Caspase-3-dependent cleavage of Bcl-xL in the stroma exosomes is required for their uptake by hematological malignant cells.

The intercellular crosstalk between hematological malignancies and the tumor microenvironment is mediated by cell-to-cell interactions and soluble factors. One component of the secretome that is gaining increasing attention is the extracellular vesicles and, in particular, the exosomes. Apart from the role as vectors of molecular information, exosomes have been shown to possess intrinsic biological activity. In this study, we found that caspase-3 is activated in L88 bone marrow stroma cell-derived exosomes and identified 1 of the substrates to be the antiapoptotic protein Bcl-xL. The cleaved Bcl-xL is found in a panel of normal and cancer cell-derived exosomes and is localized on the outer leaflet of the exosomal membrane. Incubation of the exosomes with a caspase-3 inhibitor or the pan-caspase inhibitor prevents the cleavage of Bcl-xL. Importantly, MCF-7 cell-derived exosomes that are caspase-3-deficient are enriched in full-length Bcl-xL, whereas ectopic expression of caspase-3 restores the cleavage of Bcl-xL. Chemical inhibition of Bcl-xL with ABT737 or molecular inhibition by using the D61A and D76A Bcl-xL mutant leads to a significant decrease in the uptake of exosomes by hematopoietic malignant cells. These data indicate that the cleaved Bcl-xL is required for the uptake of exosomes by myeloma and lymphoma cells, leading to their increased proliferation. In summary, we demonstrate for the first time that Bcl-xL is an exosomal caspase-3 substrate and that this processing is required for the uptake of exosomes by recipient cells.

Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells.

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor ß-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

Targeting a G-protein-coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death.

Nanotherapy using targeted magnetic nanoparticles grafted with peptidic ligands of receptors overexpressed in cancers is a promising therapeutic strategy. However, nanoconjugation of peptides can dramatically affect their properties with respect to receptor recognition, mechanism of internalization, intracellular trafficking, and fate. Furthermore, investigations are needed to better understand the mechanism whereby application of an alternating magnetic field to cells containing targeted nanoparticles induces cell death. Here, we designed a nanoplatform (termed MG-IONP-DY647) composed of an iron oxide nanocrystal decorated with a ligand of a G-protein coupled receptor, the cholecystokinin-2 receptor (CCK2R) that is overexpressed in several malignant cancers. MG-IONP-DY647 did not stimulate inflammasome of Raw 264.7 macrophages. They recognized cells expressing CCK2R with a high specificity, subsequently internalized via a mechanism involving recruitment of ß-arrestins, clathrin-coated pits, and dynamin and were directed to lysosomes. Binding and internalization of MG-IONP-DY647 were dependent on the density of the ligand at the nanoparticle surface and were slowed down relative to free ligand. Trafficking of CCK2R internalized with the nanoparticles was slightly modified relative to CCK2R internalized in response to free ligand. Application of an alternating magnetic field to cells containing MG-IONP-DY647 induced apoptosis and cell death through a lysosomal death pathway, demonstrating that cell death is triggered even though nanoparticles of low thermal power are internalized in minute amounts by the cells. Together with pioneer findings using iron oxide nanoparticles targeting tumoral cells expressing epidermal growth factor receptor, these data represent a solid basis for future studies aiming at establishing the proof-of-concept of nanotherapy of cancers using ligand-grafted magnetic nanoparticles specifically internalized via cell surface receptors.

Multitargeted therapies for multiple myeloma.

Multiple myeloma (MM) comprises 1% of all malignancies and 10% of all hematological malignancies. MM is a malignancy of plasma cells in the bone marrow where complex and dynamic interactions with the bone marrow microenvironment lead to tumor progression, skeletal destruction and angiogenesis. Despite the discovery of several novel treatments against MM, including the proteasome inhibitor bortezomib, it is considered to be an incurable disease with an average 4-5 years overall survival.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors.

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

Glucose-dependent insulinotropic polypeptide receptors in most gastroenteropancreatic and bronchial neuroendocrine tumors.

CONTEXT: Gastrointestinal peptide hormone receptors overexpressed in neuroendocrine tumors (NET), such as somatostatin or glucagon-like peptide-1 (GLP-1) receptors, are used for in vivo tumor targeting. Unfortunately, not all NET express these receptors sufficiently. OBJECTIVE: Our aim was to evaluate in vitro the expression of another incretin receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, in human tumors and compare it with that in adjacent nonneoplastic tissues and also with somatostatin and GLP-1 receptor expression. METHODS: GIP receptor protein expression was qualitatively and quantitatively investigated in 260 human tumors and in nonneoplastic human tissues with receptor autoradiography using [(125)I]GIP(1-30). Pharmacological competition experiments and mRNA analysis were performed to provide proof of specificity. Somatostatin receptor and GLP-1 receptor autoradiography were performed in adjacent sections. RESULTS: GIP receptors are expressed in the majority of pancreatic, ileal, and bronchial NET. Importantly, most of the somatostatin receptor-negative NET and GLP-1 receptor-negative malignant insulinomas are GIP receptor positive. Conversely, the epithelial and stromal gastrointestinal tumors, including gastric, colonic, and hepatocellular carcinomas, cholangiocarcinomas, and gastrointestinal stromal tumors as well as lung adenocarcinomas are usually GIP receptor negative, except for 26% of pancreatic adenocarcinomas. Pancreatic islets, but not acini, are GIP receptor positive. The rank order of potencies for receptor binding and mRNA analysis by PCR reveal specific GIP receptors. CONCLUSIONS: The numerous GIP receptors in gastroenteropancreatic and bronchial NET represent novel universal molecular targets for clinical applications, in particular for in vivo scintigraphy and targeted radiotherapy. These results may also be the basis for multiple targeting, with concomitant use of GIP, somatostatin, and GLP-1 analogs as radiotracers.