Tumor angiogenesis: past, present and the near future
Robert S. Kerbel
Sunnybrook and Women's College Health Sciences Centre, Division of Cancer
Biology Research, S-218 Research Building, 2075 Bayview Avenue, Toronto, Ontario
M4N 3M5, Canada
The concept of treating solid tumors by inhibiting tumor angiogenesis was first
articulated almost 30 years ago. For the next 10 years it attracted little
scientific interest. This situation changed, relatively slowly, over the
succeeding decade with the discovery of the first pro-angiogenic molecules such
as basic fibroblast growth factor and vascular endothelial growth factor (VEGF),
and the development of methods of successfully growing vascular endothelial
cells in culture as well as in vivo assays of angiogenesis. However, the 1990s
have witnessed a striking change in both attitude and interest in tumor
angiogenesis and anti-angiogenic drug development, to the point where a
remarkably diverse group of over 24 such drugs is currently undergoing
evaluation in phase I, II or III clinical trials. In this review I will discuss
the many reasons for this. These features, together with other recent
discoveries have created intense interest in initiating and expanding
anti-angiogenic drug discovery programs in both academia and industry, and the
testing of such newly developed drugs, either alone, or in various combinations
with conventional cytotoxic therapeutics. However, significant problems remain
in the clinical application of angiogenesis inhibitors such as the need for
surrogate markers to monitor the effects of such drugs when they do not cause
tumor regressions, and the design of clinical trials. Also of concern is that
the expected need to use anti-angiogenic drugs chronically will lead to delayed
toxic side effects in humans, which do not appear in rodents, especially in
short-term studies.
Abbreviations: -SMA, -smooth muscle action; bFGF, basic fibroblast
growth factor; TAF, tumor angiogenesis factor; VEGF, vascular endothelial growth
factor.
Roughly a decade before the inaugural issue of Carcinogenesis was published, Dr
Judah Folkman articulated several hypotheses regarding what he felt was the
critical importance of tumor angiogenesis in the development and metastatic
spread of tumors, and how therapeutic inhibition of such angiogenesis might be
exploited as a new and novel means of treating cancer (1,2). His ideas were
based not only on his own work, but also on some studies of a small number of
insightful investigators such as Algire and Chalkley (3), Greenblatt and Shubik
(4) and Warren (5). Indeed, it was Shubik who first coined the term `tumor
angiogenesis' (4). The fundamental points of Folkman's visionary hypotheses can
be summarized as follows: (i) most primary solid tumors probably go through a
prolonged state of avascular, and apparently dormant, growth in which the
maximum size attainable is ~1? mm in diameter. Up to this size, tumor cells can
obtain the necessary oxygen and nutrient supplies they require for growth and
survival by simple passive diffusion; (ii) these microscopic tumor masses can,
in some way, eventually switch on angiogenesis by recruiting surrounding mature
host blood vessels to begin sprouting new blood vessel capillaries which grow
toward, and eventually infiltrate the tumor mass, thus setting in motion the
potential for relentless expansion of the tumor mass and hematogenous metastatic
spread as well; (iii) the angiogenic switch was initially hypothesized to be
triggered by the ectopic production and elaboration by tumor cells of a growth
factor (6) called `tumor angiogenesis factor' (TAF) (7); (iv) it ought to be
possible to affect tumor growth by blocking tumor angiogenesis, e.g. by somehow
preventing TAF production (or its biologic function) or directly targeting the
vascular endothelial cells of newly formed, immature blood vessels. The latter
possibility presupposed that such tumor vessels and their constituent
endothelial cells would be sufficiently different, phenotypically speaking, from
the endothelial cells of more mature vessels such that a sufficient therapeutic
index could be achieved; (v) this kind of a therapeutic approach, if successful,
would not be curative in the usual sense, i.e. it would not eradicate all tumor
cells, but instead would either prevent any new expansion of tumor mass or, at
best, perhaps cause sustained regressions of established solid tumors to a size
of ~1? mm diameter where survival is possible without a blood vessel supply.
Such a therapeutic approach was designated as `dormancy-inducing' and meant to
control the disease in a chronic fashion.
These theories are widely accepted today; indeed, as Carcinogenesis approaches
its twentieth anniversary, tumor angiogenesis is clearly one of the most
exciting and visible areas of cancer research and therapeutics in clinical
oncology (8). Such was not the case in 1980. Back then there was, relatively,
only a handful of investigators working on tumor angiogenesis, and virtually no
companies interested in developing anti-angiogenic drugs. What has changed this
situation so dramatically such that there are now over 20 anti-angiogenic drugs
being tested in various phase clinical trials, as shown in Table I (9?2), and
numerous others in preclinical development?
Table I. Some endogenous inhibitors of angiogenesis
Name / Description
Thrombospondin-1 and internal fragments of thrombospondin-1 Thrombospondin is a
180 kDa, large, modular extracellular matrix protein (53)
Angiostatin A 38 kDa fragment of plasminogen involving either kringle domains
1?, or smaller kringle 5 fragments (58,163,164)
Endostatin A 20 kDa zinc-binding fragment of type XVIII collagen (59)
Vasostatin An N-terminal fragment (amino acids 1?0) of calreticulin (61)
Vascular endothelial growth factor inhibitor (VEGI) A 174 amino acid protein
with 20?0% homology to tumor necrosis factor superfamily (60)
Fragment of platelet factor 4 (PP4) An N-terminal fragment of PP4 (63)
Derivative of prolactin 16 kDa fragment of the hormone (57)
Restin NC10 domain of human collagen XV (165)
Proliferin-related protein (PRP) A protein related to the pro-angiogenic
proliferin molecule (166)
SPARC
cleavage product Fragments of secreted protein, acid and rich in cysteine (62)
Osteopontin cleavage product Thrombin-generated fragment ontaining an RGD
sequence (65)
Interferon /?nbsp;Well known anti-viral proteins (56)
Meth 1 and Meth 1 Proteins containing metalloprotease and thrombospondin
domains, and disintegrin domains in NH2 termini (65)
Angiopoietin-2 Antagonist of angiopoietin-1 which binds to tie-2 receptors
(39,44)
Anti-thrombin III fragment A fragment missing C-terminal loop of anti-thrombin
III (a member of the serpin family) (64)
Past and present
What follows are 10 of the most significant reasons (with no particular
significance attached to their order) for the explosive growth in tumor
angiogenesis research development of anti-angiogenic drugs for cancer treatment.
1. The discovery of basic fibroblast growth factor (bFGF) as the first
pro-angiogenic molecule
Despite the logic of Folkman's ideas (1,2,7), sceptics awaited the precise
identification of the hypothetical TAF molecule. This did not occur until the
mid 1980s, when Shing et al. (13) reported that basic fibroblast growth factor (bFGF,
also known as FGF-2) isolated from a chondrosarcoma could function as a
tumor-derived capillary growth factor and stimulate angiogenesis in various
models (14). However, a puzzling feature about bFGF is that it lacks a signal
sequence for secretion and generally remains intracellular, at least in cell
culture. This cast some doubt on its authenticity as a major inducer of tumor
angiogenesis (15). Nevertheless, the discovery provoked new interest in the
field, and there is no doubt that bFGF is recognized as a potent inducer of
angiogenesis, including in the clinic for non-cancerous diseases such as hind
limb ischemia and ischemic heart disease (16?8).
2. The discovery of vascular endothelial growth factor (VEGF), and
VEGF receptor tyrosine kinases on activated endothelial cells
VEGF was initially termed vascular permeability factor (VPF), and still retains
this term (15,19). Its first function (vascular permeability) was discovered by
Dvorak and colleagues (20,21) and was first molecularly defined by Ferrara et
al. (22,23). There are a number of families (VEGF, VEGF-B, VEGF-C, VEGF-D) of
which VEGF (or VEGF-A as it is sometimes called) is the most important; there
are several VEGF isoforms and a number (e.g. VEGF121 and VEGF165) are readily
secreted. Moreover, unlike bFGF (as the name implies), VEGF is a very specific
mitogen for vascular endothelial cells. It also functions as a potent
pro-survival (anti-apoptotic) factor for endothelial cells in newly formed
vessels (24?6) and indeed this may be one of its most significant functions.
VEGF is expressed by the vast majority of cancers (20,21), often at elevated
levels, and blocking its activity, e.g. by specific neutralizing antibodies to
VEGF or to VEGF receptors expressed by `activated' endothelial cells, can
inhibit experimental tumor growth in vivo, but not in vitro (27,28). This is
because of the highly elevated and restricted expression of two receptor
tyrosine kinases, called flk-1/KDR (also known as VEGF-receptor-2) and flt-1
(also known as VEGF receptor-1), by the endothelial cells of newly formed blood
vessels, which bind VEGF with high affinity (29). Thus tumor cells can `feed'
(induce) new blood vessels by producing VEGF which, in turn, can nourish the
tumor cells, an insidious and self-perpetuating paracrine loop. The possibility
of therapeutic disruption of this loop has stimulated intense interest in the
biotech and pharmaceutical industries, as well as academic centres, using agents
such as antibodies (28,30?2), VEGF-toxin conjugates (33), aptamers (30) and
small molecule VEGF receptor antagonists (34,35), among others.
Another significant feature of VEGF is that levels of this growth factor in
tumor cells can be significantly enhanced by hypoxia (36). This can occur
through both transcriptional and post-transcriptional mechanisms, e.g. mRNA
stabilization (37). Given the impact of hypoxia as a mediator of tumor
progression (38), and modulator of therapeutic responsiveness to agents such as
radiation, the hypoxia/VEGF connection has heightened awareness of angiogenesis
among certain segments of the cancer research and medical/radiation oncology
communities.
3. The discovery of the angiopoietins and their tyrosine kinase receptors
A second family of ligands and receptors specific for vascular endothelial cells
have been uncovered more recently (39). In this case, it was the receptors that
were discovered first, i.e. tie-2/tek and tie-1 (40,41). They remained as orphan
receptors until 1996, when the ligands for one of them (tie-2), called
angiopoietin-1 and angiopoietin-2 (ang-1 and ang-2), were discovered by
Yancopolous and colleagues (42,43). Of considerable interest is the fact that
although both ang-1 and ang-2 bind tie-2, ang-1 functions as an agonist, whereas
ang-2 behaves in a contrary manner (44). Indeed, ang-2 can cause regression of
newly formed vessels by endothelial cell apoptosis, unless VEGF is present, in
which case the two collaborate to promote angiogenesis (45). The ligand for
tie-1 remains unknown. Similar to VEGF and its receptors, the use of transgenic
and gene knockout mice has been a powerful methodology to uncover the functional
significance of the angiopoietin–angiopoietin receptor systems in both embryonic
vasculogenesis and angiogenesis (46). By way of example, embryonic mice which
are only partially VEGF deficient, i.e. express one VEGF allele, do not survive
in utero and express major defects in vasculogenesis and angiogenesis (46). Mice
made deficient in flk-1, flt-1 and tie-2 also do not survive in utero (46). In
the case of flk-1-deficient mice, endothelial cells are not produced, whereas
tie-2-deficient embryos produce endothelial cells and form primitive vascular
structures but these do not assemble into mature, stabilized vessels as a result
of a failure to recruit periendothelial support cells such as pericytes and
smooth muscle cells (46). These developmental biology and other gene knockout
studies have been particularly important contributions to the angiogenesis field
in general, and as such have had an accelerating effect on tumor angiogenesis
research as well. In particular, they have helped fuel interest in developing
drugs which target VEGF, VEGF receptors, or the tie-2 receptors (35,47).
4. The discovery of endogenous inhibitors of angiogenesis
Like the field of cancer genetics (48), where virtually all the emphasis in the
early years was placed on dominantly acting oncogenes which act as positive
acting stimulators of cell growth, research in angiogenesis over the first 15?0
years was also heavily focused on pro-angiogenic growth factor stimulators (14).
Later, in the cancer genetics field, the concept of (recessive) tumor suppressor
genes came to be accepted, i.e. genes which encode proteins that normally
function to block cell growth (or survival), and inactivation of which (e.g. by
mutation or chromosome loss) can result in a loss of this cellular `brake'
mechanism (48). It is now accepted that it is the combination of these two types
of event which promote cancer development and progression (48). The same appears
to be the case for (tumor) angiogenesis (49?1). Thus, a large, growing and
structurally diverse family of endogenous protein inhibitors of angiogenesis has
been discovered, e.g. thrombospondin-1 (52,53), interferon /?(54?6), the 16
kDa fragment of prolactin (57), angiostatin (58), endostatin (59), vascular
endothelial cell growth inhibitor (VEGI) (60), vasostatin (61), Meth-1 and
Meth-2 (62) and cleavage products of platelet factor 4 (63), or anti-thrombin
III (64), among many others (Table I ). Some of these are internal fragments of
various proteins which normally lack any anti-angiogenic activity (51,65), e.g.
angiostatin is one or more fragment(s) of plasminogen (58) and endostatin is a
fragment of type XVIII collagen (59), as summarized in Table I . Many of the
precursor proteins are components of the extracellular matrix (ECM)/basement
membranes (e.g. type XVIII collagen and thrombospondin) or members of the
clotting/fibrinolytic pathways (e.g. plasminogen and anti-thrombin III) (64).
It is now thought that the tumor angiogenic switch is triggered as a result of a
shift in the balance of stimulators to inhibitors (50). When the ratio is low,
tumor angiogenesis is blocked or modest in magnitude; in contrast, when the
ratio is high, the switch is turned to the `on' position (50). Of considerable
interest was the finding by Bouck and colleagues, that loss of wild-type p53
gene function resulted in a loss of thrombospondin expression (52). Not only did
this finding establish a possible critical link between the genetic basis of
cancer and tumor angiogenesis, it also opened up the now flourishing field of
endogenous angiogenesis inhibitors. Furthermore, it is now increasingly
recognized that oncogenes, such as mutant ras, may also contribute to tumor
angiogenesis by influencing (i.e. enhancing) the production of pro-angiogenic
molecules such as VEGF (66?9). Such effects were slow to be uncovered in the
oncogene and tumor suppressor gene fields given the predominant use of pure
tumor cell culture systems to study the function of cancer causing genetic
alterations.
Other tumor suppressor genes, in their non-mutant, wild-type form, such as VHL
(von Hippel–Lindeau) and p16, have been shown to block the production of
regulators of angiogenesis, such as VEGF (70,71), while other oncogenes, such as
the erbB family, can stimulate VEGF production (67). Given the enormous interest
in the molecular genetics of cancer, these relationships between cancer causing
genetic changes and angiogenesis have fostered an increased awareness of the
importance of angiogenesis to the development, growth and treatment of cancer.
5. The discovery of additional molecular markers in newly formed blood
vessels, especially integrins and cell adhesion molecules
The discovery of VEGF receptors and their upregulation in newly formed blood
vessels highlights the fact that indeed, there can be major phenotypic
differences between mature, quiescent vessels, and their newly formed
counterparts. Such differences are essential to avoiding unwanted toxicity to
normal vessels when using anti-angiogenic drugs, and thus achieving a sufficient
therapeutic index. A number of such differences are now known, and include a
very significant elevation of expression in ECM-binding integrin receptors, such
as v? or v? (72,73). This was first reported by Cheresh and colleagues (72)
who exploited such differences using specific neutralizing antibodies or small
molecule peptide antagonists to block angiogenesis, which occurs, at least in
part, by induction of endothelial cell apoptosis. Other `markers' that are
upregulated in activated endothelial cells include adhesion molecules such as E-selectin
(74), endoglin (75), glycoproteins such as `prostate-specific' membrane antigen
(76), the ED-B domain of fibronectin (77?9) and various proteases (80). Many of
these can be exploited not only as potential therapeutic targets but also for
detection of cancer by nuclear medicine based medical imaging techniques (78).
6. The development of quantitative assays for angiogenesis
It is of course difficult to evaluate the function of angiogenic growth
regulators in the absence of reliable assays. This was a particularly serious
problem in the early days of angiogenesis research since it had not been
possible to successfully grow vascular endothelial cells in long term culture
until Folkman's group established the appropriate conditions in 1980, and used
them to elucidate the functional/sequential steps in angiogenesis (81). Since
then a number of semi-quantitative or quantitative assays have appeared that
involve sprouting angiogenesis in tumor (cell) free systems (82), such as the
corneal micropocket assay, the subcutaneous `Matrigel plug' assay (83) and
assays involving growth of cut sections (slices) of blood vessels in three
dimensional gels of an ECM-associated material such as collagen (84). The
development of these systems has been a boost to the discovery of new
stimulators and inhibitors of angiogenesis, especially the latter (84).
7. Recognition of the prognostic significance of tumor angiogenesis
Another major finding which attracted interest in the field was reported by
Weidner et al. (85) who found that the greater the degree of angiogenesis
detected in a primary tumor, the worse the prognosis. This established a direct
relationship between metastasis and angiogenesis. It was first shown in breast
cancer and subsequently a large and diverse array of other tumors, including
melanomas, gliomas, lung, bladder and prostate cancers, and many others (86).
Tumor vascularity is measured by staining tissue sections with antibodies
specific (or highly specific) for antigens expressed by vascular endothelial
cells such as factor VIII (von Willibrand factor), CD-31 or CD-34 (86) and then
counting (under high power) the number of highlighted vessels in so-called
vascular `hotspots' i.e. localized areas where there are unusually high numbers
of vessels, as detected first under lower power magnification (85).
Aside from the prognostic implications of this work, it also served to highlight
the extent to which tumor masses can become `contaminated' by blood vessels.
Many such vessels are very small and deformed, containing bizarre tortuosities,
corkscrew structures, blind ends and abnormal branching characteristics, thus
making many of them almost impossible to detect in a normal hematoxylin and
eosin tissue section. Consequently, the degree of tumor angiogenesis had been
underestimated, and hence less appreciated, prior to publication of this type of
work. In this regard, endothelial cells are a rich source of biologically active
substances such as cytokines and proteases (87,88) which can themselves affect
the behavior of adjacent cancer cells independent of classic angiogenesis, i.e.
of providing oxygen and nutrients.
Detection of blood vessels in tissue sections has recently been modified by
Benjamin et al., so that it is now possible to discriminate between newly formed
immature vessels and those that are more established and mature (26). It is
based on the use of antibodies to -smooth muscle action ( -SMA), which appears
to stain the latter type of vessel as a result of mature vessels attracting a
`coat' of periendothelial support cells, i.e. pericytes and smooth muscle ( -SMA-antigen-positive)
cells (26). Of interest is the finding that anti-angiogenic therapeutic
procedures, such as blockade of tumor cell VEGF production, results not only in
a drop in the vessel count, but also a change in the ratio of immature/mature
vessels because of the relative vulnerability of the immature vessels to this,
and most other, forms of anti-angiogenic therapy.
8. Lack of acquired resistance to direct-acting anti-angiogenic drugs
It was first proposed in 1991 by Kerbel (89) that anti-angiogenic therapy
might bypass a major problem encountered in virtually all strategies used to
treat cancer, especially chemotherapy and hormonal ablation/antagonist
therapies, namely acquired drug resistance. The underlying rationale was that
the cellular target of anti-angiogenic drugs is a normal, and hence genetically
stable, host cell, i.e. the vascular endothelial cells which line newly formed
blood vessels in tumors. Because acquired resistance is largely a consequence of
the small and large scale genetic instabilities associated with cancer cells
(e.g. gene amplification, chromosomal translocations, chromosome loss, simple
point mutations, etc.), it should not develop when using certain anti-angiogenic
therapeutic strategies (89), just as heritable, and acquired resistance does not
emerge among the descendants of drug-sensitive normal host cells exposed to
chemotherapy, e.g. dividing bone marrow, gut mucosal or hair follicle cells
(90). Clinical `experiments of nature' provided the first indication that,
indeed, acquired resistance may not be inevitable when using certain
anti-angiogenic drugs. Thus, long term (eg. 1 year), daily treatment (using
interferon 2? of infants with life-threatening hemangiomas can result in
complete regression of these benign blood vessel tumors without any evidence of
the development of acquired resistance to this drug (56). In contrast, chronic
exposure of malignant cancer cells to the same type of drug is known to result
in acquired interferon resistance among variants of the treated cancer cells
(54)
The first preclinical/experimental evidence for circumventing acquired drug
resistance came from long term, cyclic exposure of tumor-bearing mice with the
anti-angiogenic protein drug known as endostatin (91). Unlike conventional
cyclophosphamide therapy, using maximum tolerated doses administered in an
intermittent fashion, no resistance to endostatin was ever seen and the tumors
eventually stopped growing after a certain number of cycles of therapy (91).
This work probably attracted more attention than perhaps any other single study
on tumor angiogenesis, if not cancer research, over the last decade (92,93).
Given the great importance (and effort) attached to developing drugs, or
therapeutic strategies, to reverse (or actually prevent) acquired drug
resistance, e.g. the use of P-glycoprotein antagonists such as cyclosporin
analogues to block multidrug resistance and the fact that such efforts have not
met with much clinical success, at least thus far (94?6), the results obtained
using endostatin have certainly increased interest in the pharmaceutical
industry for using this type of therapeutic approach as a means of treating
cancer. Indeed, it is also possible to use conventional cytotoxic drugs as
potent direct-acting `resistance-free' cytotoxic anti-endothelial agents by such
methods as peptide-based targeting strategies (97) or altering the dosing and
schedules of the drug so as to create an `anti-angiogenic' schedule of
chemotherapy (98,99).
Nevertheless, it should be noted that some anti-angiogenic drugs work by
blocking a particular redundant tumor cell property, such as VEGF production,
and thus may be subject to inactivation over time by classic acquired resistance
mechanisms since, for example, tumor cells can produce a number of different
pro-angiogenic growth factors. Therefore, rare cellular variants producing a
spectrum of different pro-angiogenic molecules will likely be selected by the
therapy. This could be an important factor in determining the best
anti-angiogenic drugs to use, given that treatments using such drugs are
probably going to be chronic in nature. In other words, it may be useful to
think in terms of `direct-acting' anti-angiogenic agents, such as endostatin,
and those that are `indirect acting', such as a drug that blocks a
pro-angiogenic growth factor produced by tumor cells, or even its relevant
endothelial cell receptor tyrosine kinase (90). An indirect acting
anti-angiogenic agent, such as an anti-VEGF neutralizing antibody, could be
converted to a direct-acting agent by such procedures as conjugating the
antibody with a toxic radionuclide or a toxin molecule such that binding of the
antibody to endothelial cells results in direct endothelial cell death. In
addition, there are reports showing that the majority of the VEGF produced in a
tumor mass appears to come from the normal host stromal cells infiltrating the
tumor, rather than the tumor cell population itself (100). Use of VEGF blocking
antibodies in such situations may not be compromised by the rapid development of
acquired drug resistance (90).
Finally, with respect to the issue of drug resistance, anti-angiogenic therapies
may also circumvent what may be a major mechanism of intrinsic drug resistance,
namely insufficient drug penetration into the interior of a tumor mass due to
high interstitial pressure gradients within tumors (101). Targeting the tumor
vasculature (rather than the tumor cell population) would avoid the necessity of
having to obtain intra-tumor drug delivery (101).
9. The discovery of the impact of angiogenesis on `liquid' hematologic
malignancies
Another remarkable recent development, and one that is clearly
counter-intuitive as well, is the recent realization that so-called `liquid'
hematologic malignancies are angiogenesis dependent (102?04), i.e. this is not
just a property of solid tumors. The discovery was based on findings such as
elevated levels of pro-angiogenic growth factors, i.e. bFGF and VEGF, in the
serum and urine of patients with acute lymphatic leukemia and multiple myeloma
(102). Similarly, a sharp increase in bone marrow angiogenesis, as measured by
means of vessel density in vascular hot spots, has also been detected in such
patients (102). The newly formed blood vessels detected in the marrow of
patients with acute lymphoytic leukemia or multiple myeloma could be a rich
source of growth factors and cytokines, as well as survival factors, for tumor
cells that arise in this tissue.
This work has already resulted in the initiation of early phase I clinical
trials to test putative anti-angiogenic drugs such as thalidomide (105) in
multiple myeloma patients, the results of which look very encouraging (106). If
this holds up and is found to be a consequence of an anti-angiogenic effect it
would provide major impetus to a large segment of the medical oncology community
to become much more actively engaged in angiogenesis research and
anti-angiogenic therapies to treat these types of cancer.
10. The discovery of the `accidental' anti-angiogenic effects of various
conventional or new anti-cancer drugs and treatment strategies
A number of discoveries led to the provocative conclusion that the use of
anti-angiogenic drugs and therapies—in the clinic—is, in reality, probably not a
new development. Oncologists may have been using them without realizing it for
decades, albeit in a less than optimal manner. This is based on preclinical data
showing that various cytotoxic drugs such as taxanes (107?09), topoisomerase
inhibitors (110), purine analogue anti-metabolites (111), radiation therapy
(112) and hormonal ablation therapeutic procedures (26,113,114) can all result
in either direct or indirect killing of vascular endothelial cells of newly
formed tumor blood vessels. Denekamp and colleagues were the first to have the
insight to point out these `accidental' anti-vascular effects using various
rodent tumor models and a diverse spectrum of anti-cancer agents ranging from
cytokines/biological response modifiers and ionizing radiation to
chemotherapeutic drugs and photodynamic therapy (115). Denekamp coined the term
`vascular targeting' to describe this effect (115). The possibility of
optimizing the anti-vascular targeting effects of chemotherapeutic drugs by
altering the drug doses and schedules used for therapy has been elegantly shown
by Folkman and colleagues (98), as well as Klement et al. (99).
More recently, much interest has been aroused by findings which relate to how
withdrawal of androgens may cause regression of hormone-sensitive cancers, such
as prostate cancer, at least in part through an anti-angiogenic mechanism
(26,113). The basis for this intriguing possibility is that androgens are
powerful inducers of VEGF in hormone-sensitive tissues (114) and VEGF, in turn,
is a potent powerful survival factor for endothelial cells of newly formed
immature blood vessels (24), perhaps by upregulating bcl-2 (116,117) or
activating the PI3 kinase/Akt/PKB survival/signalling pathway (118,119) in
endothelial cells. Hence, acute withdrawal of tumor cell VEGF, which could occur
in patients or animals with androgen-dependent tumors after androgen ablation
therapy, can result in rapid apoptosis of the endothelial cells comprising such
immature tumor vessels (113). This, in turn, results in a secondary, but much
more massive, wave of apoptotic cell death in the cords of tumor cells
surrounding the regressive/dying vessels (113). This secondary cell death
process leads to the regression of tumor mass (113). Unfortunately such tumors
will eventually recur due to the emergence of hormone refractory tumor cell
variants (113). Thus, this would be another example of an indirect acting
anti-angiogenic therapy, or drug, to which resistance may develop.
In addition to conventional therapeutic agents, some of the newer generation of
anti-cancer drugs making their way into the clinic (and which were never
designed with the intent of inhibiting angiogenesis) may affect tumor growth,
partly by suppression of tumor angiogenesis. An example is the so-called signal
transduction inhibitor therapies which target the products of mutant oncogenes,
such as ras, or overexpressed proto-oncogenes such as the EGF receptor tyrosine
kinase and the erbB2/Her-2 receptor tyrosine kinase (69). The drugs in question
include small molecule inhibitors of mutant ras (or a related target) such as
ras farnesyltransferase inhibitors (Ras FTIs) and monoclonal neutralizing
antibodies to the EGF receptor, such as C225, or to erbB2/Her2, e.g. Herceptin
(69). Their potential ability to block angiogenesis (69) is based on the
discovery that oncogene activation can lead to induction or upregulation of
pro-angiogenic growth factors such as VEGF, as first reported by Grugel et al.
(120) and Rak et al. (66). Thus, treatment using drugs which block oncoprotein
function may result in downregulation of VEGF, and perhaps other pro-angiogenic
growth factors (66,67). This could contribute to the drug's overall therapeutic
effect in vivo, where angiogenesis is required, but not in vitro, where
angiogenesis is irrelevant for tumor cell growth and survival, a possibility
which could help explain why such drugs may lack cytotoxic properties in vitro
but sometimes express such an effect in vivo (69). Similar reasoning is evident
with a number of other drugs and therapeutic modalities such as interleukin-12
based immunotherapy (121).
Finally, with respect to the effects of traditional/conventional therapeutics,
mention should be made of the findings of Teicher and colleagues, where it has
been shown preclinically that the combination of an anti-angiogenic drug (or
drugs) (such as TNP-470) with a conventional cytotoxic agent, such as
cisplatinum, taxol or cyclophosphamide, can significantly improve the anti-tumor
efficacy of the cytotoxic drug (122). This observation is counterintuitive since
it might be expected that anti-angiogenic agents would decrease blood flow into
tumors and, hence, limit drug delivery to tumors. However, just the opposite may
be the case (123), and this may be due to a transient normalization of the
abnormal structure of tumor vessels mediated by certain angiogenesis inhibitors,
e.g. neutralizing anti-VEGF antibodies (124), thus leading to the possibility of
a temporary increase in blood flow and, hence, drug delivery (123).
Alternatively, there is the potential for a cytotoxic drug to function directly
on endothelial cells of newly formed tumor vessels; this effect may be
exaggerated by the inclusion of an anti-angiogenesis drug which compromises
endothelial cell survival mechanisms (99). Regardless of the actual mechanism,
these combination therapy effects, which have also been observed with radiation
therapy and angiogenesis inhibitors (112), could play a significant role in the
clinical evaluation and effects of angiogenesis inhibitors, as discussed below.
The future: opportunities to be seized, problems to overcome
