Fumagillin

The understanding that the growth of tumours is dependent on angiogenesis has led to the development of new approaches to treatment and new agents directed at tumour vasculature. These have yielded striking successes in experimental models, which if translated into the clinical setting will have a substantial effect on patient survival. Such new approaches are vital because, although great strides have occurred in the treatment of certain cancers, the overall standardised mortality from most solid tumours has altered little over the past two decades.[1]

This article considers the process of tumour angiogenesis and discusses the potential of angiogenic inhibitors as therapeutic agents.

Role of angiogenesis in growth of tumours

The vascularity of tumours has been noted for many years.[2] Alguire noted that vascularisation was instigated by the developing tumour: "An outstanding characteristic of the growing tumour is its capacity to elicit the production of a new capillary endothelium from the host."[3] Tannock elegantly showed that the rate of division of tumour cells decreased in proportion to their distance from the supplying blood vessel and related this to diminishing oxygen supply.[4] Moreover, he showed that the overall rate of growth was dictated not by proliferation of tumour cells but by the lower rate of proliferation of endothelial cells, concluding that the supply of oxygen and nutrients to the tumour limited its growth.

Tumour vascularisation is a vital process for the progression of a neoplasm from a small, localised tumour to an enlarging tumour with the ability to metastasise (figure).[5,6] Anti-angiogenesis as a therapeutic concept was developed in the early 1970s based on observations that tumours that did not vascularise failed to grow beyond a few millimetres in diameter.[7] By comparing the growth of transplanted tumours in the avascular aqueous humour of a rabbit eye with those in the vascular iris, Folkman could show distinct avascular and vascular phases of tumour growth. The start of the vascular phase of growth coincided with tumours growing beyond 2-3 [mm.sup.3] and a 20-fold increase in the rate of tumour growth. Tumours in the aqueous humour were prevented from entering the vascular phase and remained dormant.[8] He concluded that vascularisation was essential to tumour growth and inferred that preventing this process was a viable therapeutic approach.

[Figure ILLUSTRATION OMITTED]

Induction of angiogenesis by tumours

Adult endothelium is essentially quiescent, but in response to physiological or pathological stimuli (such as proliferating endometrium, injury, tumour growth, or diabetic retinopathy) the endothelium can alter to a proliferating and organising population of cells. Physiological angiogenesis can also be rapidly curtailed, indicating that the process is held in check physiologically and yet can be activated in response to the appropriate stimuli, somewhat analogous to the clotting cascade. Box 1 lists some of the regulatory proteins identified.

Box 1: Proteins that may regulate angiogenesis

Promoters Inhibitors

A tumour induces this proliferative vascular response from host vessels by altering the balance of positive and negative regulators locally. This "angiogenic switch" is necessary for tumour growth and may be rate limiting.[9] Convincing evidence exists that tumours undergo a switch to an angiogenic character as they progress. In cervical carcinoma the development of vascularity can be associated with progression from a noninvasive premalignant stage to invasive carcinoma.[10] The density of microvessels in tumours is a powerful independent prognostic indicator of distant metastasis and survival, suggesting that tumour vascularisation correlates with growth and metastatic potential.[11]

Antivascular treatment

There are four key approaches to antivascular treatment (box 2). All depend on targeting endothelial cells rather than tumour cells for drug action, and destruction of the tumour cells is secondary. A theoretical advantage of these approaches is that endothelial cells are not transformed and are unlikely to acquire mutations resulting in drug resistance. Furthermore, treatment directed at endothelial cells is applicable to all solid tumours, irrespective of the origin of the tumour cells. Also, endothelial cells are uniquely exposed to bloodborne agents, circumventing the problem of delivering drugs to the centre of a tumour, which is a major hurdle in conventional treatment.

Box 2: Approaches to antivascular treatment

Neutralising angiogenic promoters

One of the favoured approaches is to interfere with the balance of angiogenic proteins produced by a tumour--turning off the "switch." This has been made possible by an understanding of the mediators of angiogenesis in normal and pathological settings. Current evidence implicates vascular endothelial growth factor (VEGF) and the family of fibroblast growth factors (FGF 1-12) as critical regulators of physiological angiogenesis. Angiopoietin 1 and 2 have been implicated as the factors controlling the recruitment of supporting cells to the developing tubule.[12,13]

Both vascular endothelial growth factor and fibroblast growth factors 1 and 2 have been implicated as important promoters of tumour angiogenesis. Vascular endothelial growth factor, a mitogen and permeability factor specific for endothelial cells, is up regulated in many different tumour types and cell lines,[14-16] often in areas of tumour hypoxia.[17] Fibroblast growth factor 2 is ubiquitous in the extracellular matrix bound in an inactive form, but in tumours the active factor is released either from the tumour or from the extracellular matrix by various mechanisms.[18] Increased serum and urine concentrations of fibroblast growth factor 2 can be detected in cancer patients.[19] Increasing the amounts of active fibroblast growth factors secreted by tumour cell lines, by transfecting DNA into cells, can markedly increase the ability of poorly tumorigenic cells to form tumours and to metastasise.[20]

Because of the evidence that vascular endothelial growth factor and fibroblast growth factor 2 are promoters of tumour angiogenesis, researchers have investigated ways of interfering with their angiogenic effects and demonstrated the ability to induce tumour regression in mice. These approaches include targeting vascular endothelial growth factor with a monoclonal antibody[21] and preventing release of active fibroblast growth factor 2 from the extracellular matrix by eradicating a binding protein necessary for its release.[22] The problem with such approaches is that they inhibit only a single positive factor. There is a range of positive angiogenic factors, and many have been shown to be up regulated in tumours. More importantly, individual tumours can express several angiogenic factors[23] and thus may have a number of routes around such a specific approach.

Angiogenic inhibitors as therapeutic agents

An alternative approach is to use an angiogenesis inhibitor in order to counter the sum effects of all angiogenic factors produced by a tumour. These agents can be synthetic inhibitors of endothelial cell proliferation--such as synthetic derivatives of fumagillin--or endogenous inhibitors of angiogenesis--often fragments of larger inactive circulatory proteins--that function physiologically in maintaining vascular quiescence or curtailing physiological angiogenesis. These endogenous agents can be highly potent inhibitors of endothelial proliferation and may be more effective than synthetic agents because they may also inhibit the capillary remodelling that is involved in expansion of tumour vessels. Angiostatin and endostarin are currently the most potent agents, both having striking antitumour activity. Others include a 140 kDa fragment of thrombospondin, a protein normally involved in platelet aggregation under the regulation of the tumour suppresser gene p53.[24 25]

Angiostatin and endostatin

The discovery of these two potent endogenous inhibitors of angiogenesis with powerful antitumour activity in mice has produced great interest in the clinical use of angiogenesis inhibitors.[26-28] Both were discovered as a result of the observation that the presence of a primary tumour can occasionally inhibit the development of metastases: when the primary tumour is removed, metastases develop rapidly. Folkman hypothesised that the primary tumour produced angiogenesis inhibitors, perhaps incidentally as a result of proteolytic degradation. These inhibitors persisted in the circulation while local angiogenic promoters were degraded and exerted no systemic effect. Angiostatin and endostatin were identified from experimental tumours that demonstrated this phenomenon.

Both are fragments of larger circulatory proteins with no angiogenic activity. Angiostatin is a 38 kDa fragment of plasminogen, and endostatin is a fragment of collagen XVIII, a type of collagen found exclusively in blood vessels. Their antitumour activity in mice is impressive, being able to cause regression in various solid tumours up to 1% of body mass. There is no evidence of drug resistance, even after multiple treatment cycles.[29] Research efforts are currently directed at elucidating the precise mechanism of action of these agents, and at their large scale purification, before they are tested in clinical trials.

Targeting endothelial cells of turnouts

Another experimental antivascular approach to treating tumours is to target tumour endothelial cells with a toxin directed at a cell marker specific for tumour endothelium and cause infarction of the tumour by inducing coagulation within the tumour vessels. This has been shown to be effective experimentally by using a neuroblastoma tumour that was generically engineered to express the class II histocompatibility antigens on tumour endothelial cells. These antigens are normally absent from endothelial cells, so they served as specific markers for tumour vessels. A toxin was constructed consisting of an antibody to class II antigens linked to a truncated form of tissue factor that would cause coagulation only when bound to an endothelial cell by the antibody. This toxin induced complete regression of the experimental tumour by thrombosis of the tumour vessels while leaving the host vasculature intact.[30]

Potential endogenous target molecules include integrin [[Alpha].sub.v][[Beta].sub.3], which is expressed only on proliferating vessels in healing wounds and in tumours. Antibodies to integrin [[Alpha].sub.v][[Beta].sub.3] promote tumour regression by inducing endothelial cell apoptosis.[31]

Future developments

Preclinical research into tumour angiogenesis has led to the identification of several antivascular treatments with impressive efficacy in animal models of human cancer. Currently, 19 antivascular agents are being assessed in clinical trials, mostly still phase I and II trials that involve treating patients with advanced metastatic disease that is resistant to other treatments. (Information about current trials can be obtained from the National Cancer Institute's website at cancertrials.nci.nih.gov.) There are occasional reports of striking clinical remissions,[32] but the real efficacy of these agents will only become apparent over the next decade as they are fully evaluated in extensive clinical studies either alone or with standard treatments.

As inhibition of angiogenesis may induce dormancy of a tumour rather than killing it, there is growing appreciation that the administration of these agents, and their assessment in clinical trials, may need to be different from that currently used for cytotoxic drugs. Indeed, it is possible that these agents could be effective in maintaining long term remission, an approach not currently used for solid tumours.

Competing interests: None declared.

Predicted developments

Research will clarify the mechanisms by which endogenous inhibitors of angiogenesis prevent tumour growth

Strategies will be developed for large scale production of antivascular drugs for clinical use

New treatment regimens will be developed to modify the balance of positive and negative angiogenic proteins in tumours

Extensive clinical evaluation of new antivascular treatments alongside traditional treatments will define their anticancer potential more clearly

New trials and treatments will focus on inducing long term remission

[1] Bailer JC, Gornik HL. Cancer undefeated. N Engl J Med 1997;336:1569-74.

[2] Bowen W. The effects of surgical interference with the blood supply on the growth of transplanted carcinomata and sarcomata. Sci Rep Imperial Cancer Res Fund 1908;3:146-58.

[3] Alguire GH. The transparent chamber technique as a tool in experimental tumor therapy. In: Symposia: approaches to tumour chemotherapy. Washington DC: American Association for the Advancement of Science, 1947:13-26.

[4] Tannock IF. The relationship between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 1968;22:258-73.

[5] Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182-6.

[6] Liotta L, Kleinerman J, Saidel G. Quantitative relationships of intravascular tumor cells, tumor vessels and pulmonary metastasis following tumor implantation. Cancer Res 1974;34:997-1004.

[7] Folkman J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 1972;175:409-16.

[8] Gimbrone M, Leapman S, Cotran R, Folkman J. Tumor dormancy in vivo by prevention of neo-vascularisation. J Exp Med 1972;136:261-76.

[9] Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:353-64.

[10] Smith-McCune KK, Weidner N. Demonstration and characterization of the angiogenic properties of cervical dysplasia. Cancer Res 1994;54:800-4.

[11] Weidner N. Current pathological methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat 1996;36:169-80.

[12] Suri C, Jones P, Patan S, Bartunkova S, Masonpierre P, Davis S, et al. Requisite role of angiopoietin-1, a ligand for the TIE-2 receptor during embryonic angiogenesis. Cell 1996 ;87:1171-80.

[13] Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997;277:55-60.

[14] Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HS. Tumor cells secrete a vascular permeability factor that promotes the accumulation of ascites fluid. Science 1983;219:983-5.

[15] Leung DW, Cachaines G, Kwang W-J, Goeddel DV, Ferrera N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306-9.

[16] Senger DR, Perruzzi CA, Feder J, Dvorak HS. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 1986;46:5629-32.

[17] Schweiki D, Itin A, Soffe D, Kehet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia initiated angiogenesis. Nature 1992;359:843-5.

[18] Vlodavsky I, Bashkin P, Ishai-Michaeli R, Chajek-Shaul T, Bar-Shavit R, Haimovitz-Friedman A, et al. Sequestration and release of basic fibroblast growth factor. Ann N Y Acad Sci 1991;638:207-20.

[19] Nguyen M, Watanabe H, Budson AE, Richie JP Hayes DF, Folkman JE Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J Natl Cancer Inst 1994;86:356-61.

[20] Mcleskey S, Kurebayashi J, Honig S, Zwiebel J, Lippman ME, Dickson R, et al. Fibroblast growth factor 4 transfection of MCF-7 cells produces cell lines that are tumorigenic and metastatic in ovarectomised or tamoxifen treated athymic nude mice. Cancer Res 1993;53:2169-77.

[21] Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 1993;362:841-4.

[22] Czubayko F, Liaudet-Coopman ED, Aigner A, Tuveson AT, Berchem GJ, Wellstein A. A secreted FGF-binding protein can serve as the angiogenic switch in human cancer. Nature Med 1997;3:1137-40.

[23] Relf M, Le Jeune S, Scott PA, Fox S, Smith K, Leek R, et al. Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res 1997;57:963-9.

[24] Good D, Rastinejad F, Polverini PJ, LeBeau MM, Lemons RS, Frazier WA, et al. A tumor suppresser dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci 1990;87:6624-8.

[25] Dameron KM, Volpert OV, Tainsky AM, Bouck N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994;265:1582-4.

[26] O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, et al. Angiostatin: a novel angiogenesis inhibitor that mediates suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:315-28.

[27] O'Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 1996;2:689-92.

[28] O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997;88:277-85.

[29] Boehm T, Folkman J, Browder T, O'Reilly M. Anti-angiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997;390:404-7.

[30] Huang X, Molema G, King S, Watkins L, Edgington TS, Thorpe PE. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science 1997;275:547-50.

[31] Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld RA, Hu T, Klier G, et al. Integrin [[Alpha].sub.v][[Beta].sub.3] antagonists promote tumour regression by inducing apoptosis of angiogenic blood vessels. Cell 1994;79:1157-64.

[32] Kudelka AP, Verschraegen CF, Loyer E. Complete remission of metastatic cervical cancer with the angiogenesis inhibitor TNP-470. N Engl J Med 1998;338:991-2.

Lombardi Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road, Washington DC, 20007, USA

AJ Hayes, surgical registrar

L Y Li, assistant professor of biochemistry

M E Lippman professor of medicine and pharmacology

Correspondence to: Dr Hayes hayesa@gunet. georgetown.edu

BMJ 1999;318:853-6

COPYRIGHT 1999 British Medical Association
COPYRIGHT 2000 Gale Group