Beatriz Arteta

Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages.

Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-β1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.

α2,3-Sialyltransferase ST3Gal IV promotes migration and metastasis in pancreatic adenocarcinoma cells and tends to be highly expressed in pancreatic adenocarcinoma tissues.

Sialyltransferases have received much attention recently as they are frequently up-regulated in cancer cells. However, the role played by each sialyltransferase in tumour progression is still unknown. α2,3-Sialyltransferases ST3Gal III and ST3Gal IV are involved in sialyl-Lewis(x) (SLe(x)) synthesis. Given that the role of ST3Gal III in pancreatic adenocarcinoma cells has been previously reported, in this study we have focused on investigating the role of ST3Gal IV in the acquisition of adhesive, migratory and metastatic capabilities and, secondly, in analyzing the expression of ST3Gal III and ST3Gal IV in pancreatic adenocarcinoma tissues versus control tissues. ST3Gal IV overexpressing pancreatic adenocarcinoma MDAPanc-28 cell lines were generated. They showed a heterogeneous increase in SLe(x), and enhanced E-selectin adhesion and migration. Furthermore, when injected into nude mice, increased metastasis and decreased survival were found in comparison with controls. The behaviour of MDAPanc-28 ST3Gal IV overexpressing cells in these processes was similar to the already reported MDAPanc-28 ST3Gal III overexpressing cells. Furthermore, pancreatic adenocarcinoma tissues tended to express high levels of ST3Gal III and ST3Gal IV together with other fucosyltransferase genes FUT3 and FUT6, all involved in the last steps of sialyl-Lewis(x) biosynthesis. In conclusion, both α2,3-sialyltransferases are involved in key steps of pancreatic tumour progression processes and are highly expressed in most pancreatic adenocarcinoma tissues.

Human antigen R contributes to hepatic stellate cell activation and liver fibrosis

RNA‐binding proteins (RBPs) play a major role in the control of messenger RNA (mRNA) turnover and translation rates. We examined the role of the RBP, human antigen R (HuR), during cholestatic liver injury and hepatic stellate cell (HSC) activation. HuR silencing attenuated fibrosis development in vivo after BDL, reducing liver damage, oxidative stress, inflammation, and collagen and alpha smooth muscle actin (α‐SMA) expression. HuR expression increased in activated HSCs from bile duct ligation mice and during HSC activation in vitro, and HuR silencing markedly reduced HSC activation. HuR regulated platelet‐derived growth factor (PDGF)‐induced proliferation and migration and controlled the expression of several mRNAs involved in these processes (e.g., Actin, matrix metalloproteinase 9, and cyclin D1 and B1). These functions of HuR were linked to its abundance and cytoplasmic localization, controlled by PDGF, by extracellular signal‐regulated kinases (ERK) and phosphatidylinositol 3‐kinase activation as well as ERK/LKB1 (liver kinase B1) activation, respectively. More important, we identified the tumor suppressor, LKB1, as a novel downstream target of PDGF‐induced ERK activation in HSCs. HuR also controlled transforming growth factor beta (TGF‐β)‐induced profibrogenic actions by regulating the expression of TGF‐β, α‐SMA, and p21. This was likely the result of an increased cytoplasmic localization of HuR, controlled by TGF‐β‐induced p38 mitogen‐activated protein kinase activation. Finally, we found that HuR and LKB1 (Ser428) levels were highly expressed in activated HSCs in human cirrhotic samples. Conclusion: Our results show that HuR is important for the pathogenesis of liver fibrosis development in the cholestatic injury model, for HSC activation, and for the response of activated HSC to PDGF and TGF‐β. (HEPATOLOGY 2012;56:1870–1882)

Colon Carcinoma Cell Interaction with Liver Sinusoidal Endothelium Inhibits Organ-Specific Antitumor Immunity Through Interleukin-1-Induced Mannose Receptor in Mice

Mannose receptor (ManR)‐mediated liver sinusoidal endothelial cell (LSEC) endocytosis plays a role in antigen presentation and innate immunity, but its role in hepatic metastasis is unknown. We studied ManR‐mediated endocytosis during C26 colorectal cancer cell interaction with LSECs and its implications in metastasis. Uptake of labeled ManR ligands (mannan and ovalbumin) and immunohistochemistry were used to study ManR endocytosis and expression. Several interleukin (IL)‐1 inhibitors and the cyclooxygenase‐2 (COX‐2) inhibitor celecoxib were used to analyze the role of IL‐1 and COX‐2 in ManR regulation. Anti‐mouse ManR antibodies and ManR knockout (ManR−/−) mice were used to identify ManR‐dependent mechanisms during antitumor immune response of liver sinusoidal lymphocytes (LSLs) interacting with tumor‐activated LSECs. ManR expression and endocytosis increased in tumor‐activated LSECs through a two‐step mechanism: (1) Release of COX‐2–dependent IL‐1–stimulating factors by lymphocyte function–associated antigen‐1–expressing C26 cells in response to intercellular adhesion molecule‐1 (ICAM‐1), which was expressed and secreted by tumor‐activated LSECs; and (2) widespread up‐regulation of ManR in LSECs through tumor‐induced IL‐1. In addition, LSLs that had interacted with tumor‐activated LSECs in vivo decreased their antitumor cytotoxicity and interferon (IFN)‐gamma secretion while they increased IL‐10 release ex vivo. IFN‐gamma/IL‐10 ratio also decreased in the hepatic blood from tumor‐injected mice. Immunosuppressant effects of tumor‐activated LSECs on LSLs were abrogated in both LSECs from ManR−/− mice and tumor‐activated LSECs given anti‐mouse ManR antibodies. Conclusion: ICAM‐1–induced tumor COX‐2 decreased antitumor activity during hepatic metastasis through IL‐1–induced ManR. ManR constituted a common mediator for prometastatic effects of IL‐1, COX‐2, and ICAM‐1. A rise in hepatic IFN‐gamma/IL‐10 ratio and antitumor cytotoxicity by way of ManR blockade is consistent with the antimetastatic effects of IL‐1, COX‐2, and ICAM‐1 inhibitors. These data support ManR and ManR‐stimulating factors as targets for hepatic colorectal metastasis therapy. Hepatology 2010;51:2172–2182

Kupffer cell products and interleukin 1? directly promote VLDL secretion and apoB mRNA up-regulation in rodent hepatocytes

Plasma VLDL accumulation in Gram-negative sepsis is partly ascribed to an increased hepatic VLDL production driven by pro-inflammatory cytokines. We previously showed that hepatocytes of the Kupffer cell (KC)-rich periportal area are major contributors to enhanced VLDL production in lipopolysaccharide (LPS)-injected rats. However, it remains to be established whether KC generated products directly affect the number (apoB) and composition of secreted VLDL. Using rat primary cells, we show here that hepatocytes respond to stimulation by soluble mediators released by LPS-stimulated Kupffer cells with enhanced secretion of apoB and triglycerides in phospholipid-rich VLDL particles. Unstimulated KC products also augmented the secretion of normal VLDL, doubling apoB mRNA abundance. IL-1β treatment resulted in concentration-dependent increases of hepatocyte apoB mRNA and protein secretion, increases that were greater, but not additive, when combined with IL-6 and TNF-α. Lipid secretion and MTP mRNA levels were unaffected by cytokines. In summary: (i) enhanced secretion of phospholipid-rich VLDL particles is a net hepatocyte response to LPS-stimulated KC products, which gives a clue about the local role of Kupffer cells in septic dyslipidemia induction; and (ii) pro-inflammatory cytokines act redundantly to enhance apoB secretion involving translational apoB up-regulation, but other humoral components or KC mediators are necessary to accomplish increased lipid association.

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro : Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.

Proangiogenic Implications of Hepatic Stellate Cell Transdifferentiation into Myofibroblasts Induced by Tumor Microenvironment

Hepatic stellate cells are perisinusoidal fibroblasts that transdifferentiate into myofibroblasts in response to paracrine factors released from cancer cells and cancer-activated endothelial cells. Tumor-associated myofibroblasts exhibit contractility, proliferation, production of extracellular matrix molecules and metalloproteases. They secrete soluble factors inducing proinflammatory and immune suppressant effects. Myofibroblasts are present in avascular micrometastasis prior to endothelial cell recruitment, and act as supporting stroma for tumor neoangiogenesis. In replacement-type cancer growth, the reticular arrangement of tumor-associated myofibroblasts provides a sinusoidal-type angiogenic pattern. In pushing-type cancer growth, fibrous tract-forming myofibroblasts support a portal-type angiogenic pattern. Additionally, tumor-activated myofibroblasts support cancer development via paracrine release of tumor invasion and proliferation-stimulating factors. In summary, this information suggests that the ability of cancer cells to activate hepatic stellate cells and to collaborate with myofibroblasts along the metastatic process may represent a key phenotypic property of liver-colonizing cancer cells. On the other hand, experimental anti-tumor and anti-angiogenic agents have inhibited intrametastatic recruitment and proangiogenic activities of hepatic myofibroblasts, suggesting that targeting tumorigenic effects of these cells may contribute to hepatic metastasis inhibition.

Ocoxin® oral solution slows down tumor growth in an experimental model of colorectal cancer metastasis to the liver in Balb/c mice

Liver metastatic disease is the main cause of death in colorectal cancer (CRC) patients. During metastatic spread of the disease an imbalance in the oxidative stress and inflammation plays a crucial role in tumor progression. In order to improve the efficacy of current therapies, new complementary therapeutic approaches are being analyzed including biologically active compounds with low side effects. The anti-inflammatory and anti-oxidant properties of Ocoxin® oral solution (OOS) prompt us to analyze its effect on the metastatic development of CRC to the liver. First, in vitro effect of OOS in tumor cell viability and migration was analyzed. Second, in vivo effect of different dosage patterns and concentrations in the development of hepatic metastasis was analyzed by intrasplenic inoculation of C26 colon carcinoma cells in Balb/c mice. Third, the expression of alpha smooth muscle actin, caspase-3 and Ki-67 expression was quantified by immunohistochemistry, then gene expression levels of inflammatory factors were measured by quantitative RT-PCR. According to our results, OOS reduced tumor cell viability and migration in vitro. Moreover, in vivo daily administration of OOS from the 7th day after tumor cell inoculation decreased the total area and size of metastatic foci in the liver. Furthermore, cell proliferation and fibroblast recruitment was decreased in tumor foci while a higher number of apoptotic cells were observed. Finally, RNA levels for the inflammatory mediators COX-2, IFNγ, IL1β, IL6 and TNFα were reduced in total liver. In conclusion, OOS reduced the metastatic development of colorectal cancer to the liver by increasing apoptosis, and decreasing tumor cell proliferation and fibroblast recruitment in the tumor foci, as well as the expression of inflammatory mediators in total liver. These results point out OOS as a potential supplement to be applied as complementary therapy for the treatment of liver metastasis from colorectal cancer.

Abstract B10: LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.

Intercellular adhesion molecule (ICAM)-1 is the main counter-receptor for the lymphocyte function associated antigen (LFA)-1. However, the role this interaction plays during liver metastasis of colorectal cancer cells and its underlying mechanisms remain unclear. Previously, we showed that mannose receptor (ManR) stimulation on liver sinusoidal endothelial cells (LSECs) resulted in a reduced cytotoxic potential of liver infiltrating lymphocytes (LIL) towards C26 murine colon carcinoma cells. Using ICAM-1 specific siRNAs in vivo, we aimed to study the role of hepatic ICAM-1 in the prometastatic shift of the liver microenvironment. Livers of control and silenced mice, 24 hours and 14 days after C26 cell inoculation, were compared for: percentage of detained C26 cells, number and volume of metastatic foci, levels of recruited immune, myofibroblastic and inflammatory cells and whole liver expression of pro-inflammatory genes. Detained cancer cells and metastatic foci number were reduced in silenced mice compared to controls, along with a decrease in myeloid suppressor cells and recruited macrophages and myofibroblasts. On the contrary, significant higher numbers of lymphocytic cells were observed in mice with reduced ICAM-1. Finally, silencing of ICAM1 resulted in altered total liver mRNA levels of proinflammatory factors in the tumor-bearing mice. Therefore, an orchestrated prometastatic program might be switched on in the liver microenvironment by LFA-1/ICAM-1 interaction which, in turn, could lead not only to a reduced antitumor citotoxicity but, also, to activation of the stromal compartment favoring tumor progression, and, opening liver9s doors to tumor cells to aggressively metastasize. Based on these results, LFA-1/ICAM-1 interaction arises as a potential therapeutic target in cancer treatment. Citation Format: Aitor Benedicto, Joana Marquez, Elvira Olaso, Beatriz Arteta. LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B10. doi:10.1158/1538-7445.CHTME14-B10

Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.

Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT‐29 human colorectal cancer cells to create a HT‐29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT‐29 adhesion to LSEC were altered. Next, HT‐29 RHGP cell library fractions with upregulated or silenced LSEC adhesion‐related genes were isolated. Around 160 clones having altered expression in LSEC adhesion‐related genes were obtained, and nine relevant protein‐coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT‐29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT‐29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT‐29 cells (ITPKC gene) and their silenced status in adherent HT‐29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT‐29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.