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Background Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. Results We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. Conclusions Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.

Non-coding RNAs (ncRNAs) contribute to the regulation of gene expression. By acting as competing endogenous RNA (ceRNA), long non-coding RNAs (lncRNAs) hijack microRNAs (miRNAs) and inhibit their ability to bind their coding targets. Viral miRNAs can compete with and target the same transcripts as human miRNAs, shifting the balance in networks associated with multiple cellular processes and diseases. Epstein-Barr virus (EBV) is an example of how a subset of viral coding RNA and non-coding RNAs can cause deregulation of human transcripts and contribute to the development of EBV-associated malignancies. EBV non-coding transforming genes include lncRNAs (i.e circular RNAs), and small ncRNAs (i.e. miRNAs). Among the latter, most ongoing research has focused on miR-BARTs whereas target many genes associated with apoptosis and epithelial-mesenchymal transition, in EBV-associated gastric cancer (GC). In this review, we propose to include the interactions between EBV ncRNAs human transcripts in the hypothesis known as "competitive viral and host RNAs". These interactions may shift the balance in biological pathways such as apoptosis and epithelial-mesenchymal transition in EBV-associated gastric cancer.

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

Background. Nine percent of Gastric Cancer (GC) is associated with Epstein-Barr virus (EBV-associated GC), with significant variations in the global prevalence rates. Emerging evidence shows that, in addition to cellular miRNAs, viral miRNAs can also bind human mRNAs. EBV miRNAs are examples of this binding and are associated with oncogenic capacities of EBV. ebv-miR-BART5-5p shares the exact seed sequence with hsa-miR-18a-5p. However, common mRNA targets have not been fully described. The aim of this study is to determine predicted targets shared by both miRNAs in EBV-associated GC. Methodology. By in house method we identified predicted mRNA targets for ebv-miR-BART5-5p and hsa-miR-18a-5p. These targets were queried in published results from the supplementary files of the STAD TCGA study and miRwalk 2.0 software. Validation was performed in 7 EBV-associated GC (cases) and 6 EBV-negative GC (controls) samples by miQ-PCR (ebv-miR-BART5-5p and hsa-miR-18a-5p) and qRT-PCR (predicted targets). Results. We identified 25 downregulated and 11 upregulated mRNAs in EBV-associated GC cases compared with EBV-negative tumors in validated databases. Interestingly CDH1, which has been associated with diffuse and hereditary GC, was among the predicted targets by miRwalk 2.0. In clinical samples of EBV-associated GC, ebv-miR-BART5-5p and hsa-miR-18a-5p were overexpressed compared with control samples (p=0.0253 and p=0.0205, respectively). Furthermore, CDH1 was downregulated in EBV-associated tumors (p=0.0290). Conclusion: We identified a common human mRNA target, the CDH1 transcript for ebv-miR-BART5-5p and hsa-miR-18a-5p in EBV-associated GC. An additive effect can be suggested between both miRNAs, which may explain the unique downregulation of CDH1 in EBV-associated GC. This work warrants future validation in vitro, as well as in a larger cohort of EBV-associated GC. Grant support: CONICYT-FONDAP-15130011, Fondecyt Regular-1191928 and Fondecyt PostDoc-3201028 Citation Format: Keila Torres, Natalia Landeros, Ignacio Wichmann, Allan Peñaloza-Otárola, Iva Polakovicova, Vinicius Maracaja-Coutinho, Maria Jose Maturana, Franz Villaroel-Espindola, Alejandro Corvalan. An additive effect of ebv-miR-BART5-5p and hsa-miR18a-5p in the downregulation of CDH1 transcript in Epstein-Barr virus associated gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1400.

Background: Gastric cancer (GC) is the third cause of cancer death in the world. In Latin America, GC is one of the leading causes of death, particularly in Chile. E-cadherin has been classified as a tumor suppressor gene that participates in cell adhesion. Reduction or loss of E-cadherin expression could promote tumor progression particularly in the sporadic diffuse subtype of GC (SDGC). In addition, inactivation germline mutations of CDH1 (E-cadherin gene) have been linked to the hereditary diffuse gastric cancer (HDGC) syndrome. Methods: We evaluated the expression of E-cadherin by Immunohistochemistry (IHC) in SDGC (n=27) and HDGC (n=22) cohorts. We also analyzed, by Sanger sequencing the full coding region of CDH1 in both cohorts. To identified miRNA-CDH1 interactions, an in silico analysis by mirWalk 2.0 software (5 or more algorithms) was performed. To validated in silico findings, 10 paired tumor and normal matched fresh gastric tissues were analyzed by miQ-PCR (miRNA) and qRT-PCR assay (CDH1 transcript) were performed. Results: The expression analysis of E-cadherin showed loss of expression in 13/27 (48%) SDGC and in 19/22 (90%) HDGC cases, respectively. The decrease of E-cadherin expression was significantly higher in the hereditary cohort (p=0.002, χ2: 9.12, 95%CI:13.0-63.3). Exome sequence analysis of the full coding region of CDH1 identified 3 and 9 mutations in SDGC and HDGC, respectively. Correlation between loss of protein expression by IHC and mutations was not found. Next, we explore the role of miRNA, as an alternative mechanism of the loss of E-cadherin protein expression. In silico analysis identified 417 miRNAs candidates with predicted binding sites. Among these candidates, all members of the polycistronic cluster miR-17/92 have seed regions against the 3'-UTR of the CDH1 transcript. Levels of expression of all 6 members of the polycistronic cluster were validated against CDH1 expression in paired normal and tumor fresh tissues of SDGC. A strong inverse correlation between the overexpression of miR-20a and downregulation of CDH1 transcript was found exclusively in tumor tissue (p= 0.0267) but not in normal counterparts. These results were validated in the STAD-TCGA cohort. Conclusions: Our results might explain the loss of expression of CDH1 in GC due to overexpression miR-20, a member of the polycistronic cluster miR-17/92. Future experimental validation of the predicted seed region will be necessary to confirm these findings. Grant support: CONICYT-FONDAP-15130011, Fondecyt-1191928 Citation Format: Natalia Landeros, Maria A. Alarcon, Graciela Molina, Pablo Santoro, Keila Torres, Wilda Olivares, Ignacio Wichmann, Franz Villarroel-Espindola, Iva Polakovicova, Enrique Norero, Alejandro H. Corvalan. Loss of expression of wild-type transcript of CDH1 gene is associated with overexpression of microRNA-20 in diffuse-type of gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2523.

Background: Gastric cancer (GC) is one of the leading causes of cancer death. Elucidating the molecular bases of GC might contribute to develop opportune diagnostic strategies. Reprimo-like (RPRML) is a poorly characterized member of the Reprimo gene family. The founding member of this family, Reprimo, is reported to be a putative tumor suppresser gene and candidate biomarker for GC diagnosis. We have recently reported the downregulation of RPRML expression in GC tissues compared to paired normal adjacent tissues, and that in vitro expression is regulated by DNA methylation. We hypothesize that RPRML promoter methylation may act as a non-invasive biomarker for GC. Methods: RPRML expression was evaluated by immunohistochemistry (IHC) in 91 GC cases from The Gastric Cancer Task Force (NCT03158571). Two pathologists scored staining by multiplying the proportion and the intensity of stained cells (range 0-3). Cox proportional hazard ratio was applied to determine an association between RPRML IHC score and global survival, after adjusting by stage. In addition, we investigated the effect of RPRML overexpression in GC cell behavior in vitro through the stable transfection and sorting of RPRML coupled to GFP or GFP alone in the AGS cell line. Functional assays included Ki67 immunofluorescence, colony formation, soft agar, transwell migration and wound healing assays. All experiments were performed in biological triplicate and Kruskal-Wallis statistical analysis applied. The Methylight assay was used to quantify methylated RPRML DNA copies in plasma samples from 25 GC cases (tumor bank repository BTHCUCH) and 25 healthy donors under an endoscopic surveillance program (PREVECAN). ROC curve analysis was performed to determine the cut-off value for the highest sensitivity and specificity. Results: Among 91 GC cases, RPRML expression showed a median IHC score of 0.024 (range 0-2.45). By using the median as a cut-off point, low RPRML IHC score was associated with poor overall survival of stage III and IV GC patients (HR=2.13, 95%CI:1.1-4.1, p=0.02). In vitro overexpression of RPRML inhibited cell proliferation (p=0.04) and resulted in reduced clonogenic capacity (p=0.001) and anchorage independent growth (p≤0.0001). Furthermore, RPRML overexpression retarded migration and wound healing in AGS cells. Methylation of RPRML DNA in plasma samples discriminated between GC patients and controls with an AUC of 0.66. Utilizing a cut-off value of 0.15 copies/ul, sensitivity was 56% (95%CI: 34.9 - 75.6) and specificity 72% (95%CI: 50.6 - 87.9). Conclusion: RPRML downregulation associated with poor survival of advanced stage GC patients. In accordance, ectopic overexpression of RPRML in vitro inhibited biological characteristics associated with tumor progression, suggesting that RPRML may have a tumor suppressor role in GC. Future analysis will determine if the methylation of RPRML is a candidate marker for the non-invasive detection of GC. Grants: CONICYT-FONDAP 15130011 and Fondecyt 1191928 Citation Format: Maria Alejandra Alarcon, Wilda Olivares, Andres Rodriguez, Maria Jose Maturana, Rocío Bustos, Iva Polakovicova, Miguel Cordova-Delgado, Matías Muñoz-Medel, Marcelo Garrido, Gareth I. Owen, Tomas De Mayo, Gonzalo Carrasco-Avino, Julio Amigo, Arnoldo Riquelme, Robinson Gonzalez, Carlos Barrientos, Edmundo Aravena, Franz Villarroel-Espíndola, Alejandro H. Corvalan. Clinical, biological and translational significance in gastric cancer of reprimo-like gene, an uncharacterized member of the reprimo gene family [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2437.

Emerging evidence suggests that chronic inflammation caused by pathogen infection is connected to the development of various types of cancer. It is estimated that up to 20% of all cancer deaths is linked to infections and inflammation. In gastric cancer, such triggers can be infection of the gastric epithelium by either Helicobacter pylori (H. pylori), a bacterium present in half of the world population; or by Epstein-Barr virus (EBV), a double-stranded DNA virus which has recently been associated with gastric cancer. Both agents can establish lifelong inflammation by evolving to escape immune surveillance and, under certain conditions, contribute to the development of gastric cancer. Non-coding RNAs, mainly microRNAs (miRNAs), influence the host innate and adaptive immune responses, though long non-coding RNAs and viral miRNAs also alter these processes. Reports suggest that chronic infection results in altered expression of host miRNAs. In turn, dysregulated miRNAs modulate the host inflammatory immune response, favoring bacterial survival and persistence within the gastric mucosa. Given the established roles of miRNAs in tumorigenesis and innate immunity, they may serve as an important link between H. pylori- and EBV-associated inflammation and carcinogenesis. Example of this is up-regulation of miR-155 in H. pylori and EBV infection. The tumor environment contains a variety of cells that need to communicate with each other. Extracellular vesicles, especially exosomes, allow these cells to deliver certain type of information to other cells promoting cancer growth and metastasis. Exosomes have been shown to deliver not only various types of genetic information, mainly miRNAs, but also cytotoxin-associated gene A (CagA), a major H. pylori virulence factor. In addition, a growing body of evidence demonstrates that exosomes contain genetic material of viruses and viral miRNAs and proteins such as EBV latent membrane protein 1 (LMP1) which are delivered into recipient cells. In this review, we focus on the dysregulated H. pylori- and EBV-associated miRNAs while trying to unveil possible causal mechanisms. Moreover, we discuss the role of exosomes as vehicles for miRNA delivery in H. pylori- and EBV-related carcinogenesis.

Background Multiple aberrant microRNA expression has been reported in gastric cancer. Among them, microRNA-335-5p (miR-335), a microRNA regulated by DNA methylation, has been reported to possess both tumor suppressor and tumor promoter activities. Results Herein, we show that miR-335 levels are reduced in gastric cancer and significantly associate with lymph node metastasis, depth of tumor invasion, and ultimately poor patient survival in a cohort of Amerindian/Hispanic patients. In two gastric cancer cell lines AGS and, Hs 746T the exogenous miR-335 decreases migration, invasion, viability, and anchorage-independent cell growth capacities. Performing a PCR array on cells transfected with miR-335, 19 (30.6%) out of 62 genes involved in metastasis and tumor invasion showed decreased transcription levels. Network enrichment analysis narrowed these genes to nine (PLAUR, CDH11, COL4A2, CTGF, CTSK, MMP7, PDGFA, TIMP1, and TIMP2). Elevated levels of PLAUR, a validated target gene, and CDH11 were confirmed in tumors with low expression of miR-335. The 3′UTR of CDH11 was identified to be directly targeted by miR-335. Downregulation of miR-335 was also demonstrated in plasma samples from gastric cancer patients and inversely correlated with DNA methylation of promoter region (Z = 1.96, p = 0.029). DNA methylation, evaluated by methylation-specific PCR assay, was found in plasma from 23 (56.1%) out of 41 gastric cancer patients but in only 9 (30%) out of 30 healthy donors (p = 0.029, Pearson’s correlation). Taken in consideration, our results of the association with depth of invasion, lymph node metastasis, and poor prognosis together with functional assays on cell migration, invasion, and tumorigenicity are in accordance with the downregulation of miR-335 in gastric cancer. Conclusions Comprehensive evaluation of metastasis and invasion pathway identified a subset of associated genes and confirmed PLAUR and CDH11, both targets of miR-335, to be overexpressed in gastric cancer tissues. DNA methylation of miR-335 may be a promissory strategy for non-invasive approach to gastric cancer.

Mast cells and basophils (MCs/Bs) play a crucial role in type I allergy, as well as in innate and adaptive immune responses. These cells mediate their actions through soluble mediators, some of which are targeted therapeutically by, for example, H1- and H2-antihistamines or cysteinyl leukotriene receptor antagonists. Recently, considerable progress has been made in developing new drugs that target additional MC/B mediators or receptors, such as serine proteinases, histamine 4-receptor, 5-lipoxygenase-activating protein, 15-lipoxygenase-1, prostaglandin D2, and proinflammatory cytokines. Mediator production can be abrogated by the use of inhibitors directed against key intracellular enzymes, some of which have been used in clinical trials (eg, inhibitors of spleen tyrosine kinase, phosphatidylinositol 3-kinase, Bruton tyrosine kinase, and the protein tyrosine kinase KIT). Reduced MC/B function can also be achieved by enhancing Src homology 2 domain-containing inositol 5' phosphatase 1 activity or by blocking sphingosine-1-phosphate. Therapeutic interventions in mast cell-associated diseases potentially include drugs that either block ion channels and adhesion molecules or antagonize antiapoptotic effects on B-cell lymphoma 2 family members. MCs/Bs express high-affinity IgE receptors, and blocking their interactions with IgE has been a prime goal in antiallergic therapy. Surface-activating receptors, such as CD48 and thymic stromal lymphopoietin receptors, as well as inhibitory receptors, such as CD300a, FcγRIIb, and endocannabinoid receptors, hold promising therapeutic possibilities based on preclinical studies. The inhibition of activating receptors might help prevent allergic reactions from developing, although most of the candidate drugs are not sufficiently cell specific. In this review recent advances in the development of novel therapeutics toward different molecules of MCs/Bs are presented.

Mast cells play crucial roles in both innate and adaptive arms of the immune system. Along with basophils, mast cells are essential effector cells for allergic inflammation that causes asthma, allergic rhinitis, food allergy and atopic dermatitis. Mast cells are usually increased in inflammatory sites of allergy and, upon activation, release various chemical, lipid, peptide and protein mediators of allergic reactions. Since antigen/immunoglobulin E (IgE)-mediated activation of these cells is a central event to trigger allergic reactions, innumerable studies have been conducted on how these cells are activated through cross-linking of the high-affinity IgE receptor (FceRI). Development of mature mast cells from their progenitor cells is under the influence of several growth factors, of which the stem cell factor (SCF) seems to be the most important. Therefore, how SCF induces mast cell development and activation via its receptor, KIT, has been studied extensively, including a cross-talk between KIT and FceRI signaling pathways. Although our understanding of the signaling mechanisms of the FceRI and KIT pathways is far from complete, pharmaceutical applications of the knowledge about these pathways are underway. This review will focus on recent progresses in FceRI and KIT signaling and chemotaxis.

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FceRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FceRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FceRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FceRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.