The endocytic receptor protein LRP-1 modulate P-glycoprotein mediated drug resistance in MCF-7 cells.

Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. A typical form of MDR is due to the overexpression of membrane transport proteins., such as Glycoprotein-P (P-gp), resulting in an increased drug efflux preventing drug cytotoxicity. P-gp is mainly localized on the plasma membrane; however, it can also be endocytosed resulting in the trafficking of P-gp in endoplasmic reticulum, Golgi, endosomes, and lysosomes. The lysosomal P-gp has been found to be capable of transporting and sequestering P-gp substrates (e.g., Doxorubicin (Dox)) into lysosomes to protect cells against cytotoxic drugs. Many translational studies have shown that low-density lipoprotein receptor-related protein-1 (LRP-1) is involved in endocytosis and regulation of signalling pathways. LRP-1 mediates the endocytosis of a diverse set of extracellular ligands that play important roles in tumor progression. Here, we investigated the involvement of LRP-1 in P-gp expression and subcellular redistribution from the cell surface to the lysosomal membrane by endocytosis and its potential implication in P-gp-mediated multidrug resistance in MCF-7 cells. Our results showed that MCF-7 resistant cells (MCF-7R) overexpressed the P-gp, LRP-1 and LAMP-1 and were 11.66-fold resistant to Dox. Our study also revealed that in MCF-7R cells, lysosomes were predominantly high density compared to sensitized cells and P-gp was localized in the plasma membrane and lysosomes. LRP-1 blockade reduced lysosomes density and level of LAMP-1 and P-gp. It also affected the subcellular distribution of P-gp. Under these conditions, we restored Dox nuclear uptake and ERK 1/2 activation thus leading to MCF-7R cell sensitization to Dox. Our data suggest that LRP-1 is able to modulate the P-gp expression and subcellular redistribution by endocytosis and to potentiate the P-gp-acquired Dox resistance.

LRP-1-dependent control of calpain expression and activity: A new mechanism regulating thyroid carcinoma cell adhesion.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP1 also regulates cell surface expression of matrix receptors by modulating both extracellular and intracellular signals, though current knowledge of the underlying mechanisms remains partial in the frame of cancer cells interaction with matricellular substrates. In this study we identified that LRP1 downregulates calpain activity and calpain 2 transcriptional expression in an invasive thyroid carcinoma cell model. LRP1-dependent alleviation of calpain activity limits cell-matrix attachment strength and contributes to FTC133 cells invasive abilities in a modified Boyden chamber assays. In addition, using enzymatic assays and co-immunoprecipitation experiments, we demonstrated that LRP1 exerts post-translational inhibition of calpain activity through PKA-dependent phosphorylation of calpain-2. This LRP-1 dual mode of control of calpain activity fine-tunes carcinoma cell spreading. We showed that LRP1-mediated calpain inhibition participates in talin-positive focal adhesions dissolution and limits ß1-integrin expression at carcinoma cell surface. In conclusion, we identified an additional and innovative intracellular mechanism which demonstrates LRP-1 pro-motile action in thyroid cancer cells. LRP-1 ability to specifically control calpain-2 expression and activity highlights a novel facet of its de-adhesion receptor status.

Discoidin Domain Receptor 1 Expression in Colon Cancer: Roles and Prognosis Impact.

Extracellular matrix components such as collagens are deposited within the tumor microenvironment at primary and metastatic sites and are recognized to be critical during tumor progression and metastasis development. This study aimed to evaluate the clinical and prognostic impact of Discoidin Domain Receptor 1 (DDR1) expression in colon cancers and its association with a particular molecular and/or morphological profile and to evaluate its potential role as a prognosis biomarker. Immunohistochemical expression of DDR1 was evaluated on 292 colonic adenocarcinomas. DDR1 was highly expressed in 240 (82.2%) adenocarcinomas. High DDR1 immunostaining score was significantly associated, on univariate analysis, with male sex, left tumor location, BRAF wild type status, KRAS mutated status, and Annexin A10 negativity. High DDR1 immunohistochemical expression was associated with shorter event free survival only. Laser capture microdissection analyses revealed that DDR1 mRNA expression was mainly attributable to adenocarcinoma compared to stromal cells. The impact of DDR1 expression on cell invasion was then evaluated by modified Boyden chamber assay using cell types with distinct mutational profiles. The invasion capacity of colon adenocarcinoma is supported by DDR1 expression. Thus, our results showed that DDR1 was highly expressed in most colon adenocarcinomas and appears as an indicator of worse event free survival.

LRP-1 Matricellular Receptor Involvement in Triple Negative Breast Cancer Tumor Angiogenesis.

BACKGROUND: LRP-1 is a multifunctional scavenger receptor belonging to the LDLR family. Due to its capacity to control pericellular levels of various growth factors and proteases, LRP-1 plays a crucial role in membrane proteome dynamics, which appears decisive for tumor progression. METHODS: LRP-1 involvement in a TNBC model was assessed using an RNA interference strategy in MDA-MB-231 cells. In vivo, tumorigenic and angiogenic effects of LRP-1-repressed cells were evaluated using an orthotopic xenograft model and two angiogenic assays (Matrigel® plugs, CAM). DCE-MRI, FMT, and IHC were used to complete a tumor longitudinal follow-up and obtain morphological and functional vascular information. In vitro, HUVECs' angiogenic potential was evaluated using a tumor secretome, subjected to a proteomic analysis to highlight LRP-1-dependant signaling pathways. RESULTS: LRP-1 repression in MDA-MB-231 tumors led to a 60% growth delay because of, inter alia, morphological and functional vascular differences, confirmed by angiogenic models. In vitro, the LRP-1-repressed cells secretome restrained HUVECs' angiogenic capabilities. A proteomics analysis revealed that LRP-1 supports tumor growth and angiogenesis by regulating TGF-ß signaling and plasminogen/plasmin system. CONCLUSIONS: LRP-1, by its wide spectrum of interactions, emerges as an important matricellular player in the control of cancer-signaling events such as angiogenesis, by supporting tumor vascular morphology and functionality.

Lactosylceramide induced by elastin-derived peptides decreases adipocyte differentiation.

Elastin, the major protein of the extracellular matrix, is specially found in cardiovascular tissues and contributing to 30-50% of the dry weight of blood vessels. Elastin regulates cell signalling pathways involved in morphogenesis, injury response and inflammation. The function of elastin is frequently compromised in damaged or aged elastic tissues. Indeed, elastin degradation, observed during ageing, and the resulting production of elastin-derived peptides (EDPs), have crucial impacts on cardiovascular disease (atherosclerosis, thrombosis) or on metabolism disease progressions (type 2 diabetes or non-alcoholic steatohepatitis). In the present study, we analysed the EDP effects on 3T3 preadipocyte cell differentiation. In a first part, we treated 3T3-L1 cells with EDP and visualized the lipid droplet accumulation by the oil red O staining and measured the expression of various transcription factors and adipocyte-specific mRNAs by real-time RT-PCR. We demonstrated that the elastin receptor complex, ERC, is activated by EDPs and decreased adipocyte differentiation by a modulation of crucial adipogenesis transcriptional factor particularly PPARγ. In a second part, we identified the signalling pathway implicated in EDP-reduced cell differentiation. The flow cytometry and immunocytochemistry approaches showed that ERC activated by EDP produced a second messenger, lactosylceramide (Lac-Cer). Moreover, this Lac-Cer production favoured the phosphorylation of ERK1-2 (p-ERK1-2), to decrease adipocyte differentiation by a modulation of adipogenesis transcriptional factor PPARγ. To conclude, the EDP/Lac-Cer/p-ERK1-2 signalling pathway may be studied further as a critical target for treating complications associated with adipocyte dedifferentiation such as obesity and diabetes insulin resistance.

LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis.

Low density lipoprotein receptor related protein-1 (LRP-1) is a large ubiquitous endocytic receptor mediating the clearance of various molecules from the extracellular matrix. Several studies have shown that LRP-1 plays crucial roles during tumorigenesis functioning as a main signal pathway regulator, especially by interacting with other cell-surface receptors. Discoïdin Domain Receptors (DDRs), type I collagen receptors with tyrosine kinase activity, have previously been associated with tumor invasion and aggressiveness in diverse tumor environments. Here, we addressed whether it could exist functional interplays between LRP-1 and DDR1 to control colon carcinoma cell behavior in three-dimensional (3D) collagen matrices. We found that LRP-1 established tight molecular connections with DDR1 at the plasma membrane in colon cancer cells. In this tumor context, we provide evidence that LRP-1 regulates by endocytosis the cell surface levels of DDR1 expression. The LRP-1 mediated endocytosis of DDR1 increased cell proliferation by promoting cell cycle progression into S phase and decreasing apoptosis. In this study, we identified a new molecular way that controls the cell-surface expression of DDR1 and consequently the colon carcinoma cell proliferation and apoptosis and highlighted an additional mechanism by which LRP-1 carries out its sensor activity of the tumor microenvironment.

Identification of LRP-1 as an endocytosis and recycling receptor for ß1-integrin in thyroid cancer cells.

LRP-1 is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP-1 was reported to control focal adhesion turnover to optimize the adhesion-deadhesion balance to support invasion. To better understand how LRP-1 coordinates cell-extracellular matrix interface, we explored its ability to regulate cell surface integrins in thyroid carcinomas. Using an antibody approach, we demonstrated that ß1-integrin levels were increased at the plasma membrane under LRP1 silencing or upon RAP treatment, used as LRP-1 antagonist. Our data revealed that LRP-1 binds with both inactive and active ß1-integrin conformations and identified the extracellular ligand-binding domains II or IV of LRP-1 as sufficient to bind ß1-integrin. Using a recombinant ß1-integrin, we demonstrated that LRP-1 acts as a regulator of ß1-integrin intracellular traffic. Moreover, RAP or LRP-1 blocking antibodies decreased up to 36% the number of ß1-integrin-containing endosomes. LRP-1 blockade did not significantly affect the levels of ß1-integrin-containing lysosomes while decreasing localization of ß1-integrin within Rab-11 positive vesicles. Overall, we identified an original molecular process in which LRP-1 acts as a main regulator of ß1-integrin internalization and recycling in thyroid cancer cells.

Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.

LRP-1--CD44, a new cell surface complex regulating tumor cell adhesion.

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.