Angiostatin and Endostatin: Key Players in a Dual Threat Approach to Cancer Treatment

John Swarthout, Ph.D.

Cancer is a complex disease characterized by unregulated proliferation, tissue invasion, and metastasis. One promising area of research toward the continued development of anticancer drugs surrounds natural and synthetic angiogenesis inhibitors. Advancing our understanding of the regulatory mechanisms central to tumor angiogenesis will provide fundamental insights into how to target this process for limiting the growth and spread of a tumor.

Numerous antiangiogenic factors have been identified, including the endogenous proteins angiostatin and endostatin.¹ Angiogenic inhibitors influence the induction of angiogenesis by growth factors, the activity of angiogenic proteinases, endothelial cell proliferation and migration, or microtubule formation. However, the exact manner by which angiostatin and endostatin impede tumorigenesis needs to be completely elucidated. Recently, angiostatin and endostatin became widely available to the research community in recombinant forms, expressed and purified from Pichia pastoris. The increased availability of these proteins will likely facilitate tumor angiogenesis research.

Throughout tumorigenesis, cells acquire a set of functional capabilities including self-sufficiency in growth signals, insensitivity to antigrowth signals, evading apoptosis, limitless replicative potential, tissue invasion and metastasis, and sustained angiogenesis.² Angiogenesis is the multistep physiological process of new capillary growth from pre-existing blood vessels,³ and is requisite for the growth and spread of cancer.² In many cancers, the balance that normally exists between angiogenic inducers and angiogenic inhibitors shifts toward the proangiogenic state resulting in the synthesis of new blood vessels (Figure 1).⁴ Initially cancers co-opt the existing vasculature. Then the angiogenic switch results in the production of factors that induce angiogenic sprouting of the vasculature. Expressed pro-angiogenic factors bind to receptors including vascular endothelial growth factor (VEGF) receptor 2 and neuropilin-1 on the vascular endothelial cells of nearby blood vessels and promote cell proliferation, migration, and invasion into the tumor.^2,4,5 This is important as avascular tumors are limited in size and require development of new blood vessels to deliver the necessary oxygen and nutrients, and for removal of cellular waste through the interstitium. Therefore, reducing or inhibiting angiogenesis removes a vital lifeline that allows cancer cells to grow, invade nearby tissue, metastasize, and form new colonies of cancer cells.

Figure 1. The “angiogenic switch” is illustrated as a balance between pro-angiogenic factors (represented by red spheres) and angiogenesis inhibitors (represented by gray spheres). When the level of angiogenic inhibitors predominates (left image), the microenvironment remains angiostatic and the tumor is quiescent. When the level of pro-angiogenic factors increases and overcomes the effects of the angiogenic inhibitors (right image), the process of constructing new vasculature is initiated.

Both angiostatin and endostatin require enzymatic cleavage from a parent molecule before they are biologically active.¹ Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen. It was originally isolated from tumor bearing mice,⁶ and has both potent antiangiogenic activity and antiproliferative activity toward endothelial and cancer cells.⁷ Plasminogen contains five kringle (K) domains of ~80 residues each. Studies using recombinant angiostatin demonstrated tumor inhibitory activity resides in a fragment of K1-3,⁸ and this region forms a central cavity that may contain a protein recognition site essential for activity.⁹

Recent evidence supports dual antitumor mechanisms for plasminogen derivatives, one affecting angiogenesis and another targeting tumor cells directly.¹⁰ Kringle 5 (K5), like angiostatin, is a byproduct of the proteolytic cleavage of plasminogen. In a recent study, Ansell et al., demonstrated K5 functions as a competitive antagonist of hepatocyte growth factor (HGF).¹¹ HGF contains kringle motifs and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiostatic role, K5 can direct a potent antitumor response with its ability to recruit tumor-associated neutrophils and Natural Killer T cells.¹²

Endostatin is 20 kDa carboxyl-terminal fragment of type XVIII collagen that is present in walls and basement membranes of blood vessels and plays an important role in endothelial cell adhesion and cytoskeletal organization.^13,14 Endogenous endostatin inhibits migration and induces apoptosis in endothelial cells, inhibits tumor growth, and impairs blood vessel maturation in wound healing.^15,16 It is thought to interfere with the proangiogenic action of growth factors such as basic fibroblast growth factor and VEGF, and is known to inhibit at least 65 different tumor types.¹⁷ Additionally, a study measuring changes in gene expression in human dermal microvascular cells following treatment with endostatin demonstrated a downregulation of several proangiogenic pathways as well as the upregulation of many antiangiogenic genes.¹⁷

There are numerous clinical trials underway employing antiangiogenic drugs for the treatment of cancers.¹⁸ However, to enhance the survival benefits, there is mounting interest in developing more effective ways to combine antiangiogenic drugs with established chemotherapies.¹⁹ Recent studies indicate a combination of antiangiogenic factors demonstrate an additive or synergistic inhibition of angiogenesis and tumor growth.^20,21 For example, the in vivo co-production of human endostatin and tissue inhibitor of metalloproteinase-1 demonstrated a synergistic antitumor growth and reduction of metastasis in murine melanoma.²²

Continued research will advance our understanding of the antiangiogenic mechanisms of these proteins as well as lead to improved efficacy in anticancer therapies.

References

1. Folkman J. 2003. Angiogenesis Inhibitors: A New Class of Drugs. Cancer Biology & Therapy. 2(sup1):126-132. https://doi.org/10.4161/cbt.212

2. Hanahan D, Weinberg RA. 2000. The Hallmarks of Cancer. Cell. 100(1):57-70. https://doi.org/10.1016/s0092-8674(00)81683-9

3. Folkman J, Shing Y. 1992. Angiogenesis. J. Biol. Chem.(267): 10931-4

4. Bergers G, Benjamin LE. 2003. Tumorigenesis and the angiogenic switch. Nat Rev Cancer. 3(6):401-410. https://doi.org/10.1038/nrc1093

5. Whitaker GB, Limberg BJ, Rosenbaum JS. 2001. Vascular Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor Complex That Is Responsible for the Differential Signaling Potency of VEGF165and VEGF121. J. Biol. Chem.. 276(27):25520-25531. https://doi.org/10.1074/jbc.m102315200

6. O'Reilly M. 1994. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma. Cell. 79(2):315-328. https://doi.org/10.1016/0092-8674(94)90200-3

7. O?Reilly MS. 1997. Angiostatin: An endogenous inhibitor of angiogenesis and of tumor growth.273-294. https://doi.org/10.1007/978-3-0348-9006-9_11

8. Gately S, Twardowski P, Stack MS, Cundiff DL, Grella D, Castellino FJ, Enghild J, Kwaan HC, Lee F, Kramer RA, et al. 1997. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proceedings of the National Academy of Sciences. 94(20):10868-10872. https://doi.org/10.1073/pnas.94.20.10868

9. MacDonald NJ, Murad AC, Fogler WE, Lu Y, Sim B. 1999. The Tumor-Suppressing Activity of Angiostatin Protein Resides within Kringles 1 to 3. Biochemical and Biophysical Research Communications. 264(2):469-477. https://doi.org/10.1006/bbrc.1999.1486

10. Schmitz V, Raskopf E, Gonzalez-Carmona M, Vogt A, Kornek M, Sauerbruch T, Caselmann WH. 2008. Plasminogen Derivatives Encoding Kringles 1-4 and Kringles 1-5 Exert Indirect Antiangiogenic and Direct Antitumoral Effects in Experimental Lung Cancer. Cancer Investigation. 26(5):464-470. https://doi.org/10.1080/07357900801970927

11. Ansell PJ, Zhang H, Davidson DJ, Harlan JE, Xue J, Brodjian S, Lesniewski R, McKeegan E. 2010. Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling. European Journal of Cancer. 46(5):966-973. https://doi.org/10.1016/j.ejca.2009.12.026

12. Perri SR, Martineau D, Francois M, Lejeune L, Bisson L, Durocher Y, Galipeau J. Plasminogen Kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways. Molecular Cancer Therapeutics. 6(2):441-449. https://doi.org/10.1158/1535-7163.mct-06-0434

13. Dixelius J, Cross M, Matsumoto T, Sasaki T, Timpl R, Claesson-Welsh L. 2002. Endostatin regulates endothelial cell adhesion and cytoskeletal organization. Cancer Res.(62):1944-7.

14. Miosge N, Sasaki T, Timpl R. 1999. Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls. FASEB j.. 13(13):1743-1750. https://doi.org/10.1096/fasebj.13.13.1743

15. O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J. 1997. Endostatin: An Endogenous Inhibitor of Angiogenesis and Tumor Growth. Cell. 88(2):277-285. https://doi.org/10.1016/s0092-8674(00)81848-6

16. Bloch W, Huggel K, Sasaki T, Grose R, Bugnon P, Addicks K, Timpl R, Werner S. 2000. The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing. FASEB j.. 14(15):2373-2376. https://doi.org/10.1096/fj.00-0490fje

17. FOLKMAN J. 2006. Antiangiogenesis in cancer therapy?endostatin and its mechanisms of action. Experimental Cell Research. 312(5):594-607. https://doi.org/10.1016/j.yexcr.2005.11.015

18. Types of Cancer in Active Phase III Treatment Clinical Trials of Angiogenesis Inhibitors.. [Internet]. National Cancer Institute: Available from: http://www.cancer.gov/cancertopics/factsheet/Therapy/angiogenesis-inhibitors

19. Ma J, Waxman DJ. Combination of antiangiogenesis with chemotherapy for more effective cancer treatment. Molecular Cancer Therapeutics. 7(12):3670-3684. https://doi.org/10.1158/1535-7163.mct-08-0715

20. Li X, Raikwar SP, Liu Y, Lee S, Zhang Y, Zhang S, Cheng L, Lee S, Juliar BE, Gardner TA, et al. 2006. Combination therapy of androgen-independent prostate cancer using a prostate restricted replicative adenovirus and a replication-defective adenovirus encoding human endostatin-angiostatin fusion gene. Mol Cancer Ther. 5(3):676-684. https://doi.org/10.1158/1535-7163.mct-05-0339

21. Sun X, Qiao H, Jiang H, Zhi X, Liu F, Wang J, Liu M, Dong D, Kanwar JR, Xu R, et al. 2005. Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors. Cancer Gene Ther. 12(1):35-45. https://doi.org/10.1038/sj.cgt.7700766

22. Shen W, Zhu J, Zhang Y, Su Q. 2010. Synergistic antitumor effects of in vivo production of human endostatin and tissue inhibitor of metalloproteinase-1 in mice after subcutaneous implantation of primary fibroblasts transfected by adenovirus-mediated gene delivery. Chin Med J (Engl). 123.922-8.

Recombinant angiostatin and endostatin proteins, sold under license from CMCC Boston. Reference US Patent Nos. 5,854,205; 6,024,688; 5,861,372.