Exosomes are emerging as important mediators of the cross-talk between tumor cells and the microenvironment. However, the mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic cancer remain largely unknown. Here, we found that hypoxic exosomes derived from pancreatic cancer cells activate macrophages to the M2 phenotype in a HIF1a or HIF2a-dependent manner, which then facilitates the migration, invasion, and epithelial-mesenchymal transition of pancreatic cancer cells. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we discovered that miR-301a-3p was highly expressed in hypoxic pancreatic cancer cells and enriched in hypoxic pancreatic cancer cell-derived exosomes. Circulating exosomal miR-301a-3p levels positively associated with depth of invasion, lymph node metastasis, late TNM stage, and poor prognosis of pancreatic cancer. Hypoxic exosomal miR-301a-3p induced the M2 polarization of macrophages via activation of the PTEN/PI3Kγ signaling pathway. Coculturing of pancreatic cancer cells with macrophages in which miR-301a-3p was upregulated or treated with hypoxic exosomes enhanced their metastatic capacity. Collectively, these data indicate that pancreatic cancer cells generate miR-301a-3p-rich exosomes in a hypoxic microenvironment, which then polarize macrophages to promote malignant behaviors of pancreatic cancer cells. Targeting exosomal miR-301a-3p may provide a potential diagnosis and treatment strategy for pancreatic cancer. These findings identify an exosomal miRNA critical for microenvironmental cross-talk that may prove to be a potential target for diagnosis and treatment of pancreatic cancer. http://cancerres.aacrjournals.org/content/canres/78/16/4586/F1.large.jpg .

Long non-coding RNAs (lncRNAs) play key roles in the progression and metastasis of some carcinomas. We previously showed that the expression of lncRNA H19 (H19) was higher in gastric cancer (GC) tissues than that in paired noncanerous tissues. However, the underlying mechanisms remain unclear.In this study, H19/miR-675 knockdown models in the MKN45 cell line and ectopic expression models in the SGC7901 cell line were established, and a co-expression network of H19 was generated to identify target genes by RIP and DLR. The results showed that overexpression of H19 promoted the features of GC including proliferation, migration, invasion and metastasis. An H19 co-expression network identified ISM1 as a binding protein of H19, and its expression was positively correlated with that of H19. CALN1 was identified as a target gene of miR-675 and its expression was negatively correlated with that of miR-675. H19 and MiR-675 function in a similar manner. However, H19 RNA actively binds to ISM1 and miR-675 targets CALN1. These differences suggest that H19 plays other roles besides encoding miR-675 in GC. Our results suggest that the effect of H19 in GC is mediated by the direct upregulation of ISM1 and the indirect suppression of CALN1 expression via miR-675.

Background & Aims-Inflammatory bowel disease (IBD) is a multifactorial disease thought to be caused by alterations in epithelial function, innate and adaptive immunity, and luminal microbiota. The specific role of epithelial barrier function remains undefined, although increased activity of intestinal epithelial myosin light chain kinase (MLCK), which is the primary mechanism of tumor necrosis factor (TNF)-induced barrier dysfunction, occurs in human IBD. We aimed to determine whether in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis.

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

BACKGROUND & AIMS Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease (IBD). However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are incompletely understood. We investigated these processes using colitis models in mice. METHODS We induced colitis by adoptive transfer of CD4+CD45RBhi cells or administration of dextran sulfate sodium (DSS) to mice, including those deficient in tumor necrosis factor receptor (TNFR) 1, TNFR2, or the long isoform of myosin light chain kinase (MLCK). Intestinal tissues and isolated epithelial cells were analyzed by immunoblot, immunofluorescence, ELISA, and real-time PCR assays. RESULTS Induction of immune-mediated colitis by CD4+CD45RBhi adoptive transfer increased intestinal permeability; epithelial expression of claudin-2, the long isoform of MLCK, and TNFR2 (but not TNFR1); and phosphorylation of the myosin II light chain (MLC). Long MLCK upregulation, MLC phosphorylation, barrier loss, and weight loss were attenuated in TNFR2−/−, but not TNFR1−/−, recipients of wildtype CD4+CD45RBhi cells. Similarly, long MLCK−/− mice had limited increases in MLC phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of cell transfer-induced colitis. However, coincident with onset of epithelial apoptosis, colitis ultimately developed. This indicates that disease progresses via apoptosis in the absence of MLCK-dependent tight junction regulation. In support of this conclusion, long MLCK−/− mice were not protected from epithelial apoptosis-mediated, damage-dependent DSS colitis. CONCLUSIONS In immune-mediated IBD models, TNFR2 signaling increases long MLCK expression, resulting in tight junction dysregulation, barrier loss and induction of colitis. At advanced stages, colitis progresses by apoptosis and mucosal damage that results in tight junction- and MLCK-independent barrier loss. Therefore, barrier loss in immune-mediated colitis occurs via two temporally and morphologically distinct mechanisms. Differential targeting of these mechanisms may lead to improved IBD therapies.

IFN-gamma primes intestinal epithelia to respond to TNF by inducing TNFR2 expression, which in turn mediates TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the molecular mechanisms underlying the tumor-promoting properties of CAFs in gastric cancer remain unclear. Here, we show that CAFs isolated from gastric cancer produce significant amounts of interleukin-6 (IL-6). CAFs enhances the migration and EMT of gastric cancer cells through the secretion of IL-6 that activates Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in gastric cancer cells, while deprivation of IL-6 using a neutralizing antibody or inhibition of JAK/STAT3 pathway with specific inhibitor AG490 markedly attenuates these phenotypes in gastric cancer cells induced by CAFs. Moreover, silencing IL-6 expression in CAFs or inhibiting JAK2/STAT3 pathway in gastric cancer cells impairs tumor peritoneal metastasis induced by CAFs in vivo. Taken together, these results suggest that CAFs in the tumor microenvironment promote the progression of gastric cancer through IL-6/JAK2/STAT3 signaling, and IL-6 targeted therapy could be a complementary approach against gastric cancer by exerting their action on stromal fibroblasts.

BACKGROUND & AIMS-LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a TNF core family member that regulates T cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT was capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling.