Claude Lalou

Evidence that limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) occurs in the normal state outside of the bloodstream.

In serum and other biological fluids, IGF-I and IGF-II are reversibly bound to six molecular species of specific binding proteins (IGFBP-1 to -6) which regulate their transport to target cells as well as their biological activities. Most of the IGFs in adult serum are bound to IGFBP-3 and associated with a 85 kDa subunit to form 150 kDa ternary complexes, very few of which cross the capillary barrier. In earlier studies we showed that during pregnancy one or more serine proteinases are responsible for limited proteolysis of IGFBP-3 in the serum. This may result in easier dissociation of the IGFs and hence an increase in their availability. In the present work, the results of Western blot analyses of the IGFBPs, using either labelled IGF or a polyclonal anti-IGFBP-3 antibody, have demonstrated that IGFBP-3 proteolysis occurs in the normal state. In all the adults investigated, serum contained both the 42-39 kDa doublet, characteristic of intact IGFBP-3, and its major degradation fragment of 30 kDa. The fragment was also found in lymph, in addition to smaller fragments of 21.5, 20 and 16 kDa which are the same sizes as those seen in pregnancy serum. Comparisons of lymph (which reflects the interstitium) and serum from the same subjects showed greater proportions of IGFBP-3 proteolysed in lymph than in serum and incubations with [125I]IGFBP-3 showed almost 8-fold higher proteolytic activity in lymph than in serum where it was minimal. These findings suggest that the initial sites of proteolysis are in the tissues. Like that in pregnancy serum, the activity was calcium-dependent and inhibited by aprotinin. The results of this study fit the hypothesis that limited proteolysis of IGFBP-3 may be an essential mechanism in controlling the bioavailability of the IGFs, both at the cellular level and, in the blood, from the 150 kDa complexes which constitute the circulating reserves of IGF.

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

Background The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. Methodology/Principal Findings We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

Differential molecular profiling between skin carcinomas reveals four newly reported genes potentially implicated in squamous cell carcinoma development.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are skin tumors with different invasive potential. In this work, we analysed mRNA differential expression between seven BCC and five SCC and their normal skin counterparts using 1176 cDNA macroarrays and verification by RT–PCR to identify genes modulated in each tumor type. We identified 37 genes commonly modulated in both tumors and four genes specifically modulated in SCC. Among these latter RhoC and EMMPRIN genes seem to be of particular interest and could participate in SCC aggressivity.

Proteolytic fragments of insulin-like growth factor binding protein-3: N-terminal sequences and relationships between structure and biological activity

Insulin-like growth factors (IGF-I and IGF-II) in biological fluids bind to high-affinity binding proteins (IGFBP-1 to -6), which transport them and regulate their activities. Limited proteolysis of certain IGFBPs plays a major role in this regulation. IGFBP-3 is proteolysed in vivo and in several cell lines by serine proteases, including plasmin. In earlier studies we reproduced this proteolysis in vitro using recombinant human non-glycosylated IGFBP-3. Two major fragments were obtained, the larger retaining weak affinity for IGF-I and weakly inhibiting IGF I mitogenic effects. The smaller fragment, though lacking affinity for IGFs, is a potent growth inhibitor. These proteolytic fragments were isolated by HPLC and their N-terminal amino acids sequenced. Both major fragments contain the N-terminal region of the intact protein, the larger form corresponding to residues 1-160, and the smaller form, to residues 1-95. Kinetics experiments using the MG-63 osteoblast-like cell line showed that the larger peptide is generated before the smaller peptide, the latter probably being a product of secondary proteolysis of the former. Our data suggest that proteolysis of IGFBP-3 is intimately linked to its biological function. We propose a model for its action at cellular level.

Insulin-Like Growth Factor Binding Protein-4 Proteolytic Degradation in Ovine Preovulatory Follicles: Studies of Underlying Mechanisms1

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161–264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition ...

Limited Proteolysis of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) : A Physiological Mechanism in the Regulation of IGF Bioavailability

Enzymes are known to play a role in activating or releasing growth factors like the FGFs and TFGs from the extra-cellular matrix (1, 2). When first discovered, in vivo proteolysis of serum IGFBPs, and particularly IGFBP-3, by pregnancy-associated protease(s) was naturally suspected to be physiologically significant (3, 4). Although the structural alteration of IGFBP-3 and its loss of affinity for the IGFs were clearly demonstrated by Western ligand blotting, immunoblotting and competitive binding studies (3 – 5), some doubt was expressed as to its physiological nature in view of the harsh experimental conditions used (SDS, acidification). Both IGFBP-3 and the acid-labile (a) subunit were found in the 150-kDa material and ternary complex formation was found to be possible with acidified pregnancy serum in the presence of a-subunit (6). Actually, in our initial work on pregnancy serum, we showed that the 150-kDa complex was not disrupted in pregnancy serum analysed by gel filtration at neutral pH, although there were also small IGFBP-3 fragments in the fractions containing low molecular weight proteins (3). It therefore seemed possible that the stability of the 150-kDa complex would be diminished and the IGFBP-3 functionally altered, even if proteolysis remained limited.

Isolation and Characterization of Proteolytic Fragments of Insulin-Like Growth Factor-Binding Protein-3

Limited proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) is a normal process in the regulation of insulin-like growth factor (IGF) activity, which we have reproduced in vitro using plasmin and recombinant human non-glycosylated IGFBP-3 in order to isolate and characterize the fragments obtained. Two major fragments of 22-25 and 16 kD were purified by RP-HPLC. The 22- to 25-kD fragment had severely reduced affinity for IGF-I, compared with intact IGFBP-3. It weakly inhibited cell proliferation stimulated by IGF-I and had no effect on insulin-induced stimulation. The 16-kD fragment, which had lost all affinity for IGFs, unexpectedly proved to be a potent inhibitor of both IGF-I-induced and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGF is a cell growth inhibitor

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavialability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of 22 and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The 22-25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo fibroblast assay where DNA synthesis was stimulated by IGF-I or insulin, the 22-25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

Biological Actions of Proteolytic Fragments of the IGF Binding Proteins

The existence of insulin-like growth factor binding protein (IGFBP) fragments was first discerned during purification of IGFBP-1 from human placenta (1) and IGFBP-3 from human (2,3) and rat serum (4,5) and human cerebrospinal fluid (6). A truncated form of IGFBP-5 was isolated in advance of the intact protein (6,7). The amino (N)-terminal sequences of these fragments were found to correspond to forms lacking the carboxy (C)-terminal third of the molecule. However, in the case of IGFBP-2, a fragment corresponding to the C-terminus of the native binding protein was isolated from the conditioned medium of a fetal rat liver cell line (8). At the same time, it was discovered that IGFBP-3 undergoes limited proteolysis during pregnancy (9–11). The resulting functional alteration was revealed by the disappearance of the characteristic 42–39-kDa doublet in Western ligand blotting and the concomitant appearance of a 30-kDa fragment in the chromatographic fragments normally containing the 140-kDa complexes that comprise IGFBP-3, IGF-I or -II, and the acid-labile subunit (ALS). From these observations, it seemed that the fragment must play some physiological role. Further studies confirmed its weak affinities for the IGFs, which would explain their facilitated dissociation from the 140-kDa complexes and their enhanced bioavailability (12,13).

Abstract 2341: The Subtilisin-like proprotein convertases blockade inhibits the invasiveness of human primary melanoma with altered P53, CDKN2A and N-Ras genes

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Altered tumor suppressor p53 and/or CDKN2A as well as the Ras genes are frequently found in primary and metastatic melanomas. These alterations are responsible for the invasive/metastatic potential acquisition through their defective regulatory control on various MMPs and urokinase genes. Using primary human melanoma M10 cells with altered P53, CDKN2A and N-Ras genes, we found that inhibition of the proprotein convertases (PCs), enzymes involved in the proteolytic activation and/or expression of various cancer-related protein precursors reduced significantly their invasiveness. Analysis of M10 cells and their gastric and ganglion derived metastatic lesions revealed the presence of all the PCs found in the secretory pathway namely Furin, PACE4, PC5 and PC7. Expression of the general PCs inhibitor α1-PDX in these cells in a stable manner (M10/PDX) had no effect on the mRNA expression levels of these PCs. In vitro digestion assays and cell transfection experiments, revealed that M10/PDX cells present reduced PCs activity and are unable to process the PCs substrates proIGF-1R and proPDGF-A. These cells showed reduced migration, and invasion of Matrigel in vitro that paralleled decreased gelatinase MMP-2 activity and increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2. Furthermore, these cells showed decreased mRNA levels of urokinase-type plasminogen activator receptor (uPAR) and increased mRNA levels of plasminogen activator inhibitor-1 (PAI-1). Taken together, these data suggest that PCs activity inhibition has the ability to interfere with primary human melanoma cells invasiveness despite their altered P53, CDKN2A and N-Ras genes, suggesting the potential use of PCs as new therapeutic targets in melanoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2341.