Sup Fig 1: Characterization of mAb 9D1 and its effects on tumor growth and NRP1/CgA1-373 interaction; Sup Fig 2: Effect of aprotinin treatment on tumor growth and CgA cleavage; Sup Fig 3: Anti-PGPQLR monoclonal and polyclonal antibodies inhibit tumor growth in various murine models; Sup Fig 4: Effect of anti-PQLR IgGs (highly specific for CgA1-373) and anti-QALRRG (C-terminal region of CgA1-439) on tumor growth; Sup Fig 5: Effect of anti-PGPQLR on WEHI-164 fibrosarcoma tumor vasculature; Sup Fig 6: Effect of CgA1-373 and FGF2 on HUVEC proliferation; Sup Fig 7: NRP1, but not NRP2, binds CgA1-373; the binding is blocked by anti-PGPQLR or anti-PQLR antibodies; Sup Fig 8: Effects of CgA1-373 and VEGF on VEGF-R2 phosphorylation at tyrosine (Y) residues 951, 996 or 1175 in HUVEC.
ARTICLE ABSTRACTThe unbalanced production of pro- and antiangiogenic factors in tumors can lead to aberrant vasculature morphology, angiogenesis, and disease progression. In this study, we report that disease progression in various murine models of solid tumors is associated with increased cleavage of full-length chromogranin A (CgA), a circulating vasoregulatory neurosecretory protein. Cleavage of CgA led to the exposure of the highly conserved PGPQLR site, which corresponds to residues 368–373 of human CgA1-373, a fragment that has proangiogenic activity. Antibodies against this site, unable to bind full-length CgA, inhibited angiogenesis and reduced tumor perfusion and growth. The PGPQLR sequence of the fragment, but not of the precursor, bound the VEGF-binding site of neuropilin-1; the C-terminal arginine (R373) of the sequence was crucial for binding. The proangiogenic activity of the CgA1-373 was blocked by anti-neuropilin-1 antibodies as well as by nicotinic acetylcholine receptor antagonists, suggesting that these receptors, in addition to neuropilin-1, play a role in the proangiogenic activity of CgA1-373. The R373 residue was enzymatically removed in plasma, causing loss of neuropilin-1 binding and gain of antiangiogenic activity. These results suggest that cleavage of the R373R374 site of circulating human CgA in tumors and the subsequent removal of R373 in the blood represent an important “on/off” switch for the spatiotemporal regulation of tumor angiogenesis and may serve as a novel therapeutic target.
This work reveals that the interaction between fragmented chromogranin A and neuropilin-1 is required for tumor growth and represents a novel potential therapeutic target.