Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogen esis. The antiangiogenic activity of TSP-1 has been mapped to the procollag en homology region and the type 1 repeats (TSR) using synthetic peptides. T o elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the TSRs, we have expressed recombinant versions of these motifs and have assayed their ability to inhibit the growth of experimental B16F10 melanomas and Lewis lung carcinomas. Recombinant proteins that cont ain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR 2) the transforming growth factor-beta (TGF beta) activating sequence (RFK) have been expressed in Drosophila S2 cells. In addition, recombinant prote ins with mutations in either the RFK sequence (TSR2+QFK) or the WSHWSPW seq uence [TSR2 (W/T)] of the second TSR have been prepared. Similar to platele t TSP-1, these proteins are potent inhibitors of endothelial cell migration , and 3TSR of human TSP-1 (3TSR/hTSP-1) and TSR2+RFK activate TGF beta. An 81% inhibition of B16F10 tumor growth is observed at 2.5 mg (135 nmol)/kg/d ay of the recombinant 3TSR/hTSP-1. A comparable level of inhibition is obse rved with 2.5 mg (360 nmol)/kg/day of TSR2+PXK. By contrast, 3TSR of mouse TSP-2 (3TSR/mTSP-2), TSR2+QFK, and TSR2 are significantly less effective. T SR2+RFK and TSR2 reduce tumor vessel density, but TSR2+RFK has a greater ef fect on B16F10 tumor cell apoptosis and proliferation. Concurrent treatment of B16F10 tumor-bearing mice with TSR2+RFK and either a soluble form of th e TGF beta receptor or an antibody to active TGF beta reduces the inhibitio n of B16FIO tumor growth to levels that are comparable with those of TSR2 a nd TSR2+QFK. By contrast, the presence of the TGF beta -activating sequence does not increase the level of inhibition of Lewis lung carcinoma experime ntal tumor growth. These data indicate that the TSRs inhibit tumor grow-th by inhibition of angiogenesis and regulation of tumor cell growth and apopt osis. The regulation of tumor cell growth and apoptosis is TGF beta depende nt, whereas the inhibition of angiogenesis is not.