Chromogranin A Is Preferentially Cleaved into Proangiogenic Peptides in the Bone Marrow of Multiple Myeloma Patients

Abstract

Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N-and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk à MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/ antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target.

Regulation of tumor growth by circulating full-length chromogranin A

“…This dose, administered at 2-3 day intervals, generates peak plasma levels of about 3-4 nM that progressively declines to 0.5-1 nM in 7-8 h and to even lower concentrations at later time points. Considering that circulating levels of CgA in normal subject is 0.2-1 nM (most consisting of fragments lacking the C-terminal region and less than 10-20% consisting of full-length CgA) [25], the biological effects observed in our models are likely patho-physiologically relevant.…”

“…This view is supported by our observation that anti-PN1 antibodies could neutralize the anti-angiogenic activity of CgA in vitro as well as the anti-tumor activity of CgA in different in vivo models. Moreover, considering that CgA is a good substrate of thrombin and plasmin [24, 25], often present in tumors, and that PN-1 is a potent inhibitor of these proteases [38], it is tempting to speculate that PN-1 may also serve to prevent the local cleavage of CgA in tumors (thereby preserving its anti-angiogenic activity) and/or other factors necessary for tumor progression and invasion. Finally, the bell-shaped dose-response curve and the consequent lack of PN1 induction by high concentrations of CgA may explain the lack of anti-tumor effects with high-dose CgA.…”

“…Furthermore, elevated serum levels of CgA have been observed in patients with renal failure, heart failure, hypertension, rheumatoid arthritis, atrophic gastritis, inflammatory bowel disease, sepsis and other inflammatory diseases [1, 2, 8, 12-23]. Using specific assays, we recently observed that CgA is present in the blood of normal subjects and cancer patients as a mixture of full-length protein and fragments, including the N-terminal fragment CgA 1-76 (vasostatin-1) and other fragments lacking part or the entire C-terminal region [24, 25]. Interestingly, the plasma levels of the fragment CgA 1-373, which is not or minimally present in normal subjects, is increased in the peripheral blood (and even more in the bone marrow plasma) of patients with multiple myeloma, while full-length CgA is decreased, pointing to increased CgA C-terminal fragmentation in these patients [25].…”

“…Using specific assays, we recently observed that CgA is present in the blood of normal subjects and cancer patients as a mixture of full-length protein and fragments, including the N-terminal fragment CgA 1-76 (vasostatin-1) and other fragments lacking part or the entire C-terminal region [24, 25]. Interestingly, the plasma levels of the fragment CgA 1-373, which is not or minimally present in normal subjects, is increased in the peripheral blood (and even more in the bone marrow plasma) of patients with multiple myeloma, while full-length CgA is decreased, pointing to increased CgA C-terminal fragmentation in these patients [25]. Thus, variable levels of full-length CgA and fragments are present in the blood of cancer patients for a variety of reasons.…”

Inhibition of chronic lymphocytic leukemia progression by full-length chromogranin A and its N-terminal fragment in mouse models

“…Structure-function studies have shown that full-length CgA contains a) a latent anti-angiogenic site in the N-terminal region 1-76, b) a latent pro-angiogenic site in the region 352-373 and c) a functional anti-angiogenic site in the C-terminal region 410-439 [14, 16, 17]. The latent anti-angiogenic site is activated by cleavage of the Q 76 -K 77 peptide bond, by unknown enzymes, whereas the pro-angiogenic site can be activated by thrombin- or plasmin-mediated cleavage of the R 373 -R 374 dibasic site [14, 18]. Furthermore, using murine models of mouse mammary adenocarcinomas and melanomas we have shown that full-length CgA and VS-1 can enhance the endothelial barrier function and reduce the trans-endothelial migration of tumor cells, thereby reducing the trafficking of cancer cells from tumor-to-blood and from blood-to-tumor/normal tissues (i.e.…”