How Does Endostatin Inhibit Breast Cancer Angiogenesis?

Institution: The Burnham Institute for Medical Research
Investigator(s): Kristiina Vuori, M.D., Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0020 Award: $292,535
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (1999)
It is now known that the growth and spread of solid tumor cancers, such as breast cancer, absolutely depends on the development of a tumor-associated vasculature by a process known as angiogenesis. Thus, there is considerable interest in finding ways to inhibit angiogenesis as an effective means for preventing tumor progression and metastasis. Our interest is an angiogenesis inhibitor protein called endostatin. It is a proteolytic cleavage product of type XVIII collagen and is one of the most potent angiogenesis inhibitors. Two important findings have increased our interest in endostatin. First, endostatin has been found to inhibit the subcutaneous growth of several tumor types in a mouse model, demonstrating that endostatin works on a wide spectrum of cancers. Secondly, repeated cycles of endostatin treatment have been shown to induce tumor dormancy, suggesting that endostatin treatment does not generate drug resistance. But, the molecular mechanism of endostatin function is not known. Our research is designed to investigate the relationship of endostatin function with certain key adhesion receptors present on endothelial cells.

Our preliminary results demonstrate that endostatin associates with two cell adhesion receptors, the avb3 and avb5 integrins. Significantly, these integrins have been previously identified as crucial molecules in controlling angiogenesis. Our hypothesis is that endostatin exerts its anti-angiogenic effects by interfering with the function of av-integrins in endothelial cells. This would disrupt endothelial cell attachment and a loss of tumor vessel function. In the first aim, we will identify the integrin binding site(s) in endostatin and characterize the significance of the integrin binding in endostatin function. Our preliminary results have identified three likely binding sites in the endostatin molecule. We plan to confirm and expand these observations by making mutant forms of endostatin in order to exactly identify the integrin-binding site. The second aim will be to characterize the effects of endostatin on endothelial physiological functions. Primary endothelial cells will be used as a model system, and the biological effects of wild-type and mutant forms of endostatin will be examined. Such cell processes as apoptosis (i.e., programmed cell death), proliferation, migration and endothelial tube formation will be studied.

Our research will expand knowledge of the structure-function relationship of endostatin, leading to possible active fragments for therapeutic development. This appears critical, since there have been reports of difficulty using the intact protein. It will also give insights into the cell biological and molecular pathways by which endostatin works on endothelial cells. Overall, these studies will take critical steps in the clinical use of endostatin in treating breast cancer.


Final Report (2001)
The growth of solid cancers, such as breast cancer, absolutely depends on the development of a tumor-associated vasculature by a process known as angiogenesis. Thus, there is considerable interest in finding ways to inhibit angiogenesis as an effective means for preventing tumor progression. Our interest is an angiogenesis inhibitor protein, called endostatin, which is currently being tested in clinical trials for its utility as cancer therapeutics. Endostatin is a proteolytic cleavage product of type XVIII collagen and is one of the most potent angiogenesis inhibitors in animal models. At present, the molecular mechanism of endostatin function is unknown.

Endothelial cells proliferate in an anchorage-dependent manner, suggesting that signals mediated by the integrin-family of adhesion receptors are of importance in the growth of new blood vessels. The integrin-dependence of tumor angiogenesis in vivo is evidenced by the fact that antagonists of the avb3 integrin, which is highly expressed in angiogenic endothelium, suppress tumor growth by inhibiting angiogenesis. Recent studies have demonstrated that the a5b1 integrin also has a crucial role in angiogenesis, as antagonists of this integrin block angiogenesis induced in vivo by several different growth factors. a5b1 antagonists were also found to inhibit tumor angiogenesis, thereby causing regression of human tumors in animal models.

Taking into consideration that endostatin is a cleavage product of collagen XVIII, itself a normal component of the basement membranes that surround the vascular tubes, we examined the possibility that sequences within endostatin might function as a binding site for integrins. We found that recombinantly produced human endostatin interacts with a5- and av-integrins on the surface of human endothelial cells. Our results further indicate that the endostatin-integrin interaction is of functional significance in vitro, as we found that immobilized endostatin supports endothelial cell survival and migration in an integrin-dependent manner. Soluble endostatin in turn inhibits integrin-dependent endothelial cell functions, such as cell migration. Taken together, these results implicate integrins as potential targets for endostatin function and support the importance of integrins in endothelial cell biology and angiogenesis.

Our studies have given insights into the cell biological and molecular pathways by which endostatin works on endothelial cells. These results are also anticipated to expand knowledge of the structure-function relationship of endostatin, and aid further development of endostatin in anti-angiogenesis therapeutics. Thus, these studies will take critical steps in the clinical use of endostatin in treating breast cancer.

Interaction of Endostatin with Integrins Implicated in Angiogenesis
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Reh, M, Veikkola T, et al, and Vuori K
Yr: 2001 Vol: 98 Nbr: 3 Abs: Pg:1024-1029