Stéphane Dedieu

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.

Myoblast migration is prevented by a calpain-dependent accumulation of MARCKS

Abstract Calpains, also called calcium activated neutral cysteine proteases are presently known to play pivotal roles in physiological and biological phenomena such as signal transduction, cell spreading and motility, apoptosis, regulation of cell cycle and regulation of muscle cell differentiation. Concerning this last point, calpains have been shown to play a crucial role during the earlier myogenesis. In this study we have analyzed the involvement of calpains during an important step of myogenesis: myoblast migration. Our findings show that myoblast migration was drastically reduced when the expression of μ‐ and m‐calpain was decreased. We have also observed that MARCKS (myristoylated alanine rich C kinase substrate), a protein localized at focal adhesion sites, was significantly accumulated when the expression levels of calpains were decreased. Also, using phorbol myristate acetate, (an activator of PKC) and plasmids carrying the full‐length cDNA of MARCKS or a cDNA fragment lacking the phosphorylation site domain, we demonstrated that normal myoblast migration is dependent on MARCKS phosphorylation and localization.

Metalloproteinase-Dependent Shedding of Low-Density Lipoprotein Receptor-Related Protein-1 Ectodomain Decreases Endocytic Clearance of Endometrial Matrix Metalloproteinase-2 and -9 at Menstruation

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

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.

LRP-1: A Checkpoint for the Extracellular Matrix Proteolysis

Low-density lipoprotein receptor-related protein-(LRP-1) is a large endocytic receptor that binds more than 35 ligands and exhibits signaling properties. Proteinases capable of degrading extracellular matrix (ECM), called matrix proteinases in this paper, are mainly serine proteinases: the activators of plasminogen into plasmin, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, and the members of the matrix metalloproteinase (MMP) family. LRP-1 is responsible for clearing matrix proteinases, complexed or not with inhibitors. This paper attempts to summarize some aspects on the cellular and molecular bases of endocytic and signaling functions of LRP-1 that modulate extra- and pericellular levels of matrix proteinases.

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.

LRP-1: a new modulator of cytoskeleton dynamics and adhesive complex turnover in cancer cells.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a large scavenger receptor mediating the internalization and catabolism of various biological components from the extracellular matrix. In the past decade, LRP-1 appeared as an attractive receptor for targeting the invasive behavior of cancer cells since this protein is able to reduce the accumulation of extracellular proteinases by endocytosis. However, recent data suggest that LRP-1 could support carcinoma cell invasion depending on the cellular environment. Indeed, in addition to its well-determined role in ligand binding and endocytosis, LRP-1 emerges as a central molecular regulator of cytoskeleton organization and adhesive complex turnover in malignant cells. This commentary reviews the functions played by LRP-1 in cancer-related events and discusses the potential mechanisms whereby LRP-1 is able to control the cellular phenotype of cancer cells.