The formation of a microvascular endothelium plays a critical role in the growth and
metastasis of established
tumors. The ability of a fragment from the first type III repeat of
fibronectin (III(1C)),
anastellin, to suppress
tumor growth and
metastasis in vivo has been reported to be related to its antiangiogenic properties, however, the mechanism of action of
anastellin remains unknown. Utilizing cultures of human dermal microvascular endothelial cells, we provide evidence that
anastellin inhibits signaling pathways which regulate the extracellular signal-regulated (ERK)
mitogen-activated protein kinase pathway and subsequent expression of
cell cycle regulatory proteins. Addition of
anastellin to primary microvascular endothelial cells resulted in a complete inhibition of serum-dependent proliferation. Growth inhibition correlated with a decrease in serum-dependent expression of
cyclin D1,
cyclin A and the
cyclin-dependent kinase, cdk4, key regulators of cell cycle progression through G(1) phase. Consistent with a block in G(1)-S transition,
anastellin inhibited serum-dependent incorporation of [(3)H]-
thymidine into S-phase nuclei. Addition of
anastellin to serum-starved microvessel cells resulted in a time-dependent and dose-dependent decrease in basal levels of phosphorylated
MEK/ERK and blocked serum-dependent activation of ERK. Adenoviral
infection with Ad.DeltaB-Raf:ER, an inducible
estrogen receptor-B-Raf fusion
protein, restored levels of active ERK in
anastellin-treated cells, rescued levels of
cyclin D1,
cyclin A, and cdk4, and rescued [(3)H]-
thymidine incorporation. These data suggest that the antiangiogenic properties of
anastellin observed in mouse models of human
cancer may be due to its ability to block endothelial cell proliferation by modulating ERK signaling pathways and down-regulating cell cycle regulatory gene expression required for G(1)-S phase progression.