Laurent Martiny

LRP-1 silencing prevents malignant cell invasion despite increased pericellular proteolytic activities

ABSTRACT The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.

CD47 update: a multifaceted actor in the tumour microenvironment of potential therapeutic interest.

CD47 is a ubiquitous 50 kDa five‐spanning membrane receptor that belongs to the immunoglobulin superfamily. This receptor, also known as integrin‐associated protein, mediates cell‐to‐cell communication by ligation to transmembrane signal‐regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also implicated in cell‐extracellular matrix interactions via ligation with thrombospondins. Furthermore, CD47 is involved in many and diverse cellular processes, including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multifaceted receptor might be a central actor in the tumour microenvironment. Solid tumours are composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms. Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with CD47 on cancer cells and stromal cells.

LRP-1 promotes cancer cell invasion by supporting ERK and inhibiting JNK signaling pathways

Background The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor mediating the clearance of various extracellular molecules involved in the dissemination of cancer cells. LRP-1 thus appeared as an attractive receptor for targeting the invasive behavior of malignant cells. However, recent results suggest that LRP-1 may facilitate the development and growth of cancer metastases in vivo, but the precise contribution of the receptor during cancer progression remains to be elucidated. The lack of mechanistic insights into the intracellular signaling networks downstream of LRP-1 has prevented the understanding of its contribution towards cancer. Methodology/Principal Findings Through a short-hairpin RNA-mediated silencing approach, we identified LRP-1 as a main regulator of ERK and JNK signaling in a tumor cell context. Co-immunoprecipitation experiments revealed that LRP-1 constitutes an intracellular docking site for MAPK containing complexes. By using pharmacological agents, constitutively active and dominant-negative kinases, we demonstrated that LRP-1 maintains malignant cells in an adhesive state that is favorable for invasion by activating ERK and inhibiting JNK. We further demonstrated that the LRP-1-dependent regulation of MAPK signaling organizes the cytoskeletal architecture and mediates adhesive complex turnover in cancer cells. Moreover, we found that LRP-1 is tethered to the actin network and to focal adhesion sites and controls ERK and JNK targeting to talin-rich structures. Conclusions We identified ERK and JNK as the main molecular relays by which LRP-1 regulates focal adhesion disassembly of malignant cells to support invasion.

De novo ceramide synthesis is responsible for the anti-tumor properties of camptothecin and doxorubicin in follicular thyroid carcinoma.

Doxorubicin and camptothecin are two cytotoxic chemotherapeutic agents triggering apoptosis in various cancer cells, including thyroid carcinoma cells. Recent studies revealed a critical role of ceramide in chemotherapy and suggested that anti-cancer drugs may kill tumor cells through sphingomyelinase activation. However, in comparison to sphingomyelin hydrolysis, the relative involvement of de novo ceramide synthesis remained poorly explored and highly controversial. Here, we evidenced that both doxorubicin and camptothecin triggered ceramide accumulation in thyroid carcinoma cells. We demonstrated that ceramide increase occurred via the de novo pathway without neither acidic nor neutral sphingomyelinase contribution. Interestingly, de novo ceramide generation was responsible for the drug-induced malignant cell apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin amount. Furthermore, blocking ceramide metabolism by inhibiting glucosylceramide synthase strengthened the camptothecin and doxorubicin-dependent effects. Altogether, we evidenced that de novo ceramide synthesis mediates the anti-tumor properties of doxorubicin and camptothecin in thyroid carcinoma and suggested that glucosylation of ceramide may contribute to the drug-resistance phenotype in thyroid malignancies.

Human thrombospondin's (TSP-1) C-terminal domain opens to interact with the CD-47 receptor: a molecular modeling study.

Thrombospondin-1 (TSP-1) interaction with the membranous receptor CD-47 involves the peptide RFYVVMWK (4N-1) located in its C-terminal domain. However, the available X-ray structure of TSP-1 describes this peptide as completely buried into a hydrophobic pocket, preventing any interaction. Where classical standard methods failed, an appropriate approach combining normal mode analysis and an adapted protocol of energy minimization identified the large amplitude motions responsible of the partial solvent exposure of 4N-1. In agreement, the obtained model of the open TSP-1 was further used for protein-protein docking experiments against a homology model generated for CD-47. Considering the multiple applications of the CD-47 receptor as a target, our results open new pharmacological perspectives for the design of TSP-1:CD-47 inhibitors and CD-47 antagonists. We also suggest a common opening mechanism for proteins sharing the same fold as TSP-1. This work also suggests the usefulness of our approach in other topics in which predictions of protein-protein interactions are of importance.

Control by thyrotropin of the production by thyroid cells of an inositol phosphate-glycan

The increased turnover of phosphatidylinositol promoted by thyrotropin (TSH) in pig thyroid tissue does not seem to be caused by an increased production of inositol tris-phosphate. We have explored another possibility, the synthesis of an inositol phosphate-glycan (IP-gly). Our results show that thyroid cells in culture produced this substance from a precursor phosphatidylinositol-glycan (Gly-PI). The obtained IP-gly seemed, by its analytical and biological properties, to be identical, or similar, to the previously described insulin mediator.

Elastin-Derived Peptides Are New Regulators of Insulin Resistance Development in Mice

Although it has long been established that the extracellular matrix acts as a mechanical support, its degradation products, which mainly accumulate during aging, have also been demonstrated to play an important role in cell physiology and the development of cardiovascular and metabolic diseases. In the current study, we show that elastin-derived peptides (EDPs) may be involved in the development of insulin resistance (IRES) in mice. In chow-fed mice, acute or chronic intravenous injections of EDPs induced hyperglycemic effects associated with glucose uptake reduction and IRES in skeletal muscle, liver, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, we propose that this IRES is due to interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex triggered by EDPs. This interplay was correlated with decreased sialic acid levels on the β-chain of the IR and reduction of IR signaling. In conclusion, this is the first study to demonstrate that EDPs, which mainly accumulate with aging, may be involved in the insidious development of IRES.

Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway.

AIMS Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. METHODS AND RESULTS Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR(-/-) mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. CONCLUSION Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in vivo.

LRP-1–CD44, a New Cell Surface Complex Regulating Tumor Cell Adhesion

ABSTRACT The low-density lipoprotein receptor-related protein 1 (LRP-1) is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. In the field of cancer, LRP-1-mediated endocytosis was first associated with antitumor properties. However, recent results suggested that LRP-1 may coordinate the adhesion-deadhesion balance in malignant cells to support tumor progression. Here, we observed that LRP-1 silencing or RAP (receptor-associated protein) treatment led to accumulation of CD44 at the tumor cell surface. Moreover, we evidenced a tight interaction between CD44 and LRP-1, not exclusively localized in lipid rafts. Overexpression of LRP-1-derived minireceptors indicated that the fourth ligand-binding cluster of LRP-1 is required to bind CD44. Labeling of CD44 with EEA1 and LAMP-1 showed that internalized CD44 is routed through early endosomes toward lysosomes in a LRP-1-dependent pathway. LRP-1-mediated internalization of CD44 was highly reduced under hyperosmotic conditions but poorly affected by membrane cholesterol depletion, revealing that it proceeds mostly via clathrin-coated pits. Finally, we demonstrated that CD44 silencing abolishes RAP-induced tumor cell attachment, revealing that cell surface accumulation of CD44 under LRP-1 blockade is mainly responsible for the stimulation of tumor cell adhesion. Altogether, our data shed light on the LRP-1-mediated internalization of CD44 that appeared critical to define the adhesive properties of tumor cells.

TIMP-1 binding to proMMP-9/CD44 complex localized at the cell surface promotes erythroid cell survival

Besides its ability to inhibit MMP activity, TIMP-1 exhibits other biological functions. We earlier reported that TIMP-1 induced UT-7 erythroid cell survival through activation of the JAK2/PI 3-kinase/Akt pathway and we now aim to determine whether the TIMP-1 anti-apoptotic effect requires MMP involvement. We first show that proMMP-9 was expressed in UT-7 cells and associated with the cell plasma membrane. Such proMMP-9 localization was crucial for TIMP-1 intracellular signalling since (i) TIMP-1 specifically bound to proMMP-9 and (ii) proMMP-9 silencing abrogated the TIMP-1 effect. We also demonstrated that TIMP-1 anti-apoptotic effect was independent on MMP inhibition since MMP-9 function blocking antibodies as well as a synthetic MMP inhibitor were unable to reproduce TIMP-1 effect. Nevertheless, these compounds prevented TIMP-1 binding to proMMP-9 and subsequently abolished TIMP-1-induced cell survival. We finally demonstrated that CD44 anchored proMMP-9 to the plasma membrane and enabled TIMP-1-mediated signal transduction. Therefore, our results indicate that the anti-apoptotic signalling of TIMP-1 depends on the formation of a ternary complex between TIMP-1, proMMP-9 and CD44 at the UT-7 erythroid cell surface.