Kringle 5 (K5) of human plasminogen has been shown to inhibit angiogenesis by inducing the apoptosis of proliferating endothelial cells. Peptide regions around the lysine-binding pocket of K5 largely mediate these effects, particularly the peptide PRKLYDY, which we show to compete with K5 for the binding to endothelial cells. The cell surface binding site for K5 that mediates these effects has not been defined previously. Here, we report that glucose-regulated protein 78, exposed on cell surfaces of proliferating endothelial cells as well as on stressed tumor cells, plays a key role in the antiangiogenic and antitumor activity of K5. We also report that recombinant K5-induced apoptosis of stressed HT1080 fibrosarcoma cells involves enhanced activity of caspase-7, consistent with the disruption of glucose-regulated protein 78-procaspase-7 complexes. These results establish recombinant K5 as an inhibitor of a stress response pathway, which leads to both endothelial and tumor cell apoptosis. (Cancer Res 2005; 65(11): 4663-72)
Metastatic cancers produce exosomes that condition pre-metastatic niches in remote microenvironments to favor metastasis. In contrast, here we show that exosomes from poorly metastatic melanoma cells can potently inhibit metastasis to the lung. These “non-metastatic” exosomes stimulate an innate immune response through the expansion of Ly6Clow patrolling monocytes (PMo) in the bone marrow, which then cause cancer cell clearance at the pre-metastatic niche, via the recruitment of NK cells and TRAIL-dependent killing of melanoma cells by macrophages. These events require the induction of the Nr4a1 transcription factor and are dependent on pigment epithelium-derived factor (PEDF) on the outer surface of exosomes. Importantly, exosomes isolated from patients with non-metastatic primary melanomas have a similar ability to suppress lung metastasis. This study thus demonstrates that pre-metastatic tumors produce exosomes, which elicit a broad range of PMo-reliant innate immune responses via trigger(s) of immune surveillance, causing cancer cell clearance at the pre-metastatic niche.
The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).
Mal II, a 19-residue peptide derived from the second type 1 properdin-like repeat of the antiangiogenic protein thrombospondin-1 (TSP-1), was inactive in angiogenesis assays. Yet the substitution of any one of three L-amino acids by their D-enantiomers conferred on this peptide a potent antiangiogenic activity approaching that of the intact 450-kDa TSP-1. Substituted peptides inhibited the migration of capillary endothelial cells with an ED50 of 8.5 nM for the D-Ile-15 substitution, 10 nM for the D-Ser-4 substitution, and 0.75 nM for the D-Ser-5 substitution. A peptide with D-Ile at position 15 could be shortened to its last seven amino acids with little loss in activity. Like whole TSP-1, the Mal II D-Ile derivative inhibited a broad range of angiogenic inducers, was selective for endothelial cells, and required CD36 receptor binding for activity. A variety of end modifications further improved peptide potency. An ethylamide-capped heptapeptide was also active systemically in that when injected i.p. it rendered mice unable to mount a corneal angiogenic response, suggesting the potential usefulness of such peptides as antiangiogenic therapeutics.
The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.
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