Renal cell carcinoma-derived exosomes deliver lncARSR to induce macrophage polarization and promote tumor progression via STAT3 pathway

Tumor-derived exosomes play a pivotal role in regulating tumor progression by mediating crosstalk between tumor cells and immune cells such as macrophages within the tumor microenvironment. Macrophages can adopt two distinct polarization statuses and switch between M1 or M2 activation phenotypes in response to the different external stimuli. However, the role of tumor derived exosomes in the macrophage phenotypic switch and tumor development have not been elucidated in renal cell carcinoma (RCC). Here we found that high macrophage infiltration was associated with worse prognosis in RCC patients, therefore we propose our hypothesis that RCC derived exosomes might directly influence macrophage polarization and thus promote tumor progression. Both cell-based in vitro models and orthotopic transplantation in vivo tumor models were constructed and ELISA, flow cytometry, and macrophage functional studies were performed to investigate whether and how RCC-derived exosomes regulate macrophage polarization and tumor growth. The results found that these exosomes promote macrophage polarization, cytokine release, phagocytosis, angiogenesis, and tumor development. Further study revealed high amount of a recently discovered lncRNA called lncARSR in RCC-derived exosomes. Overexpression of lncARSR induced phenotypic and functional changes of macrophages in vitro and promoted tumor growth in vivo, while knockdown of lncARSR by siRNA disrupted the exosomes-mediated macrophage polarization. LncARSR interacts directly with miR-34/miR- 449 to increase STAT3 expression and mediate macrophage polarization in RCC cells. Together, RCC-derived exosomes facilitate the development of tumor through inducing macrophage polarization via transferring lncARSR, suggesting that RCC-derived exosomes, lncARSR and STAT3 are the potential therapeutic targets for treatment of RCC.     

Pregnancy-associated exosomes and their modulation of T cell signaling

Exosome release by viable cells is a feature of activated cell types, including tumors, fetal cells, and cells of the immune system. Exosomes critically regulate immune activation, by mediating activation-induced cell death. Fetal cells may mimic these events to selectively delete reactive lymphocytes. In this study the presence and composition of placenta-derived exosomes are demonstrated in the maternal circulation along with their consequences on T cell activation markers. For all pregnant patients, exosomes were isolated from sera obtained between 28 and 30 wk gestation. For pregnant women, subsequently delivering at term, circulating levels of placental exosomes were 1.8 times greater than those delivering preterm (p < 0.0001). Exosomes isolated from pregnancies subsequently delivering at term expressed significantly higher levels of biologically active components, including Fas ligand (FasL) and HLA-DR, than those from pregnancies delivering preterm. Standardizing for protein concentrations, exosomes from term-delivering pregnancies exhibited greater suppression of CD3-zeta and JAK3 than those delivering preterm. The suppression of CD3-zeta and JAK3 correlated with exosome expression levels of FasL (r2= 0.92 and r2= 0.938, respectively). Fractionation of exosomes from term-delivering pregnancies by continuously eluting electrophoresis indicated that intact 42 kD FasL and an unidentified 24-kDa protein were associated with CD3-zeta suppression. Our results demonstrated that exosomes from pregnancies ultimately delivering at term are present at significantly greater concentrations than those from pregnancies delivering preterm; however, exosomes from term-delivering pregnancies also exhibit significantly greater suppression of CD3-zeta and JAK3.     

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.     

Impaired Fas signaling pathway is involved in defective T cell apoptosis in autoimmune murine arthritis

Proteoglycan (PG)-induced arthritis (PGIA) is a novel autoimmune murine model for rheumatoid arthritis induced by immunization with cartilage PG in susceptible BALB/c mice. In this model, hyperproliferation of peripheral CD4(+) T cells has been observed in vitro with Ag stimulation, suggesting the breakdown of peripheral tolerance. Activation-induced cell death (AICD) is a major mechanism for peripheral T cell tolerance. A defect in AICD may result in autoimmunity. We report in this study that although CD4(+) T cells from both BALB/c and B6 mice, identically immunized with human cartilage PG or OVA, express equally high levels of Fas at the cell surface, CD4(+) T cells from human cartilage PG-immunized BALB/c mice, which develop arthritis, fail to undergo AICD. This defect in AICD in PGIA may lead to the accumulation of autoreactive Th1 cells in the periphery. The impaired AICD in PGIA might be ascribed to an aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein, which precludes caspase-8 activation at the death-inducing signaling complex, and subsequently suppresses the caspase cascade initiated by Fas-Fas ligand interaction. Moreover, this aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein may also mediate TCR-induced hyperproliferation of CD4(+) T cells from arthritic BALB/c mice. Our data provide the first insight into the molecular mechanism(s) of defective AICD in autoimmune arthritis.     

Amphiregulin exosomes increase cancer cell invasion

Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-α (TGFα), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGFα or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGFα or HB-EGF. An average of 24?AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1?colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.     

Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

N-acetylglucosaminyltransferase V mediates cell migration and invasion of mouse mammary tumor cells 4TO7 via RhoA and Rac1 signaling pathway

In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behavior. Using small interfering RNA against GnT-V, we found that the expression of GnT-V and β1,6GlcNAc branching were significantly reduced which was particularly accompanied by the arrest in both cell migration and invasion as compared to the negative control. Moreover, the suppressed GnT-V expression by siRNA technique inactivated the signaling molecules including Rac1, cofilin, Erk and Akt, and activated RhoA levels in cells lacking GnT-V, but revealed no impact on Cdc42 activity. All these notions disclose for the first time that GnT-V and β1, 6GlcNAc branching mediate the cell migration and invasion in Rac1-positive and RhoA-negative regulatory manners. Yunxue Zhao and Jing Li contributed equally to this work.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

C6orf106 accelerates pancreatic cancer cell invasion and proliferation via activating ERK signaling pathway

Molecular and Cellular Biochemistry - The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I...     

MiR-126 suppresses colon cancer cell proliferation and invasion via inhibiting RhoA/ROCK signaling pathway

Recent data strongly suggests the profound role of miRNAs in cancer progression. Here, we showed miR-126 expression was much lower in HCT116, SW620 and HT-29 colon cancer cells with highly metastatic potential and miR-126 downregulation was more frequent in colorectal cancers with metastasis. Restored miR-126 expression inhibited HT-29 cell growth, cell-cycle progression and invasion. Mechanically, microarray results combined with bioinformatic and experimental analysis demonstrated miR-126 exerted cancer suppressor role via inhibiting RhoA/ROCK signaling pathway. These results suggest miR-126 function as a potential tumor suppressor in colon cancer progression and miR-126/RhoA/ROCK may be a novel candidate for developing rational therapeutic strategies.     

GATA1 promotes colorectal cancer cell proliferation,migration and invasion via activating AKT signaling pathway

Molecular and Cellular Biochemistry - We aimed to explore whether specific high-sucrose intake in older female rats affects myocardial electrical coupling protein, connexin-43 (Cx43), protein...     

Alternate estrogen receptors promote invasion of inflammatory breast cancer cells via non-genomic signaling

Although Inflammatory Breast Cancer (IBC) is a rare and an aggressive type of locally advanced breast cancer with a generally worst prognosis, little work has been done in identifying the status of non-genomic signaling in the invasiveness of IBC. The present study was performed to explore the status of non-genomic signaling as affected by various estrogenic and anti-estrogenic agents in IBC cell lines SUM149 and SUM190. We have identified the presence of estrogen receptor α (ERα) variant, ERα36 in SUM149 and SUM190 cells. This variant as well as ERβ was present in a substantial concentration in IBC cells. The treatment with estradiol (E2), anti-estrogenic agents 4-hydroxytamoxifen and ICI 182780, ERβ specific ligand DPN and GPR30 agonist G1 led to a rapid activation of p-ERK1/2, suggesting the involvement of ERα36, ERβ and GPR30 in the non-genomic signaling pathway in these cells. We also found a substantial increase in the cell migration and invasiveness of SUM149 cells upon the treatment with these ligands. Both basal and ligand-induced migration and invasiveness of SUM149 cells were drastically reduced in the presence of MEK inhibitor U0126, implicating that the phosphorylation of ERK1/2 by MEK is involved in the observed motility and invasiveness of IBC cells. We also provide evidence for the upregulation of p-ERK1/2 through immunostaining in IBC patient samples. These findings suggest a role of non-genomic signaling through the activation of p-ERK1/2 in the hormonal dependence of IBC by a combination of estrogen receptors. These findings only explain the failure of traditional anti-estrogen therapies in ER-positive IBC which induces the non-genomic signaling, but also opens newer avenues for design of modified therapies targeting these estrogen receptors.     

Effect of Ezrin on regulating trophoblast cell invasion via PKC signaling pathway in unexplained recurrent spontaneous abortion

Trophoblast cells are the most important cells in early pregnancy and their invasion are essential to the establishment and maintenance of pregnancy. Inadequate trophoblast cell invasion has been closely associated with several pregnancy-associated diseases including recurrent spontaneous abortion (RSA). Ezrin is an actin-associated protein, known as a marker for carcinogenesis and metastasis in solid tumors, has been proposed to play a role in the formation of microvilli in the early embryo. To further characterize its function in early pregnancy, we explored the expression of Ezrin in the trophoblast cells in early pregnancy. In this study, compared with normal pregnant women, we demonstrated that the expression of Ezrin and phosphorylated Ezrin decreased in the trophoblast cells in unexplained RSA (URSA) patients, and knockdown of Ezrin expression could suppress the invasiveness of trophoblast cells significantly. Various studies indicated that the phosphorylation of Ezrin on C-terminal threonine residue (T567) is a key event in the regulation of its activity. Our further exploration indicated that Ezrin was activated via PKC pathway. Furthermore, inhibition of the PKC pathway by a specific inhibitor suppressed invasiveness of Bewo cells. On the other hand, activation of the PKC pathway could increase the relative capacity of trophoblast cell invasion, while Ezrin knockdown reversed PKC activation induced cell invasion. These findings might provide a new fundamental mechanism for successful pregnancy and new diagnostic and therapeutic target for RSA.     

Necrotic death pathway in Fas receptor signaling

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.     

T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor

Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4(+) activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4(+) T cells enhance cholesterol accumulation in cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-alpha enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor.     

Chemokine-containing exosomes are released from heat-stressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine

Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.     

Microenvironmental innate immune signaling and cell mechanical responses promote tumor growth

1. Download : Download high-res image (106KB)
2. Download : Download full-size image

     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

The signaling network of tumor invasion

The ability of a cell to invade its surroundings is an important hallmark of malignant tumors and results from aberrant cell signaling mechanisms. The signal transduction that leads to tumor invasion can be broken down into major pathways. Even though the pathway systems are distinct in themselves, none of these pathways operate independently when it comes to transmitting signals that culminate in an invasive phenotype. That is, the malignant change in one receptor not only leads to malignant changes directly downstream but can also affect the molecules of many other pathways. Three major pathway systems involved in tumor invasion are discussed in this review: the integrin system, the insulin-like growth factor system, and the Rho family GTPases. Here we see that although the individual signaling systems can each contribute to invasion, each system is networked to others and should not be considered isolated. Each system is first reviewed as independent contributors to an invasive phenotype and then discussed in the context of interacting pathways that collectively result in tumor invasion.