Microenvironment and Immunology Chromogranin A Is Preferentially Cleaved into Proangiogenic Peptides in the Bone Marrow of Multiple Myeloma Patients Mimma Bianco 1 , Anna Maria Gasparri 1 , Barbara Colombo 1 , Flavio Curnis 1 , Stefania Girlanda 1 , Maurilio Ponzoni 1 , Maria Teresa Sabrina Bertilaccio 1 , Arianna Calcinotto 2 , Angelina Sacchi 1 , Elisabetta Ferrero 1 , Marina Ferrarini 1 , Marta Chesi 3 , P. Leif Bergsagel 3 , Matteo Bellone 2 , Giovanni Tonon 1 , Fabio Ciceri 1 , Magda Marcatti 1 , Federico Caligaris-Cappio 1,4 , and Angelo Corti 1,4 Abstract Angiogenesis has been postulated to be critical for the patho- genesis 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 antian- giogenic protein, can activate latent antiangiogenic and proan- giogenic 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 microvas- cular density. Using the Vk  MYC mouse model of multiple myeloma, we further demonstrate that exogenously adminis- tered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endo- thelial 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. Cancer Res; 76(7); 1781–91. Ó2016 AACR. Introduction Multiple myeloma is a plasma cell malignancy characterized by abnormal clonal proliferation and accumulation of plasma cells in the bone marrow (BM). This results in a variety of clinical manifestations including anemia, osteolytic bone lesions, hyper- calcemia, and renal failure. Most cases of myeloma also feature the production of a paraprotein (also called "M protein"), an abnor- mal immunoglobulin that can cause kidney problems (1). The cross-talk between myeloma and endothelial cells in the BM and the consequent activation of the angiogenesis process (i.e., the formation of new blood vessels from preexisting vessels) is critical for the pathogenesis of multiple myeloma (2–4). Accordingly, BM microvessel density increases parallel to disease progression, is an independent prognostic factor for survival, and correlates with established parameters of disease activity in patients (3–11). Physiological and pathological angiogenesis are tightly regu- lated by the coordinated action of anti- and proangiogenic factors (12–14). Among the wide range of angiogenesis regulators so far discovered, recent studies have shown that chromogranin A (CgA) may have an important role in the regulation of angiogenesis (15, 16). CgA is a glycosylated, sulfated, and phosphorylated protein, 439 residue-long, stored in the secretory vesicles of many neuro- endocrine cells and neurons (17), and exocytotically released in circulation together with the costored hormones, to reach 0.5 to 1 nmol/L levels in normal conditions (18). Tissue-specific intra- granular and extracellular proteolytic processing of CgA leads to production of various bioactive peptides involved in the regula- tion of angiogenesis, metabolism, and cardiovascular system (18). Regarding angiogenesis, it has been recently shown that CgA contains: (i) a functional antiangiogenic site in the C-termi- nal region 410–439; (ii) a latent antiangiogenic site in the N- terminal region 1 to 76, and (iii) a latent proangiogenic site in the region 352–372 (15, 19, 20). These sites are activated by proteo- lytic cleavage of Q76-K77 and R373-R374 bonds, respectively. Accordingly, full-length CgA 1-439 and the N-terminal fragment CgA 1-76 (called vasostatin-1) inhibit angiogenesis in various angiogenesis assays, whereas the fragment CgA 1-373 can stimulate angiogenesis (15). Mechanistic studies have shown that full- length CgA and the N-terminal fragment vasostatin-1 can inhibit 1 Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy. 2 Division of Immunology, San Raffaele Scientific Institute, Milan, Italy. 3 Mayo Clinic, Scottsdale, Arizona. 4 Universit a Vita-Salute San Raffaele, Milan, Italy. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Angelo Corti, Universit a Vita Salute San Raffaele, San Raffaele Scientific Institute, via Olgettina 58, Milan 20132, Italy. Phone: 00390226434802; Fax: 00390226434786; E-mail: corti.angelo@hsr.it doi: 10.1158/0008-5472.CAN-15-1637 Ó2016 American Association for Cancer Research. Cancer Research www.aacrjournals.org 1781 on May 23, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 11, 2016; DOI: 10.1158/0008-5472.CAN-15-1637