ABSTRACT
MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.
Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , MicroRNAs/metabolism , Animals , Blast Crisis , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line, Tumor , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , Proto-Oncogene Proteins c-pim-1/metabolism , RNA-Induced Silencing Complex/metabolismABSTRACT
Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis in multiple cancer types; however, sEVs in the aqueous humor (AH) of uveal melanoma (UM) patients have never previously been profiled. In this study, we used single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically the tetraspanin co-expression patterns of CD9, CD63, and CD81. sEVs were analyzed from paired pre- and post-treatment (brachytherapy, a form of radiation) AH samples collected from 19 UM patients. In post-brachytherapy samples, two subpopulations, CD63/81+ and CD9/63/81+ sEVs, were significantly increased. These trends existed even when stratified by tumor location and GEP class 1 and class 2 (albeit not significant for GEP class 2). In this initial report of single vesicle profiling of sEVs in the AH of UM patients, we demonstrated that sEVs can be detected in the AH. We further identified two subpopulations that were increased post-brachytherapy, which may suggest radiation-induced release of these particles, potentially from tumor cells. Further study of the cargo carried by these sEV subpopulations may uncover important biomarkers and insights into tumorigenesis for UM.
Subject(s)
Aqueous Humor , Brachytherapy , Extracellular Vesicles , Melanoma , Uveal Neoplasms , Humans , Uveal Neoplasms/radiotherapy , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathology , Extracellular Vesicles/metabolism , Melanoma/radiotherapy , Melanoma/metabolism , Melanoma/pathology , Aqueous Humor/metabolism , Aqueous Humor/radiation effects , Female , Male , Middle Aged , Aged , Biomarkers, Tumor/metabolism , Adult , Aged, 80 and overABSTRACT
New nitroxides based on aza-crown ethers were prepared and employed as selective sensors for the detection of inorganic and organic cations by EPR analysis of the corresponding host-guest complexes. The nitroxide unit behaves as a sensitive probe for a number of alkali and alkaline earth metal cations affording EPR spectra differing in the value of nitrogen hyperfine constants and in the appearance of splitted signals due to the non-zero nuclear spin of some metal cation upon complexation. Owing to the remarkable EPR spectral differences between the host and the corresponding cation complex the new macrocycles are likely to act as multitasking tools to recognize several cationic species. EPR behaviour of the larger nitroxide azacrown 1â when acting as a wheel in a radical synthetic bistable [2]rotaxane containing both secondary dialkylammonium and 1,2-bis(pyridinium) molecular stations, was also investigated. Reversible movements of the macrocycle between the two recognition sites in the rotaxane were promptly revealed by EPR, which shows significant changes either in nitrogen coupling constant values (aN ) or in the spectral shape in the two rotaxane co-conformations.
ABSTRACT
Breast cancer is the leading cause of cancer incidence worldwide and among the five leading causes of cancer mortality. Despite major improvements in early detection and new treatment approaches, the need for better outcomes and quality of life for patients is still high. Extracellular vesicles play an important role in tumor biology, as they are able to transfer information between cells of different origins and locations. Their potential value as biomarkers or for targeted tumor therapy is apparent. In this study, we analyzed the supernatants of MCF-7 breast cancer cells, which were harvested following 5 or 10 days of simulated microgravity on a Random Positioning Machine (RPM). The primary results showed a substantial increase in released vesicles following incubation under simulated microgravity at both time points. The distribution of subpopulations regarding their surface protein expression is also altered; the minimal changes between the time points hint at an early adaption. This is the first step in gaining further insight into the mechanisms of tumor progression, metastasis, the education of the tumor microenvironments, and preparation of the metastatic niche. Additionally, this may lighten up the processes of the rapid cellular adaptions in the organisms of space travelers during spaceflights.
Subject(s)
Breast Neoplasms , Extracellular Vesicles , Space Flight , Weightlessness , Humans , Female , Quality of Life , Weightlessness Simulation , Tumor MicroenvironmentABSTRACT
BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Chemotherapy, the treatment of choice in non-operable cases, achieves a dismal success rate, raising the need for new therapeutic options. In about 25% of NSCLC, the activating mutations of the KRAS oncogene define a subclass that cannot benefit from tyrosine kinase inhibitors (TKIs). The tumor suppressor miR-16 is downregulated in many human cancers, including NSCLC. The main objectives of this study were to evaluate miR-16 treatment to restore the TKI sensitivity and compare its efficacy to MEK inhibitors in KRAS-mutated NSCLC. METHODS: We performed in vitro and in vivo studies to investigate whether miR-16 could be exploited to overcome TKI resistance in KRAS-mutated NSCLC. We had three goals: first, to identify the KRAS downstream effectors targeted by mir-16, second, to study the effects of miR-16 restoration on TKI resistance in KRAS-mutated NSCLC both in vitro and in vivo, and finally, to compare miR-16 and the MEK inhibitor selumetinib in reducing KRAS-mutated NSCLC growth in vitro and in vivo. RESULTS: We demonstrated that miR-16 directly targets the three KRAS downstream effectors MAPK3, MAP2K1, and CRAF in NSCLC, restoring the sensitivity to erlotinib in KRAS-mutated NSCLC both in vitro and in vivo. We also provided evidence that the miR-16-erlotinib regimen is more effective than the selumetinib-erlotinib combination in KRAS-mutated NSCLC. CONCLUSIONS: Our findings support the biological preclinical rationale for using miR-16 in combination with erlotinib in the treatment of NSCLC with KRAS-activating mutations.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , Lung Neoplasms , MAP Kinase Kinase Kinases , MicroRNAs , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras) , RNA, Neoplasm , A549 Cells , Animals , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/therapy , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/therapy , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/genetics , Male , Mice , Mice, Inbred NOD , Mice, SCID , MicroRNAs/biosynthesis , MicroRNAs/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Xenograft Model Antitumor AssaysABSTRACT
As much as space travel and exploration have been a goal since humankind looked up to the stars, the challenges coming with it are manifold and difficult to overcome. Therefore, researching the changes the human organism undergoes following exposure to weightlessness, on a cellular or a physiological level, is imperative to reach the goal of exploring space and new planets. Building on the results of our CellBox-1 experiment, where thyroid cancer cells were flown to the International Space Station, we are now taking advantage of the newest technological opportunities to gain more insight into the changes in cell-cell communication of these cells. Analyzing the exosomal microRNA composition after several days of microgravity might elucidate some of the proteomic changes we have reported earlier. An array scan of a total of 754 miRNA targets revealed more than 100 differentially expressed miRNAs in our samples, many of which have been implicated in thyroid disease in other studies.
Subject(s)
Exosomes/genetics , Extraterrestrial Environment , MicroRNAs/metabolism , Thyroid Neoplasms/genetics , Weightlessness , Cell Line, Tumor , Exosomes/metabolism , Humans , MicroRNAs/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathologyABSTRACT
Space travel has always been the man's ultimate destination. With the ability of spaceflight though, came the realization that exposure to microgravity has lasting effects on the human body. To counteract these, many studies were and are undertaken, on multiple levels. Changes in cell growth, gene, and protein expression have been described in different models on Earth and in space. Extracellular vesicles, and in particular exosomes, are important cell-cell communicators, being secreted from almost all the cells and therefore, are a perfect target to further investigate the underlying reasons of the organism's adaptations to microgravity. Here, we studied supernatants harvested from the CellBox-1 experiment, which featured human thyroid cancer cells flown to the International Space Station during the SpaceX CRS-3 cargo mission. The initial results show differences in the number of secreted exosomes, as well as in the distribution of subpopulations in regards to their surface protein expression. Notably, alteration of their population regarding the tetraspanin surface expression was observed. This is a promising step into a new area of microgravity research and will potentially lead to the discovery of new biomarkers and pathways of cellular cross-talk.
Subject(s)
Exosomes/metabolism , Space Flight , Thyroid Neoplasms/metabolism , Weightlessness , Antigens, CD/metabolism , Cell Line, Tumor , Fluorescence , Humans , Interferometry , Particle SizeABSTRACT
FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.
Subject(s)
Janus Kinase 2/metabolism , Leukemia/drug therapy , Propylene Glycols/pharmacology , Protein Phosphatase 2/metabolism , Sphingosine/analogs & derivatives , Animals , Cell Line, Transformed , Cell Line, Tumor , Cells, Cultured , Class Ib Phosphatidylinositol 3-Kinase , DNA-Binding Proteins , Enzyme Activation/drug effects , Fingolimod Hydrochloride , Histone Chaperones , Humans , Immunoblotting , Immunosuppressive Agents/pharmacology , Janus Kinase 2/genetics , Kaplan-Meier Estimate , Leukemia/genetics , Leukemia/pathology , Mice , Mice, SCID , Mutation , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Phosphatase 2/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Sphingosine/pharmacology , Treatment OutcomeABSTRACT
As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Hydrazines/pharmacology , Karyopherins/antagonists & inhibitors , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Triazoles/pharmacology , Adult , Animals , Antigens, CD34/genetics , Antigens, CD34/metabolism , Apoptosis/drug effects , Cell Proliferation/drug effects , Clinical Trials, Phase I as Topic , DNA-Binding Proteins , Drug Evaluation, Preclinical , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Histone Chaperones/antagonists & inhibitors , Histone Chaperones/genetics , Histone Chaperones/metabolism , Humans , Karyopherins/genetics , Karyopherins/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Kinase Inhibitors/pharmacology , Protein Transport , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Ribonucleoproteins/antagonists & inhibitors , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Exportin 1 ProteinABSTRACT
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by expression of the p210-BCR-ABL fusion protein. We demonstrate in a murine model of p210-BCR-ABL-induced MPD that gene targeting of Rac1 and Rac2 significantly delays or abrogates disease development. Attenuation of the disease phenotype is associated with severely diminished p210-BCR-ABL-induced downstream signaling in primary hematopoietic cells. We utilize NSC23766, a small molecule antagonist of Rac activation, to validate biochemically and functionally Rac as a molecular target in both a relevant animal model and in primary human CML cells in vitro and in a xenograft model in vivo, including in Imatinib-resistant p210-BCR-ABL disease. These data demonstrate that Rac is an additional therapeutic target in p210-BCR-ABL-mediated MPD.
Subject(s)
Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Leukemic , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/metabolism , rac GTP-Binding Proteins/physiology , Aminoquinolines/pharmacology , Animals , Antigens, CD34/biosynthesis , Cell Line, Tumor , Dose-Response Relationship, Drug , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Mice , Myeloproliferative Disorders/therapy , Neoplasm Transplantation , Phenotype , Pyrimidines/pharmacology , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/metabolism , RAC2 GTP-Binding ProteinABSTRACT
Protein phosphatase 2A (PP2A), one of the main serine-threonine phosphatases in mammalian cells, maintains cell homoeostasis by counteracting most of the kinase-driven intracellular signalling pathways. Unrestrained activation of oncogenic kinases together with inhibition of tumour suppressors is often required for development of cancer. PP2A has been shown to be genetically altered or functionally inactivated in many solid cancers and leukaemias, and is therefore a tumour suppressor. For example, the phosphatase activity of PP2A is suppressed in chronic myeloid leukaemia and other malignancies characterised by aberrant activity of oncogenic kinases. Preclinical studies show that pharmacological restoration of PP2A tumour-suppressor activity by PP2A-activating drugs (eg, FTY720) effectively antagonises cancer development and progression. Here, we discuss PP2A as a druggable tumour suppressor in view of the possible introduction of PP2A-activating drugs into anticancer therapeutic protocols.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Design , Enzyme Activators/therapeutic use , Molecular Targeted Therapy , Neoplasms/drug therapy , Protein Phosphatase 2/metabolism , Animals , Enzyme Activation , Humans , Neoplasms/enzymology , Neoplasms/pathology , Signal Transduction/drug effectsABSTRACT
Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Interferon-gamma/biosynthesis , Killer Cells, Natural/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Transcription Factors/metabolism , Animals , Cells, Cultured , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins , Enzyme Activation , Gene Expression Regulation , Histone Chaperones , Humans , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Monokines/pharmacology , Signal Transduction , Transcription Factors/geneticsABSTRACT
The oncogenic BCR/ABL kinase activity induces and maintains chronic myelogenous leukemia (CML). We show here that, in BCR/ABL-transformed cells and CML blast crisis (CML-BC) progenitors, the phosphatase activity of the tumor suppressor PP2A is inhibited by the BCR/ABL-induced expression of the PP2A inhibitor SET. In imatinib-sensitive and -resistant (T315I included) BCR/ABL+ cell lines and CML-BC progenitors, molecular and/or pharmacological activation of PP2A promotes dephosphorylation of key regulators of cell proliferation and survival, suppresses BCR/ABL activity, and induces BCR/ABL degradation. Furthermore, PP2A activation results in growth suppression, enhanced apoptosis, restored differentiation, impaired clonogenic potential, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR/ABL+ cells. Thus, functional inactivation of PP2A is essential for BCR/ABL leukemogenesis and, perhaps, required for blastic transformation.
Subject(s)
Blast Crisis/metabolism , Chromosomal Proteins, Non-Histone/physiology , Fusion Proteins, bcr-abl/physiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/physiology , Transcription Factors/physiology , Animals , Antineoplastic Agents/pharmacology , Benzamides , Cell Line, Transformed , Colforsin/pharmacology , DNA-Binding Proteins , Enzyme Inhibitors/metabolism , Histone Chaperones , Humans , Imatinib Mesylate , In Vitro Techniques , K562 Cells , Leukemia/prevention & control , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mice, SCID , Neoplasm Transplantation , Phosphoprotein Phosphatases/antagonists & inhibitors , Piperazines/pharmacology , Protein Phosphatase 2 , Pyrimidines/pharmacology , Tumor Cells, Cultured , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/physiologyABSTRACT
Purpose: Although biopsy is contraindicated in retinoblastoma (RB), the aqueous humor (AH) is a robust liquid biopsy source of molecular tumor information, facilitating biomarker discovery. Small extracellular vesicles (sEVs), promising biomarker candidates across multiple cancers, were recently identified in RB AH, but relationships between sEVs and RB clinical features are unknown. Methods: We analyzed sEVs in 37 AH samples from 18 RB eyes of varying International Intraocular Retinoblastoma Classification (IIRC) groups and explored clinical correlations. Ten samples were collected at diagnosis (DX) and 27 during treatment (Tx). Unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequentially converted to percentages for analysis. Results: Comparing DX and Tx samples, a higher percentage of CD63/81+ sEVs was found in DX AH (16.3 ± 11.6% vs. 5.49 ± 3.67% P = 0.0009), with a more homogenous mono-CD63+ sEV population seen in Tx AH (43.5 ± 14.7% vs. 28.8 ± 9.38%, P = 0.0073). Among DX samples, CD63/81+ sEVs were most abundant in group E eyes (n = 2) compared to group D (n = 6) by count (2.75 × 105 ± 3.40 × 105 vs. 5.95 × 103 ± 8.16 × 103, P = 0.0006), and to group A + B (n = 2) by count (2.75 × 105 ± 3.40 × 105 vs. 2.73 × 102 ± 2.59 × 102, P = 0.0096) and percentage (32.1 ± 7.98% vs. 7.79 ± 0.02%, P = 0.0187). Conclusions: CD63/81+ sEVs enrich AH from RB eyes before treatment and those with more significant tumor burden, suggesting they are tumor-derived. Future research into their cargo may reveal mechanisms of cellular communication via sEVs in RB and novel biomarkers.
Subject(s)
Extracellular Vesicles , Retinal Neoplasms , Retinoblastoma , Humans , Retinoblastoma/diagnosis , Aqueous Humor , Biomarkers , Retinal Neoplasms/diagnosis , Tetraspanin 30ABSTRACT
The capture of tumour-derived extracellular vesicles (TEVs) by cells in the tumour microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumour cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumour cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lung macrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression.
Subject(s)
Extracellular Vesicles , Melanoma , MicroRNAs , Humans , Animals , Mice , Extracellular Vesicles/metabolism , Kinetics , MicroRNAs/metabolism , Melanoma/metabolism , Inflammation/metabolism , Tumor MicroenvironmentABSTRACT
Aqueous humor (AH), the clear fluid in front of the eye, maintains the pressure and vitality of ocular tissues. This fluid is accessible via the clear cornea which enables use of AH as a liquid biopsy source of biomarkers for intraocular disease. Extracellular vesicles are detectable in the AH and small extracellular vesicles (sEVs) are present in the AH from adults. However, EVs in AH from pediatric eyes in vivo have never previously been explored. We know very little about the heterogeneity of AH EV populations in ocular disease. Twenty-seven processing-free AH samples from 19 patients across four different pediatric ocular diseases were subjected to Nanoparticle Tracking Analysis (NTA) and Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis. NTA demonstrated the concentration of AH EV/EPs is 3.11 × 109-1.38 × 1010 particles/ml; the majority sized 76.8-103 nm. SP-IRIS revealed distinct patterns of tetraspanin expression of AH sEVs. An enriched mono-CD63+ sEV subpopulation identified in AH indicates this is a potential AH-specific biomarker. In the setting of retinoblastoma there was a more heterogeneous population of sEVs which normalized with treatment. This suggests a potential clinical application of direct measurement of sEV subpopulations in AH samples to monitor successful tumor response to therapy.
ABSTRACT
We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPbeta, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/biosynthesis , Cell Transformation, Neoplastic/metabolism , MicroRNAs/biosynthesis , Phosphoric Monoester Hydrolases/biosynthesis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cells, B-Lymphoid/metabolism , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , Cell Transformation, Neoplastic/genetics , Down-Regulation/genetics , Gene Expression Regulation, Leukemic/genetics , Inositol Polyphosphate 5-Phosphatases , Interleukin-6/metabolism , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , Mice , Mice, Transgenic , MicroRNAs/genetics , Phosphoric Monoester Hydrolases/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Signal Transduction/geneticsABSTRACT
Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.
Subject(s)
Blast Crisis/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Propylene Glycols/therapeutic use , Sphingosine/analogs & derivatives , Animals , Benzamides , Blast Crisis/genetics , Blast Crisis/metabolism , Blast Crisis/pathology , Cell Survival/drug effects , Dasatinib , Drug Resistance, Neoplasm/drug effects , Fingolimod Hydrochloride , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Gene Expression Regulation, Neoplastic , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Molecular Structure , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Piperazines/pharmacology , Propylene Glycols/chemistry , Protein Phosphatase 2 , Pyrimidines/pharmacology , Signal Transduction/drug effects , Sphingosine/chemistry , Sphingosine/therapeutic use , Thiazoles/pharmacology , Time Factors , Tumor Cells, CulturedABSTRACT
Persistence of drug-resistant quiescent leukemic stem cells (LSC) and impaired natural killer (NK) cell immune response account for relapse of chronic myelogenous leukemia (CML). Inactivation of protein phosphatase 2A (PP2A) is essential for CML-quiescent LSC survival and NK cell antitumor activity. Here we show that MIR300 has antiproliferative and PP2A-activating functions that are dose dependently differentially induced by CCND2/CDK6 and SET inhibition, respectively. MIR300 is upregulated in CML LSCs and NK cells by bone marrow microenvironment (BMM) signals to induce quiescence and impair immune response, respectively. Conversely, BCR-ABL1 downregulates MIR300 in CML progenitors to prevent growth arrest and PP2A-mediated apoptosis. Quiescent LSCs escape apoptosis by upregulating TUG1 long noncoding RNA that uncouples and limits MIR300 function to cytostasis. Genetic and pharmacologic MIR300 modulation and/or PP2A-activating drug treatment restore NK cell activity, inhibit BMM-induced growth arrest, and selectively trigger LSC apoptosis in vitro and in patient-derived xenografts; hence, the importance of MIR300 and PP2A activity for CML development and therapy.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , MicroRNAs , Humans , Killer Cells, Natural , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , MicroRNAs/genetics , Neoplastic Stem Cells , Protein Kinase Inhibitors/metabolism , Protein Phosphatase 2/genetics , Tumor Microenvironment/geneticsABSTRACT
Hypermethylation of 5'-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys(38) of p65 to inhibit nuclear factor-kappaB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC(50) of 3.5 microM, possibly through alkylation of the proximal thiolate of Cys(1226) of the catalytic domain by its gamma-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG(1) cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.