CD47 is a signaling receptor for thrombospondin-1 and the counter-receptor for signal-regulatory protein-alpha (SIRPalpha). By inducing inhibitory SIRPalpha signaling, elevated CD47 expression by some cancers prevents macrophage phagocytosis. The anti-human CD47 antibody B6H12 inhibits tumor growth in several xenograft models, presumably by preventing SIRPalpha engagement. However, CD47 signaling in nontransformed and some malignant cells regulates self-renewal, suggesting that CD47 antibodies may therapeutically target cancer stem cells (CSCs). Treatment of MDA-MB-231 breast CSCs with B6H12 decreased proliferation and asymmetric cell division. Similar effects were observed in T47D CSCs but not in MCF7 breast carcinoma or MCF10A breast epithelial cells. Gene expression analysis in breast CSCs treated with B6H12 showed decreased expression of epidermal growth factor receptor (EGFR) and the stem cell transcription factor KLF4. EGFR and KLF4 mRNAs are known targets of microRNA-7, and B6H12 treatment correspondingly enhanced microRNA-7 expression in breast CSCs. B6H12 treatment also acutely inhibited EGF-induced EGFR tyrosine phosphorylation. Expression of B6H12-responsive genes correlated with CD47 mRNA expression in human breast cancers, suggesting that the CD47 signaling pathways identified in breast CSCs are functional in vivo. These data reveal a novel SIRPalpha-independent mechanism by which therapeutic CD47 antibodies could control tumor growth by autonomously forcing differentiation of CSC. Thrombospondin-1 regulates inflammation by engaging several cell surface receptors and by modulating activities of other secreted factors. We have uncovered a novel role of thrombospondin-1 in modulating production and activation of the proinflammatory cytokine IL-1beta by human and murine macrophages. Physiological concentrations of thrombospondin-1 limit the induction by lipopolysaccharide of IL-1beta mRNA and total protein production by human macrophages. This inhibition can be explained by the ability of thrombospondin-1 to disrupt the interaction between CD47 and CD14, thereby limiting activation of NFkB/AP-1 by lipopolysaccharide. Only the CD47-binding domain of thrombospondin-1 exhibits this activity. In contrast, CD47, CD36, and integrin-binding domains of thrombospondin-1 independently enhance the inflammasome-dependent maturation of IL-1beta in human THP-1 monocyte-derived macrophages. Correspondingly, mouse bone marrow-derived macrophages that lack either thrombospondin-1 or CD47 exhibit diminished induction of mature IL-1beta in response to lipopolysaccharide. Lack of CD47 also limits lipopolysaccharide induction of IL-1beta, NLRP3, and caspase-1 mRNAs. These data demonstrate that thrombospondin-1 exerts CD47-dependent and -independent pro-and anti-inflammatory effects on the IL-1beta pathway. Therefore, thrombospondin-1 and its receptor CD47 may be useful targets for limiting the pro-inflammatory effects of lipopolysaccharide and for treating endotoxemia. Thrombospondin 1 is a glycoprotein that regulates cellular phenotype through interactions with its cellular receptors and extracellular matrix-binding partners. Thrombospondin 1 locally regulates angiogenesis and inflammatory responses that contribute to colorectal carcinogenesis in Apc(Min/+) mice. The ability of thrombospondin 1 to regulate responses of cells and tissues to a variety of stresses suggested that loss of thrombospondin 1 may also have broader systemic effects on metabolism to modulate carcinogenesis. Apc(Min/+):Thbs1(-/-) mice exhibited decreased survival and higher tumor multiplicities in the small and large intestine relative to Apc(Min/+) mice when fed a low (5%) fat western diet. However, the protective effect of endogenous thrombospondin 1 was lost when the mice were fed a western diet containing 21% fat. Biochemical profiles of liver tissue identified systemic metabolic changes accompanying the effects of thrombospondin 1 and dietary lipid intake on tumorigenesis. A high-fat western diet differentially regulated elements of amino acid, energy and lipid metabolism in Apc(Min/+):Thbs1(-/-) mice relative to Apc(Min/+):Thbs1(+/+)mice. Metabolic changes in ketone body and tricarboxylic acid cycle intermediates indicate functional interactions between Apc and thrombospondin 1 signaling that control mitochondrial function. The cumulative diet-dependent differential changes observed in Apc(Min/+):Thbs1(-/-) versus Apc(Min/+) mice include altered amino acid and lipid metabolism, mitochondrial dysfunction, eicosanoids and ketone body formation. This metabolic profile suggests that the protective role of thrombospondin 1 to decrease adenoma formation in Apc(Min/+) mice results in part from improved mitochondrial function.
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