Circulating Chromogranin A Is Cleaved Into Vasoregulatory Fragments in Patients With Pancreatic Ductal Adenocarcinoma.

Chromogranin A (CgA), a secretory protein released in the blood by the neuroendocrine system, consists of a mixture of full-length molecules and fragments endowed of vasoregulatory activity. The extent and the role of CgA fragmentation were investigated in patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC, n=172). Multivariate analysis showed that full-length CgA was associated with better progression free and overall survival, whereas CgA C-terminal fragmentation was associated with worse prognosis. In vitro studies showed that PDAC cells can promote the cleavage of CgA C-terminal region by activating plasminogen to plasmin. Limited digestion of full-length CgA with plasmin abolished its anti-angiogenic activity and generated pro-angiogenic molecules. The fragmentation of CgA C-terminal region was increased also in murine models of PDAC. In these models, the inhibition of CgA fragmentation with aprotinin, an inhibitor of plasmin and other serine proteases, or the blockade of pro-angiogenic fragments with specific antibodies inhibited the growth of PDAC implanted subcutaneously in mice. Finally, administration of full-length CgA to mice bearing orthotopic PDAC reduced tumor perfusion, as measured by contrast-enhanced ultrasound. These findings suggest that PDAC can promote the cleavage of circulating CgA C-terminal region to generate fragments that regulate the tumor vascular biology and that may represent new potential therapeutic targets.

Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to regulation of the cardiotoxic and antitumor activities of Doxo. The effects of a physiologic dose of full-length recombinant CgA on Doxo-induced cardiotoxicity and antitumor activity were investigated in rats using in vivo and ex vivo models and in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. In vivo and ex vivo mechanistic studies showed that CgA can prevent Doxo-induced heart inflammation, oxidative stress, apoptosis, fibrosis, and ischemic injury. On the other hand, CgA did not impair the anticancer activity of Doxo in all the murine models investigated. Furthermore, we observed that Doxo can reduce the intracardiac expression and release of CgA in the blood (i.e., an important cardioprotective agent). These findings suggest that administration of low-dose CgA to patients with low levels of endogenous CgA might represent a novel approach to prevent Doxo-induced adverse events without impairing antitumor effects.-Rocca, C., Scavello, F., Colombo, B., Gasparri, A. M., Dallatomasina, A., Granieri, M. C., Amelio, D., Pasqua, T., Cerra, M. C., Tota, B., Corti, A., Angelone, T. Physiological levels of chromogranin A prevent doxorubicin-induced cardiotoxicity without impairing its anticancer activity.

Spatiotemporal Regulation of Tumor Angiogenesis by Circulating Chromogranin A Cleavage and Neuropilin-1 Engagement.

The 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. SIGNIFICANCE: 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.

Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality.

The chondroitin sulfate proteoglycan 4 ( CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.-Tamburini, E., Dallatomasina, A., Quartararo, J., Cortelazzi, B., Mangieri, D., Lazzaretti, M., Perris, R. Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality.

Vasostatin-1: A novel circulating biomarker for ileal and pancreatic neuroendocrine neoplasms.

BACKGROUND: Chromogranin A (CgA) is a plasma biomarker widely used in the follow-up of patients with neuroendocrine neoplasms (NENs). However, its accuracy as a tumor biomarker is relatively low because plasma CgA can increase also in patients with other diseases or in subjects treated with proton-pump inhibitors (PPIs), a class of widely-used drugs. METHODS: In the attempt to identify a more reliable biomarker for NENs, we investigated, by ELISA, the circulating levels of full-length CgA (CgA1-439) and of various CgA-derived fragments in 17 patients with ileal or pancreatic NENs, 10 healthy controls, and 21 healthy volunteers before and after treatment with PPIs. RESULTS: Patients with ileal or pancreatic NENs showed increased plasma levels of total-CgA and CgA1-76 fragment (vasostatin-1, VS-1) compared to controls [median (25th-75th-percentiles); total-CgA: 1.85 nM (1.01-4.28) vs 0.75 nM (0.52-0.89), p = 0.004; VS-1: 2.76 nM (1.09-7.10) vs 0.29 nM (0.26-0.32), p<0.001, respectively], but not of CgA1-439 or CgA1-373 fragment. VS-1 positively correlated with total-CgA (r = 0.65, p<0.001). The Receiver Operating Characteristic area under the curve was 0.9935 for VS-1 and 0.8824 for total-CgA (p = 0.067). Treatment of patients with somatostatin analogues decreased both total-CgA and VS-1. In contrast, administration of PPIs increased the plasma levels of total-CgA, but not of VS-1. CONCLUSION: These findings suggest that plasma VS-1 is a novel biomarker for ileal and pancreatic NENs. Considering that VS-1 is a well-defined fragment not induced by proton-pump inhibitors, this polypeptide might represent a biomarker for NENs diagnosis and follow-up more accurate and easier to standardize than CgA.

Everolimus in the management of metastatic renal cell carcinoma: an evidence-based review of its place in therapy.

INTRODUCTION: Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, and its pathogenesis is strictly related to altered cellular response to hypoxia, in which mTOR signaling pathway is implicated. Everolimus, an mTOR serine/threonine kinase inhibitor, represents a therapeutic option for the treatment of advanced RCC. AIM: The objective of this article is to review the evidence for the treatment of metastatic RCC with everolimus. EVIDENCE REVIEW: Everolimus was approved for second- and third-line therapy in patients with advanced RCC according to the results of a Phase III pivotal trial that demonstrated a benefit in median progression-free survival of ~2 months compared to placebo after failure of previous lines of therapy, of which at least one was an anti-VEGFR tyrosine kinase inhibitor (TKI). The role of this drug in first-line setting has been investigated in Phase II trials, with no significant clinical benefit, even in combination with bevacizumab. Everolimus activity in non-clear cell RCC is supported by two randomized Phase II trials that confirmed the benefit in second-line setting but not in first line. Recently, two randomized Phase III trials (METEOR and CheckMate 025) demonstrated the inferiority of everolimus in second-line setting compared to the TKI cabozantinib and to the immune checkpoint inhibitor nivolumab, respectively. Moreover, a recent Phase II study demonstrated a significant benefit for the second-line combination treatment with everolimus plus lenvatinib (a novel TKI) in terms of progression-free survival and overall survival compared to the single-agent everolimus. Basing on preclinical data, the main downstream effectors of mTOR cascade, S6RP and its phosphorylated form, could be good predictive biomarkers of response to everolimus. The safety profile of the drug is favorable, with a good cost-effectiveness compared to second-line sorafenib or axitinib, and no significant impact on the quality of life of treated patients has been found. CONCLUSION: Everolimus still represents a current standard of treatment for RCC progressive to previous treatment lines with VEGFR-TKI. The evidence about two new molecules, cabozantinib and nivolumab, successfully tested head-to-head with everolimus in recently published Phase III trials, will determine the shift of everolimus to the third-line setting and subsequent lines of treatment.

Regulation of tumor growth by circulating full-length chromogranin A.

Chromogranin A (CgA), a neuroendocrine secretory protein, and its fragments are present in variable amounts in the blood of normal subjects and cancer patients. We investigated whether circulating CgA has a regulatory function in tumor biology and progression. Systemic administration of full-length CgA, but not of fragments lacking the C-terminal region, could reduce tumor growth in murine models of fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma, with U-shaped dose-response curves. Tumor growth inhibition was associated with reduction of microvessel density and blood flow in neoplastic tissues. Neutralization of endogenous CgA with antibodies against its C-terminal region (residues 410-439) promoted tumor growth. Structure-function studies showed that the C-terminal region of CgA contains a bioactive site and that cleavage of this region causes a marked loss of anti-angiogenic and anti-tumor potency. Mechanistic studies showed that full-length CgA could induce, with a U-shaped dose-response curve, the production of protease nexin-1 in endothelial cells, a serine protease inhibitor endowed of anti-angiogenic activity. Gene silencing or neutralization of protease nexin-1 with specific antibodies abolished both anti-angiogenic and anti-tumor effects of CgA. These results suggest that circulating full-length CgA is an important inhibitor of angiogenesis and tumor growth, and that cleavage of its C-terminal region markedly reduces its activity. Pathophysiological changes in CgA blood levels and/or its fragmentation might regulate disease progression in cancer patients.

Theranostic impact of NG2/CSPG4 proteoglycan in cancer.

NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be exploited in therapeutic interventions aimed at averting resistance to target therapy agents and at interfering with secondary lesion formation and/or tumour recurrence.

Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.