ABSTRACT
Several studies reported a central role of the endothelin type A receptor (ETAR) in tumor progression leading to the formation of metastasis. Here, we investigated the in vitro and in vivo anti-tumor effects of the FDA-approved ETAR antagonist, Ambrisentan, which is currently used to treat patients with pulmonary arterial hypertension. In vitro, Ambrisentan inhibited both spontaneous and induced migration/invasion capacity of different tumor cells (COLO-357 metastatic pancreatic adenocarcinoma, OvCar3 ovarian carcinoma, MDA-MB-231 breast adenocarcinoma, and HL-60 promyelocytic leukemia). Whole transcriptome analysis using RNAseq indicated Ambrisentan's inhibitory effects on the whole transcriptome of resting and PAR2-activated COLO-357 cells, which tended to normalize to an unstimulated profile. Finally, in a pre-clinical murine model of metastatic breast cancer, treatment with Ambrisentan was effective in decreasing metastasis into the lungs and liver. Importantly, this was associated with a significant enhancement in animal survival. Taken together, our work suggests a new therapeutic application for Ambrisentan in the treatment of cancer metastasis.
Subject(s)
Breast Neoplasms/drug therapy , Cell Movement , Endothelin A Receptor Antagonists/pharmacology , Liver Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Phenylpropionates/pharmacology , Pyridazines/pharmacology , Animals , Antihypertensive Agents/pharmacology , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Female , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.
Subject(s)
Alzheimer Disease/immunology , Autoantibodies/immunology , Homeostasis/immunology , Ovarian Neoplasms/immunology , Receptors, G-Protein-Coupled/immunology , Scleroderma, Systemic/immunology , Aged , Amino Acid Sequence , Animals , Autoantibodies/blood , Autoantibodies/metabolism , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Male , Mice , Middle Aged , Protein Interaction Maps/immunology , Receptor, Endothelin A/genetics , Receptor, Endothelin A/immunology , Receptor, Endothelin A/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Sequence Homology, Amino AcidABSTRACT
Microvesicles (MVs) represent a subgroup of extracellular vesicles (EVs) emerging from various cells by blebbing of their outer membrane. Therefore, they share features such as membrane composition and antigenicity with their parental cells. Released by many immune and tumor cells, MVs act as intercellular messengers, account for horizontal gene transfer and can activate the coagulation system. With the aim to investigate their relevance for tumor cell biology, we characterized MVs released by human tumor cell lines of various origins in the absence or presence of TNF-α. After stimulation, we used the combination of low and high-speed centrifugation to enrich MVs from cell culture supernatants. We analyzed the presentation of phosphatidylserine (PS) and tissue factor (TF) activity on the cell surface and investigated their potency to induce tumor cell migration. In all tumor cell lines, TNF-α stimulation enhanced the release of MVs. While the expression of PS was universally increased, an elevated activity of procoagulant TF could be detected on MVs from lung, pancreatic, and colon carcinoma, but not from breast and ovarian cancer cell lines. Functionally, TNF-α stimulation significantly increased the potency of MVs to induce tumor cell migration. In conclusion, inflammatory conditions promote the release of MVs with increased procoagulant activity from tumor cell lines in vitro. PS-containing and TF-expressing MVs may account for systemic activation of the coagulation system as seen in cancer patients and, since they induce tumor cell migration, they may serve as biomarkers for tumor progression.
Subject(s)
Extracellular Vesicles/metabolism , Inflammation/pathology , Tumor Necrosis Factor-alpha/adverse effects , Biological Assay , Caspase 3/metabolism , Cell Count , Cell Line, Tumor , Cell Movement , Extracellular Vesicles/drug effects , Extracellular Vesicles/ultrastructure , Humans , Oligopeptides/pharmacology , Phosphatidylserines/metabolism , Receptor, PAR-2/antagonists & inhibitors , Receptor, PAR-2/metabolism , Signal Transduction/drug effects , Thromboplastin/metabolismABSTRACT
Elevated levels of extracellular vesicles (EVs) have been correlated with inflammatory diseases as well as progressive and metastatic cancer. By presenting tissue factor (TF) on their membrane surface, cellular microparticles (MPs) activate both the coagulation system and cell-signaling pathways such as the PAR/ERK pathway. We have shown before that malignant effusions are a rich source of tumor cell-derived EVs. Here, we used EVs from malignant effusions from three different patients after serial low-speed centrifugation steps as recommended by the ISTH (lsEV). Significant migration of human pancreatic carcinoma cells could be induced by lsEVs and was effectively inhibited by pre-incubation with tinzaparin, a low-molecular-weight heparin. Tinzaparin induced tissue factor pathway inhibitor (TFPI) release from tumor cells, and recombinant TFPI inhibited EV-induced tumor cell migration. EVs also induced ERK phosphorylation, whereas inhibitors of PAR2 and ERK suppressed EV-induced tumor cell migration. LsEVs have been characterized by high-resolution flow cytometry and, after elimination of smaller vesicles including exosomes, by further high-speed centrifugation (hsEV). The remaining population consisting primarily of MPs is indeed the main migration-inducing population with tenase activity. Compared to other LMWHs, tinzaparin is suggested to have high potency to induce TFPI release from epithelial cells. The migration-inhibitory effect of TFPI and the interruption of tumor cell migration by inhibitors of PAR2 and ERK suggest that lsEVs induce tumor cell migration by activating the PAR2 signaling pathway. Tinzaparin might inhibit this process at least partly by inducing the release of TFPI from tumor cells, which blocks PAR-activating TF complexes. The clinical relevance of the results is discussed.
