Nonangiogenic tumors are harmless

There is an early stage of neoplastic development when tumors are not yet able to recruit new microvascular endothelial cells and cannot induce angiogenesis. As a result, most human tumors remain in situ and dormant at a microscopic size and are harmless to the host.⁴

Beginning of angiogenesis research

Hypothesis: tumor growth depends on angiogenesis

In 1971, Folkman proposed a new hypothesis that tumor growth is angiogenesis dependent.¹ This hypothesis suggested that tumor cells and vascular endothelial cells within a neoplasm may constitute a highly integrated ecosystem and that endothelial cells may be switched from a resting state to a rapid growth phase by a “diffusible” chemical signal from tumor cells. An additional speculation was that angiogenesis could be a relevant target for tumor therapy (i.e., antiangiogenic therapy). These ideas were based on experiments Folkman and Frederick Becker performed in the early 1960s, which revealed that tumor growth in isolated perfused organs was severely restricted in the absence of tumor vascularization (Figure 1).^19–23

These concepts were not widely accepted at the time. Another obstacle to research on tumor angiogenesis was the conventional wisdom at that time that any new vessels induced by a tumor, `such as new vessels in a wound, would become established and thus could not be made to involute. From this assumption, scientists concluded that antiangiogenic therapy could never regress a tumor; therefore, it would be fruitless to try to discover angiogenesis inhibitors. Eventual acceptance of Folkman’s 1971 hypothesis, and the development of angiogenesis research as a field, was subsequently facilitated by a number of advances including the ability to reproducibly culture vascular endothelial cells, the discovery of endogenous angiogenesis proteins, and the identification of drugs able to block angiogenesis in vitro and in vivo.^24–28

Throughout the 1970s, laboratory studies were devoted to demonstrating that tumor vessels were new proliferating capillaries; the sequential steps of the angiogenic process could be identified; qualitative and quantitative bioassays for angiogenesis could be developed; viable tumor cells released diffusible angiogenic factors that stimulated new capillary growth and endothelial mitosis in vivo, despite the arrest of tumor cell proliferation by irradiation; necrotic tumor products were not angiogenic per se; and angiogenesis itself could be inhibited.^29–32 Today the field has broadened to include a wide spectrum of basic science disciplines, from developmental biology to molecular genetics, as well as a variety of clinical specialties, including oncology, cardiology, dermatology, gynecology, ophthalmology, and rheumatology.

Experimental evidence

By the mid-1980s, considerable experimental evidence had been assembled to support the hypothesis that tumor growth is angiogenesis dependent. The idea could now be stated in its simplest terms: “Once tumor take has occurred, every further increase in tumor cell population must be preceded by an increase in new capillaries that converge upon the tumor”.³³ The hypothesis predicted that if angiogenesis could be completely inhibited, tumors would become dormant at a small, possibly microscopic, size.²² It forecasted that while the presence of neovascularization would be necessary but not sufficient for expansion of a tumor, the absence of neovascularization would prevent expansion of a primary tumor mass beyond 1–2 mm³ and restrict a metastasis to a microscopic dormant lesion (Figure 1).

The hypothesis that tumors are angiogenesis dependent has been supported by a large body of preclinical evidence,³⁴ including pharmacological and genetic studies, as well as clinical studies discussed in a later section. Some of the observations supporting the angiogenic hypothesis included:

1. 1. Tumors implanted into subcutaneous transparent chambers grow slowly before vascularization, and tumor volume increases linearly. After vascularization, tumor growth is rapid and tumor volume may increase exponentially.^{35, 36}
   
2. 2. Tumors grown in the vitreous of the rabbit eye remain viable but are restricted to diameters of less than 0.50 mm for as long as 100 days. Once such a tumor reaches the retinal surface, it becomes neovascularized and within 2 weeks can undergo a 19,000-fold increase in volume over the avascular tumor.³⁷
   
3. 3. The limit of oxygen diffusion is approximately 100–200 µm. Tumor cells that exceed these distances from a capillary vessel become necrotic, as determined by intravital microscopy of tumors in transparent skin chambers in mice (Figure 2).³⁸
   
4. 4. In transgenic mice that develop carcinomas of the β cells in the pancreatic islets, large tumors arise from a subset of preneoplastic hyperplastic islets but only after they have become vascularized.⁸
   
5. 5. In colon carcinomas arising in rats after carcinogen exposure, there is an early phase (tumor diameter < 3.5 mm) during which the tumor is temporarily supplied by preexisting host microvessels that are dilated and widened.³⁹ This stage is similar to “co-option” of blood vessels.⁴⁰ Subsequently, new capillary vessels sprout and proliferate (angiogenesis), which leads to increasing microvessel density and is accompanied by rapid tumor growth.
   
6. 6. A neutralizing antibody to VEGF was administered to mice bearing tumors that induced angiogenesis solely by VEGF.⁴¹ Tumor growth was inhibited by more than 90%. The antibody had no effect on the tumor cells in vitro.⁴² This observation has been replicated using a variety of different means to block VEGF, including a fusion protein engineered from VEGF receptors (VEGF trap).⁴³ Similar results were seen with an antibody directed against another angiogenic factor, bFGF.⁴⁴

The role of angiogenesis in preneoplasia and early tumorigenesis

In the experiments with isolated perfused organs by Folkman and colleagues more than four decades ago, the growth of tumors was severely restricted in diameter in the absence of angiogenesis.^{20, 45} This and other studies led to the proposal that the growth of solid tumors is dependent on new capillary sprouts (termed angiogenesis), and that without angiogenesis solid tumors might become completely dormant.¹ This raised the possibility that angiogenesis might be a therapeutic target not only for the treatment of advanced cancers, but potentially for chemoprevention as well. In recent years, angiogenesis has been validated as a therapeutic target for advanced cancers through a growing body of preclinical and clinical studies. The study of angiogenesis inhibitors for blocking the earliest steps in the development of cancer (chemoprevention) has lagged behind, despite the strong support for the concept provided by preclinical studies (reviewed in Refs ⁴⁶ and ⁴⁷).

Preclinical studies demonstrating a role for angiogenesis in early tumorigenesis

In the 1970s it was shown by Gullino⁴⁸ that precancerous tissues demonstrate signs of early angiogenesis, and it was suggested that blocking this process may be used to prevent cancer. Later, using transgenic mouse models of tumorigenesis, Hanahan and colleagues demonstrated that some premalignant lesions undergo “angiogenic switch” early in carcinogenesis, reflecting a change in the net balance between angiogenic stimulators and inhibitors.^{8, 9} VEGF and matrix metalloproteinase-9 (MMP-9) were shown to play important roles in the angiogenic switch in these models.^49–51 In a murine model of pancreatic islet carcinoma, four different antiangiogenic agents were tested (TNP-470, endostatin, angiostatin, or the MMP inhibitor BB-94) and had distinct activity profiles in terms of their ability to prevent tumor formation (chemoprevention), slow the growth of small tumors (early intervention), and regress established tumors.^{8, 52, 53} In another study, established mammary cancer cells (i.e., previously transformed cells but not primary tumors) were implanted in a dorsal skinfold window chamber in rats, and were found to induce angiogenesis long before the tumor population would have reached the limiting size of a nonneovascularized tumor, that is, 0.2–2 mm. These and other preclinical studies suggest that angiogenesis is an early and critical step in tumorigenesis, and that antiangiogenic agents may inhibit tumor progression and growth.

Preneoplasia is associated with increased angiogenesis in human tumors

Studies from human clinical specimens provide further support for the hypothesis that angiogenesis occurs early in tumor progression, typically during the preneoplastic stage. In cervix cancer, an initial mild increase in vessel density has been detected in the early dysplastic [cervical intraepithelial neoplasia (CIN) I] stage. Mid–late dysplasias (CIN II–III) exhibited a readily apparent angiogenic switch, wherein new vessels became densely apposed along the basement membrane underlying the dysplastic epithelium.^{54, 55} Biopsies from lung cancer patients and high risk individuals have shown that preneoplastic lesions ranging from hyperplasia and metaplasia to carcinoma in situ are associated with increased microvessel density in the surrounding mucosa.^56–58 A distinctive pattern known as angiogenic squamous dysplasia⁵⁷ has also been identified. The specific angiogenic stimulators in bronchial preneoplastic lesions have not been established but elevated levels of VEGF,⁵⁶ EGFR (epidermal growth factor receptor),⁵⁹ and COX-2⁶⁰ have been observed.

Role of angiogenesis in the metastatic spread of cancer

In addition to its role in enabling the growth of small premalignant or malignant lesions, angiogenesis contributes to the hematogenous metastatic spread of tumors. This spread may be facilitated by the presence of “mosaic” blood vessels in tumors, in which both endothelial and tumor cells form the luminal blood surface, facilitating the shedding of tumor cells into the circulation. In one study, approximately 15% of vessels in a colon cancer xenograft model were mosaic vessels in which tumor cells appeared to directly contact the luminal vessel surface without endothelial cells acting as a barrier.⁶¹ Similar numbers of mosaic tumor vessels were detected in human tumor biopsies. These observations suggest that the irregular architecture and function of tumor vessels may facilitate tumor cell shedding into the circulation and metastatic spread.

Micrometastases also appear to be dependent on angiogenesis in order to progress into clinically evident tumors. They may remain dormant in distant sites for an extended period of time but a small fraction of these acquire an adequate blood supply to permit the development of tumors; angiogenesis inhibitors can inhibit this process in a variety of murine models.^62–67 The continued growth of both the primary tumor and metastases thereafter depends on the maintenance of an adequate blood supply. In theory, angiogenesis inhibitors may therefore offer benefit when used for chemoprevention as well as in treatment of early stage, occult metastatic, or advanced disease.^{46, 68, 69}

Taken together, these preclinical and clinical studies provide evidence that the induction of angiogenesis is an early and important step in tumor progression, likely occurring in precancerous lesions, and is involved in metastatic spread. For these reasons, angiogenesis is a rational target for chemoprevention. Additional studies will be needed to elucidate the key regulators of early angiogenesis, and to identify the different types of antiangiogenic agents that may be optimal for chemoprevention, advanced disease, and other applications.

The discovery of diffusible factors stimulating tumor angiogenesis

The observation in the 1970s that tumors implanted into the avascular cornea or onto the vascularized chick chorioallantoic membrane induced an ingrowth of new capillaries indicated that tumors released diffusible angiogenic factors.⁹ This result motivated the development of in vitro and in vivo bioassays to guide the search for tumor-derived angiogenic factors.⁷⁰

Fibroblast growth factors

bFGF (or FGF-2) was the first angiogenic protein to be isolated and purified from a tumor (1982), followed shortly by acidic FGF (aFGF or FGF-1).^{28, 71, 72} Acidic and bFGFs stimulate endothelial cell mitosis and migration in vitro and are among the most potent angiogenic proteins in vivo. They have high affinity for heparin and heparan sulfate.⁷³ The expression of bFGF receptors is very low. A wide variety of cancer types secrete bFGF, including those of the central nervous system, sarcomas, genitourinary tumors, and even endothelial cells in the tumor vasculature. Identification of an FGF-binding protein secreted by tumors into the extracellular matrix may illuminate a mechanism of tumor mobilization of stored FGFs.^74–76 Furthermore, some tumors recruit macrophages⁷⁷ and activate them to secrete bFGF,⁷⁸ whereas others attract mast cells, which, because of their high content of heparin, could sequester bFGF. In spontaneous tumors that arise in transgenic mice, aFGF and bFGF are exported into conditioned medium by angiogenic tumor cells but not by preangiogenic cells in earlier stages of tumor progression.^{38, 79} bFGF also interferes with adhesion of leukocytes to endothelium, and it has been suggested that tumors that elaborate bFGF may produce a form of local immunologic tolerance.^80–82 In mouse models of cancer, inhibition of the FGFR signaling pathways results in inhibited tumor angiogenesis and reduced tumor growth.⁸³

Abnormally elevated levels of bFGF are found in the serum and urine of cancer patients and in the cerebrospinal fluid of patients with different types of brain tumors.^{84, 85} High bFGF levels in renal carcinoma correlated with a poor outcome.⁸⁶ Moreover, bFGF levels in the urine of children with Wilms’ tumor correlate with stage of disease and tumor grade.⁸⁷

In addition to contributing to tumor angiogenesis, multiple FGFR fusion proteins have been identified on diverse cancers including lung squamous cell, bladder, glioblastoma, and thyroid cancer, and appear to be oncogenic drivers as pharmacologic inhibition of FGFR suppresses the growth of FGFR fusion-positive tumor models.⁸⁸ Additional reports have shown FGFR1 to be amplified in a subset of small cell lung cancers among other malignancies, and tumor cells harboring FGFR amplification are sensitive to target inhibition.⁸⁹ In these settings, inhibition of FGFRs will therefore have both direct antitumor cell effects and indirect tumor cell effects through targeting of the tumor vasculature.

Vascular endothelial growth factor (VEGF) family

Dvorak first proposed that tumor angiogenesis is associated with increased microvascular permeability.⁹⁰ This led to the identification of vascular permeability factor (VPF).^91–93 VPF was subsequently sequenced by Ferrara and colleagues and in 1989 was reported to be a specific inducer of angiogenesis called VEGF.^92–94 At the same time, a novel angiogenic protein was first isolated and purified from a tumor (sarcoma 180) in the Folkman lab, and in a collaboration with Ferrara was shown to be VEGF.⁹⁴ Since then, more than 40 angiogenic inducers have been identified, most as tumor products (Table 1).^{3, 95} VEGF is an endothelial cell mitogen and motogen that is angiogenic in vivo.^96–98 Its expression correlates with blood vessel growth during embryogenesis and is essential for development of the embryonic vascular system.^{99, 100} VEGF expression also correlates with angiogenesis in the female reproductive tract, and in tumors.^{49, 101–103} VEGF is a 40–45 kDa homodimeric protein with a signal sequence secreted by a wide variety of cells and the majority of tumor cells. VEGF exists as five different isoforms of 121, 145, 165, 189, and 206 amino acids, of which VEGF₁₆₅ is the predominant molecular species produced by a variety of normal and neoplastic cells. Two receptors for VEGF are found mainly on vascular endothelial cells, the 180 kDa fms-like tyrosine kinase (Flt-1)¹⁰⁴ and the 200 kDa human kinase insert domain-containing receptor (KDR) and its mouse homolog, Flk-1.¹⁰⁵ VEGF binds to both receptors, but KDR/Flk-1 transduces the signals for endothelial proliferation and chemotaxis.^106–109 Other structural homologs of the VEGF family include VEGF-B, VEGF-C, VEGF-D, and VEGF-E.^{110, 111} VEGF-C and VEGF-D bind to Flt-4, which is preferentially expressed on lymphatic endothelium.^{112, 113}

Klagsbrun et al. discovered that neuropilin-1, a neuronal guidance molecule, is a coreceptor for VEGF₁₆₅, but not for VEGF.^{114, 115} This finding provides a molecular mediator that coordinates growth in the vascular system with the nervous system. Other neuronal guidance proteins and/or their receptors are also angiogenesis regulators. For example, neuropilin is a receptor for VEGF and for semaphorin. Semaphorin repels neurite outgrowth and is also an angiogenesis inhibitor. Ephrins are neural guidance molecules, but also genetically determine arteries and veins during embryogenesis. EphrinB2 is expressed by tumor vascular endothelium. Perhaps in part because of the overlap between regulators of the nervous and vascular systems, angiogenesis in brain tumors has distinctive characteristics such as high interstitial fluid pressure (IFP) and low oxygen tension that contribute to the malignant behavior of these tumors.¹¹⁶

Neuropilin is not a tyrosine kinase receptor and is expressed on nonendothelial cells, including tumor cells. This allows VEGF that is synthesized by tumor cells to bind to their surface. Surface-bound VEGF could make endothelial cells chemotactic to tumor cells, or it could act in a juxtacrine manner to mediate co-option of microvessels by tumor cells. Neuropilin also binds placenta growth factor-2 (PlGF-2), and heparin is essential for the binding of VEGF₁₆₅ and PlGF-2 to neuropilin-1.^{40, 117} The natural cell surface polysaccharide in vivo is heparan sulfate, not heparin. Heparan sulfate may act as a template to accelerate the interaction of VEGF or PlGF-2 with VEGF. VEGF expression by tumors is upregulated by hypoxia and is often elevated near areas of tumor necrosis.^118–121 Hypoxia activates hypoxia-inducible factor-1 (HIF-1), which binds to the hypoxia response element (HRE sequence) in the VEGF promoter, which leads to VEGF mRNA transcription.^{106, 120} Independent of this, hypoxia stabilizes the VEGF mRNA.

VEGF signal transduction

While VEGF-A binds VEGFR1 with a higher affinity than VEGFR-2, the majority of biological effects of VEGF-A on tumor endothelium are thought to be mediated through VEGFR-2. Upon ligand binding, VEGFR-2 dimerizes, resulting in activation of the tyrosine kinase and autophosphorylation of residues including Tyr⁹⁵¹, Tyr⁹⁹⁶, Tyr¹⁰⁵⁴, Tyr¹¹⁷⁵, and Tyr^1214.122 Phosphorylation of these residues induces the activation of signal transduction molecules including PI3K, phospholipase C-γ (PLC-γ), Akt, Src, Ras, and MAPK (Figure 3). Phosphorylation of Tyr¹¹⁷⁵ results in the binding and phosphorylation of PLC-γ, which subsequently promotes the release of Ca²⁺ from internal stores and activation of protein kinase C (PKC). PKC activation and Ca²⁺ mobilization are considered to be critical for VEGF-A-induced cell proliferation and nitric oxide production, respectively.¹²³

The PI3K pathway is paramount in the regulation of cell proliferation, survival, and migration. VEGF-A has been shown to promote phosphorylation of the p85 subunit of PI3K and enhance PI3K enzymatic activity. The mechanism by which VEGF-A results in activation of PI3K remains unclear, although studies have implicated a role for Src kinases, β-catenin, and VE-cadherin.^{124, 125} VEGFR-2-induced activation of PI3K results in accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP₃), which induces phosphorylation of Akt/PKB. Once activated, Akt/PKB phosphorylates and thus inhibits proapoptotic proteins BAD and caspase-9.

Members of the Src family kinases, Src, Fyn, and Yes, are expressed in endothelial cells. Following VEGFR-2 autophosphorylation, T cell-specific adapter (TSAd) binds Tyr⁹⁵¹ and then associates with Src. Src kinases control actin stress fiber organization and may mediate VEGF-A-induced PI3K activation. Ligand binding to VEGFR-2 also initiates activation of the Ras pathway, triggering signaling through the Raf-1–MEK–ERK signal cascade¹²² known to be important in growth factor-induced cell proliferation. This activation may occur through multiple routes.^{122, 126}

Biological function of VEGF

In its initial discovery, VEGF was identified as a mediator of vessel permeability.^{110, 127} This capacity to render small veins and venules hyperpermeable is a critical function of VEGF. VEGF is indeed one of the most potent regulators of vascular permeability, and the observation that tumor-associated blood vessels are hyperpermeable is attributed to tumor-secreted VEGF. While the mechanisms by which VEGF increases microvascular permeability are incompletely understood, it may be at least in part due to VEGF-induced endothelial fenestrations,¹⁰⁰ opening of junctions between adjacent endothelial cells,¹²⁸ and through a calcium-dependent pathway involving nitric oxide (NO) production.^129–131

In addition to its effect on vascular permeability, VEGF is a survival factor for endothelial cells, inhibiting endothelial cell apoptosis through activation of the PI3K–Akt pathway¹³² and increases in the antiapoptotic protein bcl-2.¹³³ In vivo, VEGF blockade has been demonstrated to cause increases in apoptosis of immature, nonpericyte-covered vessels.¹³⁴ VEGF is an endothelial cell mitogen though VEGFR-2 mediates signal transduction through Erk1/2, JNK/SAPK, and possibly PKC.^{133, 135} While VEGF is not as potent an endothelial cell mitogen as other factors such as bFGF, it has a broader range of activity for processes critical for angiogenesis. VEGF induces expression of MMPs and serine proteinases involved in degradation of the basement membrane, which is necessary for endothelial cell sprouting and invasion.¹³³ In addition, VEGF facilitates endothelial cell migration through FAK and p38 MAPK-induced actin reorganization.^{3, 128, 133, 136}

Circulating VEGF may be one of the angiogenic signals by which tumors recruit bone marrow-derived cells, including endothelial progenitors and myeloid cells whose recruitment is thought to be mediated by VEGFR-2 and VEGFR-1, respectively.^137–141 A growing body of evidence suggests that VEGFR-bearing bone marrow-derived cells contribute to initiating tumor formation, by creating a “metastatic niche”, and/or help promote tumor angiogenesis^142–146 although there appear to be VEGFR-1-independent mechanisms as well.¹⁴⁷ Circulating endothelial and myeloid cells are further being studied as potential biomarkers, as noted later in the chapter, and may contribute to resistance to VEGF pathway inhibitors.^{148, 149} It is worth noting that not all VEGF in the circulation may be tumor derived. VEGF serum concentrations closely correlate with platelet counts in cancer patients. VEGF is stored in platelets, and is transported by and released from them.¹⁵⁰ Furthermore, Pinedo and colleagues report that higher platelet counts correlate with a worse prognosis for cancer patients.^{151, 152} Therefore, it is possible that for those types of tumors that recruit bone marrow-derived endothelial cells, communication from tumor to bone marrow may be mediated in part by the VEGF in circulating platelets.

Angiopoietins

Angiopoietin-1 (Ang1) is a 70 kDa ligand that binds to a specific tyrosine kinase expressed only on endothelial cells, called Tie2 (also called Tek). Ang1 ligand binds Tie2 expressed on ECs, and Tie2 subsequently dimerizes and is phosphorylated. Activated Tie2 triggers activation of PI3K and Ras/Raf/MEK, promoting EC survival and proliferation/migration (Figure 4).¹⁵³ A ligand for Tie1 has not been elucidated.^{114, 154–157} Like VEGF, Ang1 is an endothelial cell-specific growth factor. Ang1, however, is not a direct endothelial mitogen in vitro. Rather, it induces endothelial cells to recruit pericytes and smooth muscle cells to become incorporated in the vessel wall. Pericyte and smooth muscle recruitment is mediated by endothelial production of PDGF-BB (and probably other factors) when Tie2 is activated by Ang1.¹⁵⁸ There is increased vascularization in mice that overexpress Ang1 in the skin.¹⁵⁷ The vessels are significantly larger than normal and the skin is reddened. The vessels are not leaky and there is no skin edema, in contrast to dermal vessels of mice overexpressing VEGF. In double transgenic mice expressing both Ang1 and VEGF in the skin, dermal angiogenesis is increased in an additive manner, but the vessels do not leak.¹⁵⁹ This model closely approximates angiogenesis in healing wounds (i.e., relatively nonleaky vessels with pericytes and some perivascular smooth-muscle cells contained in the vascular wall). In contrast, tumor vessels are leaky and thin walled with a paucity of pericytes. Angiopoietin-2, produced by vascular endothelium in a tumor bed, blocks the Tie-2 receptor and acts to repel pericytes and smooth muscle.¹⁵⁴ Nevertheless, tumor vessels remain thin “endothelium-lined tubes” even though some of these microvessels reach the diameter of venules. High levels of Ang2 are associated with metastases in melanoma patients, and in preclinical models, Ang2 promotes metastatic formation.^{160, 161} A key point is that angiopoietins and VEGF together play a role in angiogenesis, and the activity of both is context dependent with different activities observed in angiogenic versus mature vessels. Because VEGF plays a key role in the proangiogenic activity of Ang2, using combination inhibitors targeting both the Ang/Tie2 and VEGF receptor pathways may provide clinical benefit, a concept that has begun to be explored. However, given that angiopoietins can apply either pro- or antitumorigenic effects, depending on the context, the best targeting approach remains unclear.¹⁵³

Other factors regulating angiogenesis

Interleukin-8 (IL-8) is a proinflammatory chemotactic cytokine produced by monocytes, macrophages, and tumor cells.¹⁶² IL-8 induces endothelial cell proliferation and chemotaxis as well as promotes endothelial cell survival.^{162, 163} The effects of IL-8 are mediated through interactions with two cell-surface G protein-coupled receptors, CXCR1 and CXCR2, and activation of subsequent downstream signaling molecules including PI3K and MAPK.¹⁶⁴ Hepatocyte growth factor/scatter factor (HGF/SF) is the ligand for c-Met.¹⁶⁵ Originally identified as a mitogen for primary cultured hepatocytes, HGF has since been shown to facilitate tumor angiogenesis. c-Met is expressed on endothelial cells, and activation by tumor-secreted HGF/SF augments matrix degradation and endothelial cell invasion. NOTCH proteins and ligands are also elevated in several cancers, and NOTCH signaling has been shown to both promote and suppress tumor growth depending on the tumor type.¹⁶⁶ Interactions between NOTCH and its ligand DLL4 promote tumor angiogenesis through an intricate cross-regulation of NOTCH and VEGF/VEGFR signal transduction pathways.^{167, 168}

Endogenous inhibitors of angiogenesis

Endogenous angiogenesis inhibitors block vascular endothelial cells from proliferating, migrating, or increasing their survival in response to a spectrum of proangiogenic proteins, including VEGF, bFGF, interleukin-8, PDGF, and PD-ECGF. Natural angiogenesis inhibitors include interferon-α, interleukin-12, platelet factor 4, TSP-1, angiostatin, endostatin, arrestin, canstatin, tumstatin, PEX (MMP-2), pigment epithelium-derived factor, and antiangiogenic antithrombin III (an internal fragment of antithrombin III, named aaAT).^{27, 169–175}

The first clue to the existence of endogenous angiogenesis inhibitors came from the discovery that interferon-α inhibited endothelial cell migration and that platelet factor 4 inhibited endothelial proliferation.^176–178 Both were subsequently shown to inhibit angiogenesis.^176–180 However, Bouck and her colleagues were the first to show that a tumor could generate an angiogenesis inhibitor; they subsequently proposed that the angiogenic phenotype was the result of a net balance of endogenous inhibitors and stimulators of angiogenesis.¹⁸¹ A nontumorigenic hamster cell line became tumorigenic in association with the loss of a suppressor gene and concomitant with the onset of angiogenic activity. The nontumorigenic line secreted high levels of an angiogenesis inhibitor, a truncated form of TSP-1, that decreased by about 96% in the tumorigenic cells.¹⁸² TSP-1 was shown to be regulated by the wild-type tumor suppressor p53 in fibroblasts and in mammary epithelial cells.^{183, 184} Loss of p53 function in the transformed derivatives of these cells dramatically decreased the level of the angiogenesis inhibitor. Restoration of p53 upregulated TSP-1 and raised the antiangiogenic activity of the tumor cells. Deletion of TSP-1 led to accelerated growth of breast cancers that arise spontaneously in neu-transgenic mice.¹⁸⁵ The demonstration that the switch to an angiogenic phenotype involved a negative regulator of angiogenesis generated by the tumor per se suggested to Folkman a unifying angiogenic mechanism to explain a well-recognized but previously unsolved clinical and experimental phenomenon: the inhibition of tumor growth by the tumor mass. In this phenomenon, “the removal of certain tumors, for example, breast carcinomas, colon carcinomas, and osteogenic sarcomas can be followed by rapid growth of distant metastases”.^{186, 187} Postoperative chemotherapy was introduced mainly to prevent or delay the growth of secondary metastases. Several studies in terminally ill patients demonstrated the suppression of a secondary tumor by a primary tumor.¹⁸⁸ In melanoma, partial spontaneous regression of the primary tumor may be followed by rapid growth of metastases, and when ionizing radiation is employed to regress a small cell lung cancer, distant metastases may undergo rapid growth.^{173, 189} Once it was demonstrated that a tumor could generate a negative regulator of angiogenesis, then it became clear that a primary tumor, while stimulating angiogenesis in its own vascular bed, could possibly inhibit angiogenesis in the vascular bed of a distant metastasis.¹⁸² However, at least two conditions would be necessary: first, the primary tumor (i.e., the first tumor to grow) would need to generate an angiogenic promoter in excess of an inhibitor in its own vascular bed, and, second, the putative inhibitor would need to have a longer half-life in the circulation than the angiogenic promoter. After arriving in the Folkman laboratory in 1991, O’Reilly and his colleagues validated this hypothesis by discovering angiostatin, endostatin, and antiangiogenic antithrombin over the next 8 years.^{171–173, 190, 191}

Angiostatin

Angiostatin is a 38 kDa internal fragment of plasminogen that was purified from the serum and urine of mice bearing a subcutaneous Lewis lung carcinoma that suppressed growth of its lung metastases by inhibiting their angiogenesis (Figure 5).¹⁷¹ Angiostatin is not secreted by tumor cells but is generated through proteolytic cleavage of circulating plasminogen by a series of enzymes released from the tumor cells. At least one of these tumor-derived enzymes, urokinase plasminogen activator (uPA), converts plasminogen to plasmin, whereas a phosphoglycerate kinase from hypoxic tumor cells then reduces the plasmin so that it can be converted to angiostatin by one of several different metalloproteinases.¹⁹² Several potential mechanisms of its antiangiogenic activity have been reported. These include induction of endothelial apoptosis^193–195; suppression of endothelial cell migration induced by plasmin binding to α_vβ₃-integrin^{196, 197}; inhibition of HGF-induced signaling via c-MET, Akt, and ERK-1/2¹⁹⁸; and downregulation of VEGF expression in tumor cells.¹⁹⁹ This implies that angiostatin may act as a direct and an indirect inhibitor of angiogenesis. In support of this, when angiostatin was delivered to mice bearing a variety of tumors, it showed significant efficacy either alone or in combination with endostatin, as a fusion protein of angiostatin and endostatin, or in combination with interleukin-12.^199–202

Other pathways regulating angiogenesis

Oncogenes as regulators of angiogenesis

It is widely established that activation of proto-oncogenes can induce tumorigenesis. In tissue culture, expression of activated oncogenes increases cell proliferation and decreases apoptosis.²³⁹ While these changes contribute to tumorigenesis by altering the equilibrium between cell proliferation and apoptosis, there is considerable evidence that this alone is not sufficient to produce expansive tumor growth.^{240, 241} Rather, tumors must also acquire an adequate vascular supply to grow beyond 1–2 mm in diameter. In support of this concept, published reports have demonstrated that transfection of tumor cells with oncogenes results in enhanced production of proangiogenic molecules,²⁴² and the in vivo growth of oncogene-driven tumors can be restricted with angiogenesis inhibitors.²⁴³ In lung cancer patients, for example, mutations in K-Ras, p53, and EGFR are among the oncogenes that have been linked to angiogenesis. Oncogenes shown to play a role in regulating angiogenic factors include:

Angiogenesis inhibitors versus vascular targeting agents

Angiogenesis is the formation of new vessels from preexisting vasculature. Angiogenesis inhibitors are therefore typically targeted at the early stages in this process, including endothelial sprouting and survival mechanisms, which are often VEGF dependent. Vascular targeting agents (VTAs), also known as vascular disrupting agents (VDAs), differ from angiogenesis inhibitors in that they target established abnormal tumor vasculature.²⁶³ VTAs can induce rapid collapse of tumor vasculature, and their effects on normal vasculature can cause a host of side effects including acute coronary syndromes, thrombophlebitis, and tumor pain. None of these agents are currently in routine clinical use for cancer but several have undergone clinical testing. Vadimezan (ASA404) initially demonstrated positive results in combination with chemotherapy as treatment for patients with lung cancer in a phase II trial.²⁶⁴ Unfortunately, these findings were not validated in a subsequent phase III study evaluating vadimezan in combination with carboplatin/paclitaxel as first-line treatment in NSCLC, as no significant differences were demonstrated in overall survival (OS) between vadimezan and placebo arms [median OS 13.4 vs 12.7 months, respectively (hazard ratio; HR = 1.01; p = 0.535)]. Given the lack of clinical benefit, the development of vadimezan in lung cancer has been discontinued by Novartis.²⁶⁵ ABT-751 is another VDA with preclinical anticancer activity, which has failed to demonstrate an improvement in progression-free survival (PFS) in a phase I/II study in combination with docetaxel as second-line treatment for NSCLC.²⁶⁶

Antiangiogenic effects of chemotherapy and other therapeutic agents

Multiple preclinical studies have suggested that several “classical” chemotherapeutic agents may also have potent antiangiogenic or vascular-targeting effects, which may be enhanced by low dose, frequent dosing schedules (metronomic dosing).^267–269 Several of these regimens are undergoing clinical evaluation. It also appears that certain chemotherapy agents, particularly taxanes and vinca alkaloids, may have relatively more potent antiangiogenic effects than other drugs,²⁶⁹ which may help explain why there are differences in the degree of enhancement observed when antiangiogenic agents such as bevacizumab are added to chemotherapy. This prompted further examination of a wide variety of drugs which were initially thought to target primarily tumor cells. Many of these were subsequently found to also have antiangiogenic effects, prompting the term “accidental antiangiogenics”.²⁷⁰

Targeting VEGF pathway

VEGF is the prototypic member of a family of structurally related, homodimeric growth factors that includes PlGF, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. As described earlier, VEGF family members bind to a family of transmembrane receptor tyrosine kinases that include VEGFR-1 (Flt-1), VEGFR-2 (KDR, Flk-1), and VEGFR-3 (Flt-4) (Figure 3). The effects of VEGF or VEGFR on vascular permeability and endothelial proliferation, migration, and survival are thought to be primarily mediated by VEGFR-2, while VEGFR-3 is primarily expressed on lymphatic endothelium (reviewed in Ref. ). Agents targeting the VEGF pathway include monoclonal antibodies that bind the ligand (i.e., bevacizumab) or block the receptor (ramucirumab). In addition, many small molecule receptor tyrosine kinase inhibitors (RTKIs) have been developed to target critical signaling pathways involved in angiogenesis. These RTKs, which include vatalanib (PTK787), vandetanib (ZD6474), sunitinib (SU11248), axitinib (AG-013736), and nintedanib (BIBF 1120), have been the focus of many preclinical and clinical studies. Because of the structural similarity of the different receptor tyrosine kinase domains, RTKIs typically inhibit multiple receptors such as PDGFR, c-KIT, FGFR, and Axl in addition to VEGFR. These multitargeting agents have demonstrated higher anticancer activity compared to agents with single targets. Furthermore, they are orally available, and thus more convenient for patients. Conversely, the off-target effects may have additive toxicities. These profiles of receptor specificity for each inhibitor, as well as their pharmacokinetics and potency for receptor inhibition, are likely to be the key determinants of their clinical activity. Representative agents targeting the VEGF pathway that are currently FDA approved are listed in Table 2.

AML, acute myelogenous leukemia; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; FDA, Food and Drug Administration; EU, European Union; GBM, glioblastoma multiforme; NSCLC, non-small cell lung cancer; PlGF, placental growth factor; PDGFR, platelet-derived growth factor receptor; RCC, renal cell carcinoma; RTKI, small molecule receptor tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

Combinations of antiangiogenics with chemotherapy: mechanisms for enhanced antitumor activity

Preclinical and clinical studies have demonstrated that antiangiogenic therapy improves the outcome of cytotoxic therapies.^{271, 272} This finding is paradoxical. It was initially expected that targeting the tumor vasculature would drastically diminish the delivery of oxygen and therapeutics to the solid tumor, producing hypoxia that would cause many chemotherapeutics, as well as radiation, to be less effective.^{271, 272} Tumor vasculature is known to be structurally and functionally abnormal, with tortuous, highly permeable vessels. Blood flow within these intratumoral vessels is nonuniform. Proliferating tumor cells compress blood and lymphatic vessels resulting in a microenvironment typified by interstitial hypertension (elevated hydrostatic pressure outside the blood vessels), acidosis, and hypoxia.^{273, 274} This deficient vascular network and interstitial hypertension impair drug delivery to tumor cells. Moreover, hypoxia renders tumor cells resistant to radiation and several cytotoxic agents, and increases genetic instability selecting for tumor cells that have a greater metastatic potential. In addition to the direct effects on tumor cells, hypoxia leads to vascular abnormalization by signaling via PHD2 in tumor endothelial cells²⁷⁵ and, along with the low pH within the tumor microenvironment, weakens the cytotoxic functions of tumor infiltrating immune cells. Collectively, the abnormal vasculature within solid tumors creates a significant barrier to delivery and efficacy of cancer therapeutics.

One proposed mechanism to explain the enhancement in efficacy of chemotherapy by antiangiogenic therapy is that these agents have the potential to “normalize” tumor vessels. In animal models of cancer, inhibition of VEGF signaling results in a vasculature network that more closely resembles vessels within normal tissue. This “normalized” vasculature is less leaky, is less dilated, and has less tortuous vessels with a more normal basement membrane and increased pericyte coverage. Concurrent with these changes in vascular morphology, within the tumor there is a decrease in IFP, increased oxygenation, and improved delivery of concurrently administered chemotherapeutics (Figure 6).^276–284 Evidence from a Phase I/II clinical trial in locally advanced rectal carcinoma patients receiving bevacizumab and chemotherapy (with radiation) corroborates preclinical findings. Bevacizumab treatment was associated with a decrease in tumor IFP and an increase in mature, pericyte-covered vessels.^{285, 286}

Antiangiogenic agents in combination with radiotherapy

A growing body of evidence suggests that antiangiogenic therapy may enhance the efficacy of radiotherapy for solid tumors. There are several proposed mechanisms through which this enhancement may occur. First, radiotherapy may act by “normalizing” the disorganized and hyperpermeable vasculature in tumors.^{271, 287} Vessel normalization would permit a more effective delivery of oxygen to tumor tissue, resulting in a reduction in tumor hypoxia and augmenting radiation-induced cytotoxicity in part by increasing the formation of oxygen-free radicals. This reduction in hypoxia may be transient, however, as prolonged use of antiangiogenic agents may eventually also reduce the “normalized” vessels within tumors resulting in an inadequate intratumoral vascular supply, such that the tumor would again be hypoxic with reduced radiosensitivity. This concept is supported by mouse xenografts showing the existence of a period of time (“normalization window”) during which radiation therapy used in conjunction with an antiangiogenic agent is most effective.^{283, 288}

Antiangiogenic therapy may act to enhance the antiendothelial effects of radiotherapy. While it was initially assumed that the antitumor effect of radiotherapy was due to direct action on tumor cells, more recent evidence has demonstrated that radiotherapy also induces endothelial cell apoptosis.^288–290 Other studies have confirmed these results.²⁹¹ Thus, the exact mechanism(s) of interaction between antiangiogenic agents and radiation remains unclear.

Nevertheless, several preclinical studies demonstrate that antiangiogenic agents can synergize with or potentiate the effects of radiotherapy.^292–296 Blockade of VEGF signaling on endothelial cells may render the tumor-associated vasculature more sensitive to radiotherapy. In addition, radiation induces VEGF expression, which contributes to radioresistance by blocking radiation-induced endothelial cell apoptosis.^{297, 298}

One possible cause for concern in testing combinations of antiangiogenic therapy and radiotherapy is the observation from preclinical studies that at least some of the toxicities of radiotherapy such as intestinal radiation damage may also be due to endothelial apoptosis.²⁹⁹ In lung cancer patients treated with the combination of bevacizumab, chemotherapy, and radiotherapy, tracheoesophageal fistulas have been observed.³⁰⁰ Clearly additional studies will be needed to assess the feasibility and efficacy of these combinations.

Renal cell cancer

One tumor type for which VEGF pathway inhibitors are particularly useful, even when used as monotherapy, is metastatic RCC. These tumors are often marked by inactivation of the von Hippel-Lindau gene leading to overexpression of VEGF and other angiogenesis mediators (reviewed in Ref. 306). In randomized studies, bevacizumab, sorafenib, and pazopanib have been shown to significantly prolong time to progression compared to placebo controls.^307–309 Sunitinib and axitinib also demonstrated substantial antitumor activity in metastatic RCC, with 25–45% ORR in phase III testing.^{304, 310, 311}

Cytokine therapies (interferon-α- and interleukin-2-based therapies) had long been the mainstay of therapy for RCC, so it was rational to evaluate anti-VEGF therapy in the context of such agents. Phase III trials examining four small molecule RTKIs of VEGF receptors (sorafenib, sunitinib, pazopanib, and axitinib) and monoclonal anti-VEGF therapy (bevacizumab) have been reported, and consistently favor the use of anti-VEGF therapy compared to interferon.

Colorectal cancer

Advanced colorectal cancer represented the first human cancer in which a Phase III trial demonstrated clinical benefit, and to this day represents among the best studied of human solid tumors with regard to antiangiogenic therapy. In 2004, results of a phase III, randomized, placebo-controlled study were reported comparing standard IFL chemotherapy (irinotecan, fluorouracil, and leucovorin) with or without bevacizumab in patients with previously untreated metastatic colorectal cancer.³⁰¹ Patients treated with IFL plus bevacizumab had a significantly longer OS and PFS as well as a higher ORR. This trial provided definitive evidence that the addition of an angiogenesis inhibitor to chemotherapy could prolong survival and, based on the results of this trial, bevacizumab received approval from the US Food and Drug Administration (FDA) for use in combination with fluorouracil-containing chemotherapy as first-line treatment for metastatic colorectal cancer.

Subsequent studies have built upon this original finding. In a trial of previously treated patients with colorectal cancer, patients treated with bevacizumab combined with FOLFOX4 chemotherapy had a prolonged survival compared to those treated with FOLFOX4 alone,³³² although the magnitude of this benefit (2.1 months) appeared to be smaller than that observed in the first-line trial (4.7 months), presumably related to the more advanced nature of the disease.

First-line studies of bevacizumab or placebo in combination with either FOLFOX or XELOX failed to demonstrate an improvement in OS (21.3 vs 19.9 months) despite improvements in PFS (9.4 vs 8.0 months).³³³ The mechanism for the discrepancy between this study and the pivotal IFL study has been attributed to the different cytotoxic backbone or practice patterns of treatment until progression of disease versus regimen interruptions for cumulative oxaliplatin toxicities. Several studies have evaluated the role of bevacizumab after an initial course of oxaliplatin- and fluoropyrimidine-based first-line chemotherapy. These “maintenance” regimens have demonstrated the benefit of bevacizumab when combined with fluoropyrimidines. The CAIRO-3 randomized study demonstrated that continued bevacizumab either alone or in combination with capecitabine provided a survival benefit compared to observation alone.

Cohort studies had suggested a benefit from continued VEGF inhibition after progression on first-line therapy with bevacizumab. The TML study evaluated second-line therapy with FOLFIRI or FOLFOX (as determined by the treating physician) with or without bevacizumab for patients who had previously progressed on first-line bevacizumab-containing regimen. The study demonstrated a modest but statistically significant survival advantage of 1.4 months, resulting in the addition of bevacizumab continuation to treatment guidelines.

A similar study was conducted with ziv-aflibercept, a fusion protein utilizing the extracellular domains of VEGFR1 and VEGFR2, thereby providing inhibition of both VEGF-A and PlGF. Patients who had been previously treated with an oxaliplatin regimen (which may have included bevacizumab) were randomized to FOLFIRI with bevacizumab or placebo.³³⁴ The primary endpoint of OS was met with a 1.4 month improvement (13.5 months vs 12.1 months), with an improvement in PFS and response rate. Grade 3 and 4 diarrhea and stomatitis were substantially higher in the ziv-aflibercept arm, consistent with the established role for PlGF in mucosal lining repair.³³⁵ The result of these two studies is the addition of new treatment options for second-line antiangiogenic therapy in combination with chemotherapy for patients who have previously progressed on bevacizumab-based first-line regimens.

Biomarker analyses of colorectal cancer trials have been largely unavailing. Expression levels of VEGF-A, thrombospondin, and microvessel density performed in a subset analysis of the original Hurwitz trial were not associated with clinical outcome.³³⁶

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