells [32].

There are several inhibitors of FGF signaling in clinical trials, including FP-1039 (FGFR1:Fc), a soluble fusion protein consisting of the extracellular domain of human fibroblast growth factor receptor 1c (FGFR1) linked to the Fc portion of human IgG1. FP-1039 prevents FGFR1 ligands from binding to any of their cognate receptors within the family of seven FGF receptors, and may mediate both direct antitumor and antiangiogenic effects. Both E-3810 and TKI258 are dual VEGFR and FGFR tyrosine kinase inhibitors [30].

11.3.1.2 Notch

The Notch signaling pathway is important for cell-cell communication, involving gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch signaling has been directly implicated in tumor angiogenesis and in the process of activating dormant tumors. VEGFA also induces expression of the endothelium-specific Notch ligand Delta-like 4 (DLL4). When DLL4 activates the Notch signaling pathway in adjacent cells, the effect is to inhibit dorsal sprouting. When expressed in tumor cells DLL4 can activate Notch signaling in host stromal cells, thereby improving vascular function [33]. Inhibition of DLL4-mediated Notch signaling promotes a hyperproliferative response in endothelial cells, a process that leads to an increase in angiogenic sprouting and branching. Despite this increase in vascularity, tumors are poorly perfused, hypoxia increases, and cancer growth is inhibited. Neutralizing anti-Dll4 antibodies have been demonstrated to inhibit cancer growth in vivo [34]. These findings point to the Notch pathway as a potential therapeutic target.

11.3.1.3 TGFβ

TGFβ is a paracrine polypeptide with three homologous forms (TGFβ1, TGFβ2, and TGFβ3). TGFβ is produced in latent form as a zymogen and after secretion a latency associated peptide is proteolytically cleaved to release active TGFβ. Active TGFβ signals by binding to constitutively active type 2 receptors (TGFBR2) to activate type 1 receptors (TGFBR1) in a heteromeric complex that controls transcription through the action of a family of SMAD proteins [35]. TGF-β is a strong proangiogenic agent despite the fact that TGFβ causes growth arrest and apoptosis of endothelial cells in vitro. This paradoxical behavior may be explained by the fact that TGFβ activates the secretion of fibroblast growth factor 2, which acts as an autocrine signal to stimulate the expression of VEGF. VEGF, in turn, acts in an autocrine manner through its receptor VEGFR-2 to activate the MAPK pathway (specifically p38MAPK). However, TGFβ will reverse the protective action of VEGF, promoting apoptosis, which occurs in the pruning process, to form the final vascular network [36]. Endothelial cells subsequently become refractory to TGFβ-mediated apoptosis and TGFβ then directly promotes capillary lumen formation. Therapeutic approaches for targeting TGFβ signaling include antagonism of TGFβ ligand binding to the heteromeric receptor complex with isoform-selective antibodies, such as lerdelimumab (TGFβ2) and metelimumab (TGFβ1) or the pan-neutralizing antibody GC-1008, and intracellular inhibition of the type I TGFβ receptor kinase with small-molecule inhibitors, such as LY550410, SB-505124, or SD-208 [37].

11.3.1.4 Angiopoietin/Tie Receptors

Angiopoietins are another family of endothelial cell-specific molecules that play an important role in vessel growth, maintenance, and stabilization by binding to Tie receptors [38]. There are four types of angiopoietins: Ang-1, Ang-2, Ang-3, and Ang-4. The Tie1 receptor is highly expressed in embryonic vascular endothelium, angioblasts, and endocardium, and in adult tissues expressed strongly in lung capillaries tissues [39]. The Tie2 receptor takes part in vessel maturation by mediating survival signals for endothelial cells. Ang-1 is an agonist that promotes vessel stabilization in a paracrine fashion, whereas Ang-2 is an autocrine antagonist that induces vascular destabilization. Ang-2 is increased during vascular remodeling and is implicated in tumor-induced angiogenesis and progression [40]. AMG 386 is an investigational peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of Ang-1 and Ang-2 with their receptor, Tie2, and is being studied in clinical trials [41].

11.3.1.5 Epidermal Growth Factor

The epidermal growth factor (EGF ) family consists of 11 members which bind to one of four epidermal growth factor receptors (EGFR) [42]. All of the receptors, except HER3, contain an intracellular tyrosine kinase domain [43]. Activation of EGFR contributes to angiogenesis in xenograft models [44], in addition to cellular proliferation, survival, migration, adhesion, differentiation, and cancer metastasis [43]. Activation of the EGFR pathway upregulates the production of proangiogenic factors such as VEGF, and therefore the EGFR pathway is more of an indirect regulator of angiogenesis. There are three FDA-approved EGFR inhibitors: cetuximab and panitumumab, which are monoclonal antibodies, and erlotinib, a tyrosine kinase inhibitor that specifically targets EGFR [18].

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