The human urinary exosome as a potential metabolic effector cargo

Exosomes are nanovesicles, derived from the endocytic pathway, released by most cell types and found in many body fluids, including urine. A variety of exosomal functions have been reported, including transfer of RNA, cell communication, control of apoptosis and protein lifespan. Exosomes from mesenchymal stem cells can rescue bioenergetics of injured cells. Here the urinary exosome proteome, non-urinary exosome proteome and urinome are compared. A consistent number of identified proteins cluster to metabolic functions. Cytoscape software analysis based on biological processes gene ontology database shows that metabolic pathways such as aerobic glycolysis and oxidative phosphorylation have a high probability (p ≤ 0.05) of being expressed and therefore functional. A metabolic function appears to be associated with human urinary exosomes, whose relevance experimental studies can assess.     

Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) shows promise as a chemotherapeutic agent. However, many human cancer cells are resistant to killing by TRAIL. We have previously demonstrated that reovirus infection increases the susceptibility of human lung (H157) and breast (ZR75-1) cancer cell lines to TRAIL-induced apoptosis. We now show that reovirus also increases the susceptibility of human ovarian cancer cell lines (OVCAR3, PA-1 and SKOV-3) to TRAIL-induced apoptosis. Reovirus-induced increases in susceptibility of OVCAR3 cells to TRAIL require virus uncoating and involve increased activation of caspases 3 and 8. Reovirus infection results in the down-regulation of cFLIP (cellular FLICE inhibitory protein) in OVCAR3 cells. Down-regulation of cFLIP following treatment of OVCAR3 cells with antisense cFLIP oligonucleotides or PI3 kinase inhibition also increases the susceptibility of OVCAR3 cells to TRAIL-induced apoptosis. Finally, over-expression of cFLIP blocks reovirus-induced sensitization of OVCAR3 cells to TRAIL-induced apoptosis. The combination of reovirus and TRAIL thus represents a promising new therapeutic approach for the treatment of ovarian cancer.     

Exosome-mediated transduction of mechanical force regulates prostate cancer migration via microRNA

Physical cues in the extracellular microenvironment regulate cancer cell metastasis. Functional microRNA (miRNA) carried by cancer derived exosomes play a critical role in extracellular communication between cells and the extracellular microenvironment. However, little is known about the role of exosomes loaded miRNAs in the mechanical force transmission between cancer cells and extracellular microenvironment. Herein, our results suggest that stiff extracellular matrix (ECM) induced exosomes promote cancer cell migration. The ECM mechanical force regulated the exosome miRNA cargo of prostate cancer cells. Exosome miRNAs regulated by the ECM mechanical force modulated cancer cell metastasis by regulating cell motility, ECM remodeling and the interaction between cancer cells and nerves. Focal adhesion kinase mediated-ECM mechanical force regulated the intracellular miRNA expression, and F-actin mediate-ECM mechanical force regulated miRNA packaging into exosomes. The above results demonstrated that the exosome miRNA cargo promoted cancer metastasis by transmitting the ECM mechanical force. The ECM mechanical force may play multiple roles in maintaining the microenvironment of cancer metastasis through the exosome miRNA cargo.     

Oncogenic and Non‐Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre‐metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non‐malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non‐malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre‐metastatic niche (S100A4, F3, ITGβ5, ANXA1), clinically‐relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.     

Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.     

Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer

Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC‐labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin‐bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular fractions. A total of 1318 proteins with at least 1.5‐fold change were deregulated in highly metastatic KM12SM cells respect to KM12C cells. The protein network organization, protein complexes and functional pathways associated to CRC metastasis was revealed with spatial resolution. Although 92% of the differentially expressed proteins showed the same deregulation in all subcellular compartments, a subset of 117 proteins (8%) showed opposite changes in different subcellular localizations. The chaperonin CCT, the Eif2 and Eif3 initiation of translation and the oxidative phosphorylation complexes together with an important number of guanine nucleotide‐binding proteins, were deregulated in abundance and localization within the metastatic cells. Particularly relevant was the relationship of deregulated protein complexes with exosome secretion. The knowledge of the spatial proteome alterations at subcellular level contributes to clarify the molecular mechanisms underlying colorectal cancer metastasis and to identify potential targets of therapeutic intervention.     

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10±0.42 and 2.65±1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immuno-staining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.     

Cancer Liquid Biopsy Using Integrated Microfluidic Exosome Analysis Platforms

Liquid biopsies serve as both powerful noninvasive diagnostic tools for early cancer screening and prognostic tools for monitoring cancer progression and treatment efficacy. Exosomes are promising biomarkers for liquid biopsies, since these nano‐sized extracellular vesicles (EVs) enrich proteins, lipids, mRNAs, and miRNAs from cells of origin, including cancer cells. Although exosomes are abundantly present in various bodily fluids, conventional exosome isolation and detection methods that rely on benchtop equipment are time‐consuming, expensive, and involve complicated non‐portable procedures. As an alternative, recently developed microfluidic platforms can perform effective exosome separation and detection for liquid biopsies using a single device. Such methods offer advantages of integrity, speed, cost‐efficiency, and portability over conventional benchtop and early microfluidic‐based single‐functional methods which can only separate or detect exosomes separately. These advances have made exosome‐based point‐of‐care (POC) applications possible. This review outlines recent integrated microfluidic‐based exosomal detection strategies to guide future development of such devices for use in liquid biopsies for early cancer screening, prognostic monitoring, and other potential POC applications.     

Up-regulation of long intergenic noncoding RNA 01296 in ovarian cancer impacts invasion,apoptosis and cell cycle distribution via regulating EMT

BackgroundRecently, long intergenic non-coding RNA 01296 (LINC01296) has been demonstrated to regulate the initiation and progression of several cancers, but the functions of LINC01296 in ovarian cancer still remain unclear. The objective of our study was to determine the expression, biological roles, and clinical significance of LINC01296 in ovarian cancer.MethodsLINC01296 expression was measured in ovarian cancer tissues or cell lines. Next, the relationships between LINC01296 levels and the clinical factors of ovarian cancer, such as progression-free survival and overall survival were analyzed. Additionally, cell proliferation, migration and invasion capacities, apoptosis, cell cycle distribution were investigated after silencing of LINC01296. To confirm whether LINC01296 mediates EMT initiation in ovarian cancer cells, the effect of LINC01296 silence on E-cadherin, N-cadherin and vimentin was assessed in SKOV3 and OVCAR3 cells.ResultsWe found that LINC01296 was over-expressed in ovarian cancer tissues and cell lines, when comparing with adjacent normal tissue samples and normal cells. Higher LINC01296 expression was significantly correlated with shorter progression-free survival and overall survival. For the functional experiments, knockdown of LINC01296 suppressed cell proliferation, inhibited colony formation ability, abrogated cell migration and invasion potential, and enhanced cell apoptosis. Cell cycle analysis suggested that LINC01296 positively regulated cell cycle progression in ovarian cancer cells. Moreover, western blotting analysis displayed that knockdown of LINC01296 significantly increased E-cadherin, but reduced N-cadherin and vimentin expressions in SKOV3 and OVCAR3 cells, compared with no-transfection cells.ConclusionsLINC01296 plays an important role in promoting the progression of ovarian cancer. Over-expression of LINC01296 might function as an indicator for diagnosis and prognosis of ovarian cancer patients.     

The novel estrogen receptor GPER regulates the migration and invasion of ovarian cancer cells

G protein-coupled estrogen receptor (GPER) was identified as a new member of the estrogen receptor family in recent years. It has become apparent that GPER mediates the non-genomic signaling of 17β-estradiol (E₂) in a variety of estrogen-related cancers. Our previous study has found that GPER was overexpressed in human epithelial ovarian cancer and was positively correlated with the expression of matrix metalloproteinase 9 (MMP-9), which suggested GPER might promote the metastasis of ovarian cancer. However, the mechanisms underlying GPER-dependent metastasis of ovarian cancer are still not clear. In the present study, estrogen receptor α (ERα)-negative/GPER-positive OVCAR5 ovarian cancer cell line was used to investigate the role of GPER in the migration and invasion of ovarian cancer. Wound healing assay and transwell matrigel invasion assay were performed to determine the potentials of cell migration and invasion, respectively. The production and activity of MMP-9 in OVCAR5 cells were examined by Western blot and gelatin zymography analysis. The results showed that E₂ and selective GPER agonist G-1 increased cell motility and invasiveness, and upregulated the production and proteolytic activity of MMP-9 in OVCAR5 cells. Small interfering RNA (siRNA) targeting GPER and G protein inhibitor pertussin toxin (PTX) inhibited the migration and invasion of OVCAR5 cells, and also reduced the expression and activity of MMP-9. Our data suggested that GPER promoted the migration and invasion of ovarian cancer cells by increasing the expression and activity of MMP-9. GPER might play an important role in the progression of ovarian cancer.     

miRNA-20a促进卵巢癌细胞OVCAR3的转移

目的检测miRNA．20a对卵巢癌细胞系OVCAR3转移能力的影响。方法通过实时定量RT-PCR验证反义寡核苷酸与小干扰RNA封闭与过表达的效果,然后利用MTF、软琼脂集落形成和transwell侵袭实验检测封闭和过表达miRNA．20a对OVCAR3细胞增殖及转移能力的影响。结果封闭内源性miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显降低。过表达miRNA-20a后,细胞活性基本不受影响,但集落形成能力和细胞的转移能力明显升高。结论miRNA-20a可能参与了卵巢癌细胞OVCAR3的转移。     

Tumor-derived extracellular vesicles and microRNAs: Functional roles,diagnostic, prognostic and therapeutic options

In the last few years cancer research more and more highlighted the importance of cell to cell communication in tumor progression. Among many other functional mechanisms, results evidenced the importance of miRNAs loaded into exosomes and their actions as mediators in intercellular communication, either in the tumor microenvironment or at distant sites. Deregulation of miRNA levels is a prerogative of cancer cells and is reflected in the miRNA cargo of tumor derived exosomes. Thus, learning of circulating miRNA activities add the missing piece we need to understand some unclear aspects of cancer biology.Here we summarized the current knowledge on exosome transfer capabilities between cancer cells and all the cells constituting tumor microenvironment with a particular focus on their miRNA cargos and regulatory functions. The clinical relevance of these molecular aspects is emphasized by numerous cell interactions that ultimately result in normal cell function defeat, relevant to increase tumor malignancy. The quantitative and qualitative evaluation of circulating miRNAs offers new perspective for better diagnosis and prognosis of cancer patients, eventually improving their management.     

The farnesyltransferase inhibitor,FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.     

PI3K Inhibition Sensitize the Cisplatin-resistant Human Ovarian Cancer Cell OVCAR3 by Induction of Oxidative Stress

Background:This study evaluates the effect of simultaneous AKT inhibition and cisplatin therapy in changes of Reactive Oxygen Species (ROS) production, apoptosis induction, and cell survival in cisplatin-resistant OVCAR3 cell.Methods:OVCAR3 cancer cells were treated with cisplatin, Ly 294002 (LY), and cisplatin+Ly to investigate the cytotoxicity effect of the mentioned groups via MTT assay. Then, DCFH-DA (2′, 7′-dichlorodihydro fluorescein diacetate) assay kit is used to assess the potential of treated groups in intracellular ROS generation. Protein expression levels of caspase-3, cleaved caspase 3, PI3K, Akt, p-Akt, XIAP, and Survivin are estimated through immunoblotting assay in all three experimental groups.Results:The results showed that all three treated groups, including cisplatin and Ly alone and co-administration of cisplatin+Ly, could reduce the cell vitality of OVCAR3 cancer cells, induced intracellular production of ROS and increased the expression level of activated caspase 3 and Akt protein, whereas down-regulated the phosphorylation of Akt protein. However, the effect of combination therapy was more tangible compared to single therapy and control groups. In contrast, the expression amount of XIAP, Survivin, and PI3K did not show detectable changes in comparison with the control group.Conclusion:The results showed that the AKT inhibition by Ly could sensitize the OVCAR3 cancer cells to the cisplatin and lower the effective dose of cisplatin through hyperactivation of oxidative stress.Key Words: Caspase-3, Cisplatin, Ovarian cancer, PI3K/Akt signaling     

Exosome and epithelial–mesenchymal transition: A complex secret of cancer progression

This short communication will enlighten the readers about the exosome and the epithelial-mesenchymal transition (EMT) related to several complicated events. It also highlighted the therapeutic potential of exosomes against EMT. Exosome toxicology, exosome heterogeneity, and a single exosome profiling approach are also covered in this article. In the future, exosomes could help us get closer to cancer vaccine and precision oncology.     

Msx1 gene overexpression induces G1 phase cell arrest in human ovarian cancer cell line OVCAR3

Recent evidence suggested an involvement of homeobox genes in tumorigenesis. Here we investigated whether one of homeobox-containing genes, Msx1, might be involved in the regulation of cell proliferation and cell cycle using Msx1 overexpressing human ovarian cancer cell line, OVCAR3. Overexpression of Msx1 in OVCAR3 cells inhibited cell proliferation by markedly increasing the length of the G1 phase of the cell cycle over control cells. Consistent with this result, dramatic suppression of cyclins D1, D3, E, cyclin-dependent kinase 4, c-Jun, and Rb was observed. Elevated expression of genes involved in the growth arrest and apoptosis (GADD153 and apoptotic cystein protease MCH4) and suppression of proliferation associated protein gene (PAG) in Msx1-overexpressing cells by cDNA expression array analysis provide further evidence for a potential repressor function of Msx1 in cell cycle progression.     

Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types

Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases.