Fetuin-A triggers the secretion of a novel set of exosomes in detached tumor cells that mediate their adhesion and spreading

Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells. The data show that in the absence of fetuin-A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin-A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin-A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression.     

Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.     

Triiodothyronine induces a transferable factor which suppresses TSH secretion in cultured mouse thyrotropic tumor cells

Modulation of TSH release from mouse thyrotropic tumor cells was studied. T₃ (1 nM) inhibited basal TSH release, while 6 nM T₃ blocked TRH-induced TSH release. Prior exposure of cells to actinomycin or cycloheximide prevented T₃ from suppressing basal and TRH-induced TSH release. The TSH-suppressive activity from T₃-treated cells was extracted and exposure of untreated thyrotropic cells to this material resulted in suppression of TSH release. The data suggest that T₃ suppression of TSH is mediated by formation of an inhibitory protein in thyrotropic cells.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

Kinetics of adhesion and spreading of animal cells

The adhesion and spreading of cells at surfaces are well established phenomena which have been extensively observed using light or electron microscopy. The kinetics of both processes can be quantitatively measured by allowing the cells to attach themselves to the surface of a planar waveguide allows the number of cells per unit area and a parameter uniquely characterizing their shape, such as the area in contact with the surface, to be determined. Cells suspended in nutrient medium or pure phosphate buffer were allowed to attach to and spread on a metal oxide surface or a layer of fibronectin, and the kinetics of attachment and spreading have been measured. Attachment kinetics are the same in all cases, but spreading on the metal oxide requires nutrient medium, without which it does not occur. Spreading of cells attached to a layer of fibronectin in the absence of nutrient medium occurs at about a tenth of the rate of cells spreading on metal oxide in the presence of nutrient. (c) 1994 John Wiley & Sons, Inc.     

Loss of cellular adhesion to matrix induces p53-independent expression of PTEN tumor suppressor

Background  

The tumor suppressor gene PTEN has been found mutated in many types of advanced tumors. When introduced into tumor cells that lack the wild-type allele of the gene, exogenous PTEN was able to suppress their ability to grow anchorage-independently, and thus reverted one of the typical characteristics of tumor cells. As these findings indicated that PTEN might be involved in the regulation of anchorage-dependent cell growth, we analyzed this aspect of PTEN function in non-tumor cells with an anchorage-dependent phenotype.     

Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity- purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.     

Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.

Very late Ag (VLA)-3, VLA-4, and VLA-5, belonging to the beta-1 subfamily of integrins, have been recently identified as receptors for different binding regions of fibronectin (FN). We have detected VLA-4 and VLA-5, but not VLA-3, on fresh CD3-, CD16+, CD56+ human NK cells by flow cytometry and immunochemical analyses using mAb directed against beta-1, alpha-3, alpha-4, and alpha-5 subunits. Binding assays, performed on FN-coated plates, showed that NK cells specifically adhere to FN and their binding capacity is increased by MgCl2 but not by CaCl2. Using as inhibitory probes a polyclonal antibody against the beta-1 chain of the human FN receptor, the synthetic peptide GRGDSP, which is able to inhibit cellular adhesion mediated by VLA-5, the CS1 fragment, which contains the principal adhesion site in the IIICS domain recognized by VLA-4, and functional mAb directed against alpha-4 or alpha-5 subunits, we show that both VLA-4 and VLA-5 mediate the adhesion of human NK cells to FN. The expression of these integrin receptors may be relevant for NK interaction with extracellular matrix components and other cell types.     

Curcumin reverses breast tumor exosomes mediated immune suppression of NK cell tumor cytotoxicity

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin-proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.     

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8⁺ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4⁺ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.     

Fc-receptor cross-linking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes

In this study it was demonstrated that cross-linking of FcR on human monocytes induces the secretion of the cytotoxic and immunoregulatory cytokine TNF. Both soluble and insoluble immune complexes, solid-phase antibody and antibody-coated phagocytizable particles were used to cross-link FcR on monocytes. It was observed that monocytes secreted large amounts of TNF in each of these instances. Kinetic studies performed with soluble immune complexes showed that TNF was secreted very rapidly, e.g., within 2 h after addition of immune complexes to monocytes. These findings are relevant for the understanding of FcR-mediated immune responses by monocytes and macrophages, for example antibody-dependent cellular cytotoxicity and phagocytosis, and for disease states associated with circulating or tissue-fixed immune complexes.     

S-Allylcysteine reduces breast tumor cell adhesion and invasion

Previous studies show that aqueous garlic extract and its derivatives (e.g. S-allylcysteine [SAC]) prevent carcinogen-induced breast tumorigenesis. However, investigations testing the effect of SAC on later stages of breast tumorigenesis and/or metastasis have produced mixed results. Here we show that SAC significantly reduced anchorage-dependent and -independent growth of MDA-MB-231 breast tumor cells in a dose- and time-dependent fashion, and sub-lethal SAC-treatment altered mammary tumor cell adhesion and invasion through components of the extracellular matrix. We provide evidence to suggest increased expression of E-cadherin and reduced MMP-2 expression and activity are partially responsible for inhibition of mammary tumor cell invasion by SAC. Because E-cadherin and MMP-2 are important in cancer metastasis, these results suggest a link between SAC induction of E-cadherin and reduction of MMP2 activity with the inhibition of cell motility and invasion; thus providing evidence that events leading to breast cancer metastasis are repressed by sub-lethal SAC-treatment.     

Perturbation of cellular calcium induces secretion of luminal ER proteins

The endoplasmic reticulum (ER) contains a family of luminal proteins (reticuloplasmins) that are normally excluded from the secretory pathway. However, reticuloplasmins are efficiently secreted when murine fibroblasts are treated with calcium ionophores. The secreted and cellular forms of endoplasmin are clearly distinguishable on the basis of gel mobility and endoglycosidase H sensitivity. Reticuloplasmin secretion leads to the depletion of the proteins from the ER and their accumulation in the Golgi apparatus. The stress response to calcium ionophore induces reaccumulation of reticuloplasmins in the ER and suppresses their secretion. Secretion is also associated with changes in the structure and distribution of the ER. These observations show that perturbation of cellular calcium levels leads to the breakdown of the mechanism for ER retention of reticuloplasmins and suggest a role for calcium ions in their sorting from secretory proteins.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.     

Reduced expression of neural cell adhesion molecule induces metastatic dissemination of pancreatic beta tumor cells

As in the development of many human cancers, in a transgenic mouse model of beta-cell carcinogenesis (Rip1Tag2), expression of neural cell adhesion molecule (NCAM) changes from the 120-kDa isoform in normal tissue to the 140/180-kDa isoforms in tumors. NCAM-deficient RiplTag2 mice, generated by crossing Rip1Tag2 mice with NCAM knockout mice, develop metastases, a tumor stage that is not seen in normal Rip1Tag2 mice. In contrast, overexpression of NCAM 120 in NCAM-deficient Rip1Tag2 mice prevents tumor metastasis. The results indicate that the loss of NCAM-mediated cell adhesion is one rate-limiting step in the actual metastatic dissemination of beta tumor cells.     

Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid-derived suppressor cells which promote primary tumor growth

Purpose

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA).

Experimental design

Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15⁺CD11b⁺) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b⁺Gr-1⁺) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1⁺CD11b⁺), effector T cells, and tumor cytokine levels.

Results

Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8⁺ T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10.

Conclusions

MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.     

Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The α_vβ₃ integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of α_vβ₃ integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, α_vβ₃ integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.