Commentary: Granins, Secretory Granule Biogenesis, and Transport

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Immunomodulatory Agents with Antivascular Activity in the Treatment of Non-Small Cell Lung Cancer: Focus on TLR9 Agonists, IMiDs and NGR-TNF

Standard treatments for nonsmall cell lung cancer (NSCLC), such as surgery, chemotherapy, and radiotherapy, often lead to disappointing results. Unfortunately, also the various immunotherapeutic approaches so far tested have not produced satisfactory results to be widely applied in the clinical practice. However, the recent development of new immunomodulatory agents may open promising therapeutic options. This paper focuses on PF3512676, lenalidomide, and NGR-TNF, that is, drugs belonging to three different classes of immunomodulatory agents, that are also capable to affect tumor blood vessels with different mechanisms, and discusses the potential role of such agents in NSCLC treatment strategy

Coupling Tumor Necrosis Factor-α with αV Integrin Ligands Improves Its Antineoplastic Activity

Despite the impressive results obtained in animal models, the clinical use of tumor necrosis factor-α (TNF) as an anticancer drug is limited by severe toxicity. We have shown previously that targeted delivery of TNF to aminopeptidase N (CD13), a marker of angiogenic vessels, improved the therapeutic index of this cytokine in tumor-bearing mice. To assess whether the vascular-targeting approach could be extended to other markers of tumor blood vessels, in this work, we have fused TNF with the ACDCRGDCFCG peptide, a ligand of αV integrins by recombinant DNA technology. We have found that subnanogram doses of this conjugate are sufficient to induce antitumor effects in tumor-bearing mice when combined with melphalan, a chemotherapeutic drug. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the Arg-Gly-Asp moiety interacts with cell adhesion receptors, including αVβ3 integrin, as originally postulated. In addition, ACGDRGDCFCG-mouse TNF conjugate induced cytotoxic effects in standard cytolytic assays, implying that ACGDRGDCFCG-mouse TNF conjugate can also bind TNF receptors and trigger death signals. These results indicate that coupling TNF with αV integrin ligands improves its antineoplastic activity and supports the concept that vascular targeting is a strategy potentially applicable to different endothelial markers, not limited to CD13

Bone marrow-derived CD13+ cells sustain tumor progression: A potential non-malignant target for anticancer therapy

Non-malignant cells found within neoplastic lesions express alanyl (membrane) aminopeptidase (ANPEP, best known as CD13), and CD13-null mice exhibit limited tumor growth and angiogenesis. We have recently demonstrated that a subset of bone marrow-derived CD11b+CD13+ myeloid cells accumulate within neoplastic lesions in several murine models of transplantable cancer to promote angiogenesis. If these findings were confirmed in clinical settings, CD11b+CD13+ myeloid cells could become a non-malignant target for the development of novel anticancer regimens

Spontaneous Formation of L-Isoaspartate and Gain of Function in Fibronectin

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-L-isoAsp-O- methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn(263) at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an alpha(v)beta(3)-integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates alpha(v)beta(3) antagonists ( named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins

Chromogranin A fragments modulate cell adhesion. Identification and characterization of a pro-adhesive domain.

Although several functions have been suggested for chromogranin A, a glycoprotein secreted by many neuroendocrine cells, the physiological role of this protein and of its proteolytic fragments has not been established. We have found that mixtures of chromogranin A fragments can inhibit fibroblast adhesion. The anti-adhesive activity was converted into pro-adhesive activity by limited trypsin treatment. Pro-adhesive effects were observed also with recombinant N-terminal fragments corresponding to residues 1–78 and 1–115 and with a synthetic peptide encompassing the residues 7–57. These fragments induced adhesion and spreading of fibroblasts on plates coated with collagen I or IV, laminin, fetal calf serum (FCS) but not on bovine serum albumin. The long incubation time required for adhesion assays (4 h) and the FCS requirements for optimal adhesion suggest that the adhesive activity is likely indirect and requires other proteins present in the FCS or made by the cells. These findings suggest that chromogranin A and its fragments could play a role in the regulation of cell adhesion. Since chromogranin A is concentrated and stored within granules and rapidly released by neuroendocrine cells and neurons after an appropriate stimulus, this protein could be important for the local control of cell adhesion by stimulated cells

The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases.

Previous studies have shown that the region of human urokinase-type plasminogen activator (uPA) responsible for receptor binding resides in the amino-terminal fragment (ATF, residues 1-135) (Stoppelli, M.P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R.K. (1985) Proc. Natl. Acad. Sci. U.S. A. 82, 4939-4943). The area within ATF responsible for specific receptor binding has now been identified by the ability of different synthetic peptides corresponding to different regions of the amino terminus of uPA to inhibit receptor binding of 125I-labeled ATF. A peptide corresponding to human [Ala19]uPA-(12-32) resulted in 50% inhibition of ATF binding at 100 nM. Peptides uPA-(18-32) and [Ala13]uPA-(9-20) inhibit at 100 and 2000 microM, respectively. The human peptide uPA-(1-14) and the mouse peptide [Ala20]uPA-(13-33) have no effect on ATF receptor binding. This region of uPA is referred to as the growth factor module since it shares partial amino acid sequence homology (residues 14-33) to epidermal growth factor (EGF). Furthermore, this region of EGF is responsible for binding of EGF to its receptor (Komoriya, A. Hortsch, M., Meyers, C., Smith, M., Kanety, H., and Schlessinger, J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1351-1355). However, EGF does not inhibit ATF receptor binding. Comparison of the sequences responsible for receptor binding of uPA and EGF indicate that the region of highest homology is between residues 13-19 and 14-20 of human uPA and EGF, respectively. In addition, there is a conservation of the spacings of four cysteines in this module whereas there is no homology between residues 20-30 and 21-33 of uPA and EGF. Thus, residues 20-30 of uPA apparently confer receptor binding specificity, and residues 13-19 provide the proper conformation to the adjacent binding region

Cleavage of chromogranin A N-terminal domain by plasmin provides a new mechanism for regulating cell adhesion

It has been proposed that chromogranin A (CgA), a protein secreted by many normal and neoplastic neuroendocrine cells, can play a role as a positive or a negative modulator of cell adhesion. The mechanisms that regulate these extracellular functions of CgA are unknown. We show here that plasmin can regulate the anti/pro-adhesive activity of CgA by proteolytic cleavage of the N-terminal domain. Limited proteolytic processing decreased its anti-adhesive activity and induced pro-adhesive effects in fibronectin or serum-dependent fibroblast adhesion assays. Cleavage of Lys(77)-Lys(78) dibasic site in CgA(1-115) was relatively rapid and associated with an increase of pro-adhesive effect. In contrast, antibodies against the region 53-90 enhanced the anti-adhesive activity of CgA and CgA(1-115). Structure-activity relationship studies showed that the conserved region 47-64 (RILSILRHQNLLKELQDL) is critical for both pro- and anti-adhesive activity. These findings suggest that CgA might work on one hand as a negative modulator of cell adhesion and on the other hand as a precursor of positive modulators, the latter requiring proteolytic processing for activation. Given the importance of plasminogen activation in tissue invasion and remodeling, the interplay between CgA and plasmin could provide a novel mechanism for regulating fibroblast adhesion and function in neuroendocrine tumors