Adverse Effects of Oseltamivir Phosphate Therapy on the Liver of LDLR-/- Mice Without Any Benefit on Atherosclerosis and Thrombosis.

ABSTRACT: Desialylation, governed by sialidases or neuraminidases, is strongly implicated in a wide range of human disorders, and accumulative data show that inhibition of neuraminidases, such as neuraminidases 1 sialidase, may be useful for managing atherosclerosis. Several studies have reported promising effects of oseltamivir phosphate, a widely used anti-influenza sialidase inhibitor, on human cancer cells, inflammation, and insulin resistance. In this study, we evaluated the effects of oseltamivir phosphate on atherosclerosis and thrombosis and potential liver toxicity in LDLR-/- mice fed with high-fat diet. Our results showed that oseltamivir phosphate significantly decreased plasma levels of LDL cholesterol and elastin fragmentation in aorta. However, no effect was observed on both atherosclerotic plaque size in aortic roots and chemically induced thrombosis in carotid arteries. Importantly, oseltamivir phosphate administration had adverse effects on the liver of mice and significantly increased messenger RNA expression levels of F4/80, interleukin-1ß, transforming growth factor-ß1, matrix metalloproteinase-12, and collagen. Taken together, our findings suggest that oseltamivir phosphate has limited benefits on atherosclerosis and carotid thrombosis and may lead to adverse side effects on the liver with increased inflammation and fibrosis.

Introgression reshapes recombination distribution in grapevine interspecific hybrids.

KEY MESSAGE: In grapevine interspecific hybrids, meiotic recombination is suppressed in homeologous regions and enhanced in homologous regions of recombined chromosomes, whereas crossover rate remains unchanged when chromosome pairs are entirely homeologous. Vitis rotundifolia, an American species related to the cultivated European grapevine Vitis vinifera, has a high level of resistance to several grapevine major diseases and is consequently a valuable resource for grape breeding. However, crosses between both species most often lead to very few poorly fertile hybrids. In this context, identifying genetic and genomic features that make cross-breeding between both species difficult is essential. To this end, three mapping populations were generated by pseudo-backcrosses using V. rotundifolia as the donor parent and several V. vinifera cultivars as the recurrent parents. Genotyping-by-sequencing was used to establish high-density genetic linkage maps and to determine the genetic composition of the chromosomes of each individual. A good collinearity of the SNP positions was observed between parental maps, confirming the synteny between both species, except on lower arm of chromosome 7. Interestingly, recombination rate in V. rotundifolia × V. vinifera interspecific hybrids depends on the length of the introgressed region. It is similar to grapevine for chromosome pairs entirely homeologous. Conversely, for chromosome pairs partly homeologous, recombination is suppressed in the homeologous regions, whereas it is enhanced in the homologous ones. This balance leads to the conservation of the total genetic length of each chromosome between V. vinifera and hybrid maps, whatever the backcross level and the proportion of homeologous region. Altogether, these results provide new insight to optimize the use of V. rotundifolia in grape breeding and, more generally, to improve the introgression of gene of interest from wild species related to crops.

Dynamized ultra-low dilution of Ruta graveolens disrupts plasma membrane organization and decreases migration of melanoma cancer cell.

Cutaneous melanoma is a cancer with a very poor prognosis mainly because of metastatic dissemination and therefore a deregulation of cell migration. Current therapies can benefit from complementary medicines as supportive care in oncology. In our study, we show that a dynamized ultra-low dilution of Ruta Graveolens leads to an in vitro inhibition of migration on fibronectin of B16F10 melanoma cells, as well as a decrease in metastatic dissemination in vivo. These effects appear to be due to a disruption of plasma membrane organization, with a change in cell and membrane stiffness, associated with a disorganization of the actin cytoskeleton and a modification of the lipid composition of the plasma membrane. Together, these results demonstrate, in in vitro and in vivo models of cutaneous melanoma, an anti-cancer and anti-metastatic activity of ultra-low dynamized dilution of Ruta graveolens and reinforce its interest as complementary medicine in oncology.

Chrysin-Induced Regression of Angiogenesis via an Induction of DNA Damage Response and Oxidative Stress in In Vitro and In Vivo Models of Melanoma.

Despite the progress made in treatments, melanoma is one of the cancers for which its incidence and mortality have increased during recent decades. In the research of new therapeutic strategies, natural polyphenols such as chrysin could be good candidates owing to their capacities to modulate the different fundamental aspects of tumorigenesis and resistance mechanisms, such as oxidative stress and neoangiogenesis. In the present study, we sought to determine whether chrysin could exert antitumoral effects via the modulation of angiogenesis by acting on oxidative stress and associated DNA damage. For the first time, we show a link between chrysin-induced antiproliferative effects, the activation of the DNA damage pathway, and its ability to limit angiogenesis. More specifically, herein, we show that chrysin induces single- and double-stranded DNA breaks via the activation of the DNA damage response pathway: ATM (ataxia-telangiectasia-mutated)/Chk2 (checkpoint kinase 2) and ATR (ataxia telangiectasia and Rad3-related)/Chk1 (checkpoint kinase 1) pathways. Strong activation of this DNA damage response was found to be partly involved in the ability of chrysin to limit angiogenesis and may partly involve a direct interaction between the polyphenol and DNA G-quadruplex structures responsible for the replication fork collapse. Moreover, these events were associated with a marked reduction in melanoma cells' capacity to secrete proangiogenic factor VEGF-A. The disruption of these key protein actors in tumor growth by chrysin was also confirmed in a syngeneic model of B16 melanoma. This last point is of importance to further consider the use of chrysin as a new therapeutic strategy in melanoma treatment.

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.

Anti-Tumoral and Anti-Angiogenic Effects of Low-Diluted Phenacetinum on Melanoma.

Melanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. If primary cutaneous melanoma is mostly treated with a curative wide local excision, malignant melanoma has a poor prognosis and needs other therapeutic approaches. Angiogenesis is a normal physiological process essential in growth and development, but it also plays a crucial role in crossing from benign to advanced state in cancer. In melanoma progression, angiogenesis is widely involved during the vertical growth phase. Currently, no anti-angiogenic agents are efficient on their own, and combination of treatments will probably be the key to success. In the past, phenacetin was used as an analgesic to relieve pain, causing side effects at large dose and tumor-inducing in humans and animals. By contrast, Phenacetinum low-dilution is often used in skin febrile exanthema, patches profusely scattered on limbs, headache, or flushed face without side effects. Herein are described the in vitro, in vivo, and ex vivo anti-angiogenic and anti-tumoral potentials of Phenacetinum low-dilution in a B16F1 tumor model and endothelial cells. We demonstrate that low-diluted Phenacetinum inhibits in vivo tumor growth and tumor vascularization and thus increases the survival time of B16F1 melanoma induced-C57BL/6 mice. Moreover, Phenacetinum modulates the lung metastasis in a B16F10 induced model. Ex vivo and in vitro, we evidence that low-diluted Phenacetinum inhibits the migration and the recruitment of endothelial cells and leads to an imbalance in the pro-tumoral macrophages and to a structural malformation of the vascular network. All together these results demonstrate highly hopeful anti-tumoral, anti-metastatic, and anti-angiogenic effects of Phenacetinum low-dilution on melanoma. Continued studies are needed to preclinically validate Phenacetinum low-dilution as a complementary or therapeutic strategy for melanoma treatment.

Role of Endocytosis Proteins in Gefitinib-Mediated EGFR Internalisation in Glioma Cells.

EGFR (epidermal growth factor receptor), a member of the ErbB tyrosine kinase receptor family, is a clinical therapeutic target in numerous solid tumours. EGFR overexpression in glioblastoma (GBM) drives cell invasion and tumour progression. However, clinical trials were disappointing, and a molecular basis to explain these poor results is still missing. EGFR endocytosis and membrane trafficking, which tightly regulate EGFR oncosignaling, are often dysregulated in glioma. In a previous work, we showed that EGFR tyrosine kinase inhibitors, such as gefitinib, lead to enhanced EGFR endocytosis into fused early endosomes. Here, using pharmacological inhibitors, siRNA-mediated silencing, or expression of mutant proteins, we showed that dynamin 2 (DNM2), the small GTPase Rab5 and the endocytosis receptor LDL receptor-related protein 1 (LRP-1), contribute significantly to gefitinib-mediated EGFR endocytosis in glioma cells. Importantly, we showed that inhibition of DNM2 or LRP-1 also decreased glioma cell responsiveness to gefitinib during cell evasion from tumour spheroids. By highlighting the contribution of endocytosis proteins in the activity of gefitinib on glioma cells, this study suggests that endocytosis and membrane trafficking might be an attractive therapeutic target to improve GBM treatment.

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.

Contribution of the Low-Density Lipoprotein Receptor Family to Breast Cancer Progression.

The low-density lipoprotein receptor (LDLR) family comprises 14 single-transmembrane receptors sharing structural homology and common repeats. These receptors specifically recognize and internalize various extracellular ligands either alone or complexed with membrane-spanning co-receptors that are then sorted for lysosomal degradation or cell-surface recovery. As multifunctional endocytic receptors, some LDLR members from the core family were first considered as potential tumor suppressors due to their clearance activity against extracellular matrix-degrading enzymes. LDLRs are also involved in pleiotropic functions including growth factor signaling, matricellular proteins, and cell matrix adhesion turnover and chemoattraction, thereby affecting both tumor cells and their surrounding microenvironment. Therefore, their roles could appear controversial and dependent on the malignancy state. In this review, recent advances highlighting the contribution of LDLR members to breast cancer progression are discussed with focus on (1) specific expression patterns of these receptors in primary cancers or distant metastasis and (2) emerging mechanisms and signaling pathways. In addition, potential diagnosis and therapeutic options are proposed.

Low-diluted Phenacetinum disrupted the melanoma cancer cell migration.

Dynamic and reciprocal interactions generated by the communication between tumor cells and their matrix microenvironment, play a major role in the progression of a tumor. Indeed, the adhesion of specific sites to matrix components, associated with the repeated and coordinated formation of membrane protrusions, allow tumor cells to move along a determined pathway. Our study analyzed the mechanism of action of low-diluted Phenacetinum on murine cutaneous melanoma process in a fibronectin matrix environment. We demonstrated a reduction of dispersed cell migration, early and for as long as 24 h, by altering the formation of cell protrusions. Moreover, low-diluted Phenacetinum decreased cell stiffness highly on peripheral areas, due to a disruption of actin filaments located just under the plasma membrane. Finally, it modified the structure of the plasma membrane by accumulating large ordered lipid domains and disrupted B16 cell migration by a likely shift in the balance between ordered and disordered lipid phases. Whereas the correlation between the excess of lipid raft and cytoskeleton disrupting is not as yet established, it is clear that low-diluted Phenacetinum acts on the actin cytoskeleton organization, as confirmed by a decrease of cell stiffness affecting ultimately the establishment of an effective migration process.

LRP1 expression in colon cancer predicts clinical outcome.

LRP1 (low-density lipoprotein receptor-related protein 1), a multifunctional endocytic receptor, has recently been identified as a hub within a biomarker network for multi-cancer clinical outcome prediction. As its role in colon cancer has not yet been characterized, we here investigate the relationship between LRP1 and outcome. MATERIALS AND METHODS: LRP1 mRNA expression was determined in colon adenocarcinoma and paired colon mucosa samples, as well as in stromal and tumor cells obtained after laser capture microdissection. Clinical potential was further investigated by immunohistochemistry in a population-based colon cancer series (n = 307). LRP1 methylation, mutation and miR-205 expression were evaluated and compared with LRP1 expression levels. RESULTS: LRP1 mRNA levels were significantly lower in colon adenocarcinoma cells compared with colon mucosa and stromal cells obtained after laser capture microdissection. Low LRP1 immunohistochemical expression in adenocarcinomas was associated with higher age, right-sided tumor, loss of CDX2 expression, Annexin A10 expression, CIMP-H, MSI-H and BRAFV600E mutation. Low LRP1 expression correlated with poor clinical outcome, especially in stage IV patients. While LRP1 expression was downregulated by LRP1 mutation, LRP1 promoter was never methylated. CONCLUSIONS: Loss of LRP1 expression is associated with worse colon cancer outcomes. Mechanistically, LRP1 mutation modulates LRP1 expression.

Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

Biobanking of Fresh-Frozen Human Adenocarcinomatous and Normal Colon Tissues: Which Parameters Influence RNA Quality?

Medical research projects become increasingly dependent on biobanked tissue of high quality because the reliability of gene expression is affected by the quality of extracted RNA. Hence, the present study aimed to determine if clinical, surgical, histological, and molecular parameters influence RNA quality of normal and tumoral frozen colonic tissues. RNA Quality Index (RQI) was evaluated on 241 adenocarcinomas and 115 matched normal frozen colon tissues collected between October 2006 and December 2012. RQI results were compared to patients' age and sex, tumor site, kind of surgery, anastomosis failure, adenocarcinoma type and grade, tumor cell percentage, necrosis extent, HIF-1α and cleaved caspase-3 immunohistochemistry, and BRAF, KRAS and microsatellites status. The RQI was significantly higher in colon cancer tissue than in matched normal tissue. RQI from left-sided colonic cancers was significantly higher than RQI from right-sided cancers. The RNA quality was not affected by ischemia and storage duration. According to histological control, 7.9% of the samples were unsatisfactory because of inadequate sampling. Biobanked tumoral tissues with RQI ≥5 had lower malignant cells to stromal cells ratio than samples with RQI <5 (p <0.05). Cellularity, necrosis extent and mucinous component did not influence RQI results. Cleaved caspase-3 and HIF-1α immunolabelling were not correlated to RQI. BRAF, KRAS and microsatellites molecular status did not influence RNA quality. Multivariate analysis revealed that the tumor location, the surgical approach (laparoscopy versus open colectomy) and the occurrence of anastomotic leakage were the only parameters influencing significantly RQI results of tumor samples. We failed to identify parameter influencing RQI of normal colon samples. These data suggest that RNA quality of colonic adenocarcinoma biospecimens is determined by clinical and surgical parameters. More attention should be paid during the biobanking procedure of right-sided colon cancer or laparoscopic colectomy specimen. Histological quality control remains essential to control sampling accuracy.

Original insights on thrombospondin-1-related antireceptor strategies in cancer.

Thrombospondin-1 (TSP-1) is a large matricellular glycoprotein known to be overexpressed within tumor stroma in several cancer types. While mainly considered as an endogenous angiogenesis inhibitor, TSP-1 exhibits multifaceted functionalities in a tumor context depending both on TSP-1 concentration as well as differential receptor expression by cancer cells and on tumor-associated stromal cells. Besides, the complex modular structure of TSP-1 along with the wide variety of its soluble ligands and membrane receptors considerably increases the complexity of therapeutically targeting interactions involving TSP-1 ligation of cell-surface receptors. Despite the pleiotropic nature of TSP-1, many different antireceptor strategies have been developed giving promising results in preclinical models. However, transition to clinical trials often led to nuanced outcomes mainly due to frequent severe adverse effects. In this review, we will first expose the intricate and even sometimes opposite effects of TSP-1-related signaling on tumor progression by paying particular attention to modulation of angiogenesis and tumor immunity. Then, we will provide an overview of current developments and prospects by focusing particularly on the cell-surface molecules CD47 and CD36 that function as TSP-1 receptors; including antibody-based approaches, therapeutic gene modulation and the use of peptidomimetics. Finally, we will discuss original approaches specifically targeting TSP-1 domains, as well as innovative combination strategies with a view to producing an overall anticancer response.

Identification of TAX2 peptide as a new unpredicted anti-cancer agent.

The multi-modular glycoprotein thrombospondin-1 (TSP-1) is considered as a key actor within the tumor microenvironment. Besides, TSP-1 binding to CD47 is widely reported to regulate cardiovascular function as it promotes vasoconstriction and angiogenesis limitation. Therefore, many studies focused on targeting TSP-1:CD47 interaction, aiming for up-regulation of physiological angiogenesis to enhance post-ischemia recovery or to facilitate engraftment. Thus, we sought to identify an innovative selective antagonist for TSP-1:CD47 interaction. Protein-protein docking and molecular dynamics simulations were conducted to design a novel CD47-derived peptide, called TAX2. TAX2 binds TSP-1 to prevent TSP-1:CD47 interaction, as revealed by ELISA and co-immunoprecipitation experiments. Unexpectedly, TAX2 inhibits in vitro and ex vivo angiogenesis features in a TSP-1-dependent manner. Consistently, our data highlighted that TAX2 promotes TSP-1 binding to CD36-containing complexes, leading to disruption of VEGFR2 activation and downstream NO signaling. Such unpredicted results prompted us to investigate TAX2 potential in tumor pathology. A multimodal imaging approach was conducted combining histopathological staining, MVD, MRI analysis and µCT monitoring for tumor angiography longitudinal follow-up and 3D quantification. TAX2 in vivo administrations highly disturb syngeneic melanoma tumor vascularization inducing extensive tumor necrosis and strongly inhibit growth rate and vascularization of human pancreatic carcinoma xenografts in nude mice.

Rapid and multiregional adaptation to host partial resistance in a plant pathogenic oomycete: evidence from European populations of Plasmopara viticola, the causal agent of grapevine downy mildew.

Crop pathogens evolve rapidly to adapt to their hosts. The use of crops with quantitative disease resistance is expected to alter selection of pathogen life-history traits. This may result in differential adaptation of the pathogen to host cultivars and, sometimes, to the erosion of quantitative resistance. Here, we assessed the level of host adaptation in an oomycete plant pathogenic species. We analysed the phenotypic and genetic variability of 17 Plasmopara viticola isolates collected on Vitis vinifera and 35 isolates from partially resistant varieties (Regent and genotypes carrying the Rpv1 gene). Cross-inoculation experiments assessed two components of aggressiveness and a life-history trait of the pathogen: disease severity, sporangial production and sporangia size. The results contribute evidence to the emergence of P. viticola aggressive isolates presenting a high level of sporulation on the partially resistant Regent. By contrast, no adaptation to the Rpv1 gene was found in this study. The erosion of Regent resistance may have occurred in less than 5years and at least three times independently in three distant wine-producing areas. Populations from resistant varieties showed a significant increase in sporangia production capacity, indicating an absence of fitness costs for this adaptation. The increase in the number of sporangia was correlated with a reduction in sporangia size, a result which illustrates how partial plant disease resistance can impact selection of the pathogen's life-history traits. This case study on grapevine downy mildew shows how new plant pathogen populations emerge in agro-ecosystems by adapting to partial host resistance. This adaptive pattern highlights the need for wise management of plant partial disease resistance to ensure its sustainability over time.

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.

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.