ADAMTS-1 has nuclear localization in cells with epithelial origin and leads to decreased cell migration.

In the study of tumorigenesis, the involvement of molecules within the extracellular matrix (ECM) is crucial. ADAMTSs (A Disintegrin and Metalloproteinase with Thrombospondin motifs), a group of secreted proteases known for their role in ECM remodeling, were primarily considered to be extracellular proteases. However, our research specifically detected ADAMTS-1, a member of this family, predominantly within the nucleus of mammary cells. Our main objective was to understand the mechanism of ADAMTS-1 translocation to the nucleus and its functional significance in this cellular compartment. Our investigation uncovered that nuclear ADAMTS-1 was present in cells exhibiting an epithelial phenotype, while cells of mesenchymal origin contained the protease in the cytoplasm. Moreover, disruption of ADAMTS-1 secretion, induced by Monensin treatment, resulted in its accumulation in the cytoplasm. Notably, our research indicated that alterations in the secretory pathways could influence the protease's compartmentalization. Additionally, experiments with conditioned medium from cells containing nuclear ADAMTS-1 demonstrated its internalization into the nucleus by HT-1080 cells and fibroblasts. Furthermore, heightened levels of ADAMTS-1 within the ECM reduced the migratory potential of mesenchymal cells. This highlights the potential significance of nuclear ADAMTS-1 as a critical component within the tumor microenvironment due to its functional activity in this specific cellular compartment.

Laminin-derived peptide C16 regulates Tks expression and reactive oxygen species generation in human prostate cancer cells.

Laminin peptides influence cancer biology. We investigated the role of a laminin-derived peptide C16 regulating invadopodia molecules in human prostate cancer cells (DU145). C16 augmented invadopodia activity of DU145 cells, and stimulated expression Tks4, Tks5, cortactin, and membrane-type matrix metalloproteinase 1. Reactive oxygen species generation is also related to invadopodia formation. This prompted us to address whether C16 would induce reactive oxygen species generation in DU145 cells. Quantitative fluorescence and flow cytometry showed that the peptide C16 increased reactive oxygen species in DU145 cells. Furthermore, significant colocalization between Tks5 and reactive oxygen species was observed in C16-treated cells. Results suggested that the peptide C16 increased Tks5 and reactive oxygen species in prostate cancer cells. The role of C16 increasing Tks and reactive oxygen species are novel findings on invadopodia activity.

ADAMTS-1 disrupts HGF/c-MET signaling and HGF-stimulated cellular processes in fibrosarcoma.

Extracellular matrix (ECM) serves as a reservoir for biologically active factors, such as growth factors and proteases that influence the tumor cell behavior. ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a secreted protease that has the ability to modify the ECM during physiological and pathological processes. Here, we analyzed the role played by ADAMTS-1 regulating HGF and TGF-ß1 activities in the high-grade fibrosarcoma cell line (HT1080). We generated HT1080 and HEK293T cells overexpressing ADAMTS-1. HT1080 cells overexpressing ADAMTS-1 (HT1080-MPA) exhibited a significant decrease in cell proliferation and migration velocity, both in presence of HGF. We obtained similar results with ADAMTS-1-enriched conditioned medium from other cell type. However, ADAMTS-1 overexpression failed to affect TGF-ß1 activity associated with HT1080 cell proliferation and migration velocity. Immunoblotting showed that ADAMTS-1 overexpression disturbs c-Met activation upon HGF stimulation. Downstream ERK1/2 and FAK signaling pathways are also influenced by this protease. Additionally, ADAMTS-1 decreased the size of the fibrosarcospheres, both under normal conditions and in the presence of HGF. Likewise, in presence of HGF, ADAMTS-1 overexpression in HT1080 disrupted microtumors formation in vivo. These microtumors, including individual cells, presented characteristics of non-invasive lesions (rounded morphology). Our results suggest that ADAMTS-1 is involved in regulating HGF-related functions on fibrosarcoma cells. This protease may then represent an endogenous mechanism in controlling the bioavailability of different growth factors that have a direct influence on tumor cell behavior.

The laminin-derived peptide C16 regulates GPNMB expression and function in breast cancer.

Breast cancer is an important public health problem, and its progression may be related to the extracellular matrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECM proteins regulating tumor biology. The laminin-derived peptide C16 regulates different properties of tumor cells. Here we analyzed C16-induced differential gene expression in MDA-MB-231 breast cancer cells. MCF-10A normal-like breast cells served as control. Among different cancer-related genes, C16 induced overexpression of GPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non-metastatic B), involved with malignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene and protein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancer cells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highly aggressive MDA-MB-231 cancer cells. We addressed regulatory mechanisms involved in C16-mediated increase of GPNMB protein levels in MDA-MB-231 cells, and observed that C16 stimulates ß1 integrin and Src phosphorylation. Furthermore, Src inhibition decreases peptide-induced GPNMB expression levels. To contextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer human tissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed in breast cancer compared to normal tissue. We concluded that laminin-derived peptide C16 regulates gene and protein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion of highly aggressive MDA-MB-231 cells, probably through Src signaling. GPNMB presented increased expression in breast cancer in vivo compared to normal breast tissue.

Expression of molecules related to AKT pathway as putative regulators of ameloblastoma local invasiveness.

BACKGROUND: Ameloblastoma is an odontogenic neoplasm with local invasiveness and high recurrence. We previously suggested that growth factors, matrix metalloproteinases (MMPs), and TIMPs influence ameloblastoma invasiveness (Pathol. Res. Pract., 208, 2012, 225; Oral. Surg. Oral. Med. Oral. Pathol. Oral Radiol. Endod., 111, 2011, 474). Signals generated by this molecular network would be transduced by ERK 1/2 pathway (Oral. Surg. Oral. Med. Oral. Pathol. Oral Radiol. Endod., 111, 2011, 474). Others signaling pathways may influence ameloblastoma biology. Here, we studied expression of AKT and related molecules in ameloblastoma. METHODS: Fourteen cases of solid/multicystic ameloblastomas were examined. Immunohistochemistry was carried out to detected AKT (phospho-AKT), NF-қB (phospho-NF-қB), ß-catenin, cyclin-D1, and COX-2 in ameloblastoma samples. These molecules were evaluated in neoplastic cells and stroma. RESULTS: All proteins were detected in ameloblastoma. Expression of these markers was quantified and compared. Spearman's rank test was carried out to address positive correlations between proteins (P < 0.05). Ameloblastoma had a significant positive correlation of AKT (phospho-AKT) with ß-catenin. ß-catenin correlated with Cyclin-D1 and COX-2 in neoplastic cells. AKT (phospho-AKT) correlated with ß-catenin; ß-catenin with Cyclin-D1; AKT (phospho-AKT) with NF-қB (phospho-NF-қB); and NF-қB (phospho-NF-қB) with COX-2 in stromal cells. CONCLUSIONS: Results suggest that proteins studied are present and probably involved in a functional pathway in neoplastic cells and stroma and may therefore influence the local invasiveness of ameloblastoma.

Decreased expression of ADAMTS-1 in human breast tumors stimulates migration and invasion.

BACKGROUND: ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines. RESULTS: In a series of primary breast tumors, we observed variable levels of ADAMTS-1 mRNA expression but lower levels of ADAMTS-1 protein expression in human breast cancers as compared to normal tissue, with a striking decrease observed in high-malignancy cases (triple-negative for estrogen, progesterone and Her-2). This result prompted us to analyze the effect of ADAMTS-1 knockdown in breast cancer cells in vitro. MDA-MB-231 cells with depleted ADAMTS-1 expression demonstrated increased migration, invasion and invadopodia formation. The regulatory mechanisms underlying the effects of ADAMTS-1 may be related to VEGF, a growth factor involved in migration and invasion. MDA-MB-231 cells with depleted ADAMTS-1 showed increased VEGF concentrations in conditioned medium capable of inducing human endothelial cells (HUVEC) tubulogenesis. Furthermore, expression of the VEGF receptor (VEGFR2) was increased in MDA-MB-231 cells as compared to MCF7 cells. To further determine the relationship between ADAMTS-1 and VEGF regulating breast cancer cells, MDA-MB-231 cells with reduced expression of ADAMTS-1 were pretreated with a function-blocking antibody against VEGF and then tested in migration and invasion assays; both were partially rescued to control levels. CONCLUSIONS: ADAMTS-1 expression was decreased in human breast tumors, and ADAMTS-1 knockdown stimulated migration, invasion and invadopodia formation in breast cancer cells in vitro. Therefore, this series of experiments suggests that VEGF is involved in the effects mediated by ADAMTS-1 in breast cancer cells.

Oestrogen receptor ß in adenoid cystic carcinoma of salivary glands.

AIMS: This study aimed to describe the expression of oestrogen receptor (ER)α, ERß and aromatase in salivary gland adenoid cystic carcinoma (ACC). METHODS AND RESULTS: ERα, ERß and aromatase expression was analysed by immunohistochemistry in tissue microarray blocks from 38 cases of ACC and seven normal salivary glands. The intracellular localization and amount of total protein expression were investigated by immunofluorescence and western blotting in an ACC cell line. Western blotting analysis showed overexpression of ERα, ERß and aromatase in the ACC cell line; however, with immunofluorescence, only ERß was shown to be expressed in the nucleus. Immunohistochemistry revealed positive nuclear expression of ERß, positive cytoplasmic expression of aromatase and a lack of ERα expression as compared with normal salivary glands. CONCLUSIONS: The nuclear expression of ERß indicates that oestrogen may be active in ACC and possibly able to mediate E2-targeted gene transcription. This study strongly suggests that ERß may be involved in tumour progression, playing a role in tumour development, and thus corroborating the indication for ER antagonists in the clinical control of ACC. This study opens a new perspective on the potential use of anti-oestrogens and aromatase inhibitors as therapeutic agents against ACC.

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line.

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. ß1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through ß1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity.

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.

A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

Matrix metalloproteinases, TIMPs and growth factors regulating ameloblastoma behaviour.

AIMS: Ameloblastoma is an odontogenic neoplasm with local invasiveness and recurrence. We have previously suggested that growth factors and matrix metalloproteinases (MMPs) influence ameloblastoma invasiveness. The aim was to study expression of MMPs, tissue inhibitor of metalloproteinases (TIMPs) and growth factors in ameloblastoma. METHODS AND RESULTS: Thirteen cases of solid/multicystic ameloblastoma were examined. As a control, calcifying cystic odontogenic tumour (CCOT), a non-invasive odontogenic neoplasm with ameloblastomatous epithelium was also studied. Immunohistochemistry detected MMPs, TIMPs and growth factors in ameloblastoma and CCOT. The labelling index (LI) of MMP-9 and TIMP-2 was significantly higher in ameloblastoma compared with CCOT. The LI of epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and epidermal growth factor receptor (EGFR) was also increased in ameloblastoma. This neoplasm showed greater expression of MMPs, TIMPs and growth factors compared with CCOT. We then analysed these molecules in ameloblastoma cells and stroma. Ameloblastoma cells exhibited increased LI of MMP-1, -2 and EGFR. We found a positive correlation between EGF and TIMP-1, and between TGF-alpha and TIMP-2. It is known that signals generated by growth factors are transduced by the ERK pathway. Ameloblastoma stroma exhibited the phosphorylated (activated) form of ERK. CONCLUSIONS: These results suggest an interplay involving growth factors MMPs and TIMPs that may contribute to ameloblastoma behaviour. Signals generated by this molecular network would be transduced by ERK 1/2 pathway.

Progesterone decreases ovarian cancer cells migration and invasion.

The progression of cancer depends on the interaction between the cells and their microenvironment. Progesterone is a steroid and progestogen sex hormone produced by the corpus luteum, which is a transitory endocrine gland in female mammals and prepares the endometrium for implantation. Also, progesterone is involved in antitumorigenic process in different types of cancer. Our goal is to investigate the role of progesterone in cell invasion and migration. Ovarian cells were treated with different concentrations of progesterone. 500 nM or 1 µM progesterone decreased the migration of the cells in 24 h or less without affecting the viability. Immunoblot showed that treatment with 1 µM progesterone decreased the phosphorylated forms of Src and FAK, and the cells were less polarized. Our results suggest that progesterone interferes with migration and invasion of ovarian cells. Inhibitory experiments inferred the progesterone receptor playing a role in migration and invasion. Decreased phosphorylation of molecules involved in these processes was also found.

High type I collagen density fails to increase breast cancer stem cell phenotype.

Breast cancer is a highly frequent and lethal malignancy which metastasis and relapse frequently associates with the existence of breast cancer stem cells (CSCs). CSCs are undifferentiated, aggressive and highly resistant to therapy, with traits modulated by microenvironmental cells and the extracellular matrix (ECM), a biologically complex and dynamic structure composed mainly by type I collagen (Col-I). Col-I enrichment in the tumor-associated ECM leads to microenvironment stiffness and higher tumor aggressiveness and metastatic potential. While Col-I is also known to induce tumor stemness, it is unknown if such effect is dependent of Col-I density. To answer this question, we evaluated the stemness phenotype of MDA-MB-231 and MCF-7 human breast cancer cells cultured within gels of varying Col-I densities. High Col-I density increased CD44+CD24- breast cancer stem cell (BCSC) immunophenotype but failed to potentiate Col-I fiber alignment, cell self-renewal and clonogenicity in MDA-MB-231 cells. In MCF-7 cells, high Col-I density decreased total levels of variant CD44 (CD44v). Common to both cell types, high Col-I density induced neither markers related to CSC nor those related with mechanically-induced cell response. We conclude that high Col-I density per se is not sufficient to fully develop the BCSC phenotype.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment.

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted Hs578T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta1 integrin.

We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.

Secretion of collagen I and tenascin is modulated by laminin-111 in 3D culture of human adenoid cystic carcinoma cells.

Adenoid cystic carcinoma is a frequent malignant salivary gland neoplasm with high levels of recurrence and metastasis. This neoplasm expresses prominent extracellular matrix (ECM). We are studying regulatory mechanisms underlying secretion of ECM molecules in adenoid cystic carcinoma. We have previously demonstrated that laminin modulates the phenotype of a human adenoid cystic carcinoma (CAC2) cell line. Thus, this molecule would be a good candidate to regulate secretion of ECM molecules in these cells. Here we analysed the role played by laminin-111 [formerly laminin-1; Aumailley et al. (2005). Matrix Biol. 24, 326] stimulating secretory activity of CAC2 cells. Three-dimensional cultures of cells in laminin-111 (treated) or agarose (controls) were studied by light and electron microscopy. Ultrastructural analysis of CAC2 cells grown within laminin-111 showed pseudocysts filled with secretory-like material. Cells exhibited prominent and dilated endoplasmic reticulum and coated and uncoated vesicles. Ultrastructural findings suggested that laminin-111 induced secretory activity in CAC2 cells. We further investigated this point by light microscopy, immunofluorescence and confocal microscopy. Histochemistry showed periodic acid-Schiff (PAS)-positive diastase-resistant material in CAC2 cells treated by laminin-111. This material could represent laminin-induced secretion of ECM molecules. We searched for collagen I and tenascin in CAC2 cells treated by laminin-111. Confocal microscopy and immunoblot showed that laminin-111 enhanced secretion of collagen I and tenascin in CAC2 cells. We suggest that laminin-111 modulates secretion of collagen I and tenascin in cells derived from human adenoid cystic carcinoma.

Technical challenges of working with extracellular vesicles.

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies.

Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.

Progesterone acts via the progesterone receptor to induce adamts proteases in ovarian cancer cells.

BACKGROUND: Ovarian carcinomas, usually associated with sex hormones dysregulation, are the leading cause of gynecological neoplastic death. In normal ovaries, hormones play a central role in regulating cell proliferation, differentiation, and apoptosis. On the other hand, hormonal alterations also play a variety of roles in cancer. Stimulation by sex hormones potentially affects gene expression, invasiveness, cell growth and angiogenesis. Proteases of the "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) family are secreted by different cell types and become involved in collagen processing, cleavage of the proteoglycan matrix, and angiogenesis. We evaluated whether sex hormones affect ADAMTS 1 and 4 expression in ovarian cancer cells. METHODS: We analysed mRNA and protein levels in human ovarian tumor cells with different degrees of malignancy, NIH-OVCAR-3 and ES-2, that were treated or not with estrogen, testosterone and progesterone. RESULTS: Our results suggest that progesterone increases ADAMTS protein and mRNA levels in the lysates from ES-2 cells, and it increases ADAMTS protein in the lysates and conditioned media from NIH-OVCAR-3. Progesterone effects were reversed by RU486 treatment. CONCLUSION: We conclude that progesterone acts via the progesterone receptor to modulate ADAMTS 1 and 4 levels in ovarian cancer cell lines.