Christophe Schneider

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.

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.

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.

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.

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.