Cezary Marcinkiewicz - US grants

DESCRIPTION (provided by applicant): The growing of solid tumors highly depends on angiogenesis. In this new vessel formation process the integrins, expressed on endothelial cells are important mediators. Inhibition of function of these integrins is currently one of the directions in cancer research. Moreover, integrins expressed on cancer cells are involved in migration of these cells during metastasis. In the proposed research plan, we will investigate ct 1131 integrin (VLA-1), which is recognized as a specific collagen IV receptor. We will focus our research on two inhibitors of VLA-1, obtustatin and viperisrastatin. Both of them belong to disintegrin family, and are low molecular mass inhibitors (4.2 kDa) of VLA-1. The activity of these disintegrins has been localized within their integrin-binding loop as an active sequence KTS. Obtustatin showed potent angiostatic activity in the chicken CAM model in vivo, and in tube EC formation assay in vitro. Moreover, obtustatin inhibited Lewis lung cancer development in syngeneic mouse model. The activity of KTS-disintegrins will be further tested on growing tumor induced by mouse melanoma B 16 and M-3 cell lines in the syngeneic mouse, and human melanoma cell lines, MV3, HS.939T, A-375, C32, and A2058 in nude mice. The participation VLA-1 in metastasis of these cell lines to the lung will be investigated in vivo using syngeneic and nude mice. To explain the mechanism of the angiostatic effect of KTS containing disintegrins, series of experiments will be performed to investigate their effect on microvascular EC. The preliminary data showed that obtustatin induced apoptosis in these cells and inhibited their proliferation. Based on the previous reports indicating involvement of VLA-1 in signal transduction pathway dependent on MAPK, the effect of KTS-disintegrins on activation of this pathway will be evaluated. Moreover, radial migration assay in collagen gel will be proposed. That in vitro assay imitates movement of EC during early stage of neovascularization after dissolution of the basement membrane. The effect of both disintegrins in EC motility in this model will be tested. Obtustatin and its naturally occurring analog viperisrastatin may be models for designing of synthetic or recombinant compounds, which may be useful in cancer therapy. In summary, the work with KTS containing disintegrins may lead to the better understanding of the involvement of VLA-1 in cancer progression and metastasis, as well as contribute to an understanding of the role of VLA-1 in angiogenesis.

DESCRIPTION (provided by applicant): Previous studies revealed that certain integrins expressed on endothelial cells play a significant role in the progression of angiogenesis and are an attractive target for the development of angiostatic drugs that may have an application in the therapy of various tumors. Using immunohistochemistry we detected the expression of a9[unreadable]1 integrin on endothelial cells forming blood capillaries in all organs of the body, and an up-regulation of its level in certain tumors including gliomas. However, following isolation the majority of primary endothelial cells in the culture stop expressing a9[unreadable]1 integrin. This sensitivity of a9[unreadable]1 integrin on endothelial cells in vitro was the reason that researchers never considered this integrin as an important receptor for modulation of the neovascularization process, especially during oncogenesis. In the proposed research plan we will investigate a role for this integrin in angiogenesis following its interaction with thrombospondin-1 (TSP-1), in the context of brain tumor vascularization. Recently, we evaluated that TSP-1 is a ligand for a9[unreadable]1 integrin, which has a binding site on the N-terminal (NoC1) domain of this extracellular matrix protein. Based on previously published work that TSP-1 is up-regulated during brain tumor progression, we proposed a general hypothesis that the interaction of a9[unreadable]1 with TSP-1 is an important element of pathological angiogenesis occurring during diffusive glioma development. To verify this hypothesis we propose a series of experiments in vitro and in vivo that may lead to an explanation of a9[unreadable]1 integrin's and TSP-1's role in the promotion of pathological angiogenesis induced in brain tumors. We will isolate a9[unreadable]1 integrin-positive primary glioma human microvascular endothelial cells (gHMVEC) from cancer tissue obtained following surgery, by immuno-sorting in first passage cells expressing this integrin and typical endothelial cell markers such as CD31. We will investigate pro-angiogenic activities of these cells such as proliferation and migration, induced by TSP-1 and its recombinant NoC1 domain, as well as evaluate the signaling pathway that is activated inside the cell in these processes. We will confirm our expectation that the interaction of a9[unreadable]1 with TSP-1 extensively occurs on endothelial cells during glioma progression using double fluorescent color immunohistochemistry of paraffin sections obtained from normal brain and different grades of astrocyte-derived tumors. In animal experiments, we intend to prove that the blocking of a9[unreadable]1 integrin by a specific monoclonal antibody or by a MLD-disintegrin, VLO5 will suppress the development of experimental glioma by blocking the vascularization process. Also, we will transfect glioma cells with the NoC1 domain and we expect to observe a higher ratio of tumor growth following intracranial implantation of these transfectants into rats. In another part of the study, we will perform a structure/function analysis of the NoC1 domain to localize the a9[unreadable]1 integrin binding site on TSP-1. This work will be performed by chemical synthesis of peptides spanning the N-terminal module of the NoC1 domain and by site-directed mutagenesis of recombinant fragments of this part of TSP-1. We will investigate the up-regulation of NoC1-like domain in clinical and experimental glioma tissues in comparison with normal brain using a specific monoclonal antibody, 2D11 that recognizes NoC1 in Western blot. Further, the structural characterization of tumoral NoC1-like domain will be performed using proteomic approaches. PUBLIC HEALTH RELEVANCE: Glioma is one of the most frequently occurring and difficult to treat brain tumors. This tumor belongs to the most vascularized cancers and angiostatic treatment, which will block vessel growth in pathological tissue, appears to be effective in its therapy. In this context, we propose an investigation a receptor, a9[unreadable]1 integrin that is present on the endothelial cells, which are the major structural cells in vessel wall. Investigation of this receptor may be beneficial for cancer as well as for cardiovascular disease having patients, because regulation of vascularization process is important in these pathologies.