Orphan nuclear receptor TR2, a mediator of preadipocyte proliferation, is differentially regulated by RA through exchange of coactivator PCAF with corepressor RIP140 on a platform molecule GRIP1

Orphan nuclear receptor TR2 is a preadipocyte proliferator. Knockdown of TR2 in 3T3-L1 preadipocytes reduced their proliferation efficiency, whereas specific elevation of TR2 in these cells facilitated their proliferation. All-trans retinoic acid (RA) stimulates cellular proliferation in 3T3-L1 preadipocytes by activating TR2 through an IR0-type RA response element, which further activates c-Myc expression. In post-differentiated adipocytes, RA becomes a repressive signal for TR2 and rapidly down-regulates its expression. The biphasic effect of RA on TR2 expression in 3T3-L1 is mediated by differential RA-dependent coregulator recruitment to the receptor/Glucocorticoid Receptor-Interacting Protein 1 (GRIP1) complex that binds IR0 on the TR2 promoter. RA induces the recruitment of histone acetyl transferase-containing/GRIP1/p300/CBP-associated factor (PCAF) complex to the TR2 promoter in undifferentiated cells, whereas it triggers recruitment of histone deacetylase-containing/GRIP1/receptor-interacting protein 140 (RIP140) complex in differentiated cells. GRIP1 directly interacts with RIP140 through its carboxyl terminal AD2 domain. GRIP1 interacts with PCAF and RIP140 directly and differentially, functioning as a platform molecule to mediate differential RA-induced coregulator recruitment to TR2 promoter target. This results in a biphasic effect of RA on the expression of TR2 in undifferentiated and differentiated cells, which is required for RA-stimulated preadipocyte proliferation

C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

<p>Abstract</p> <p>Background</p> <p>The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLe^x) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 <it>N</it>-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLe^x(C2-O-sLe^x). Our goal was to determine the expression profiles of C2-O-sLe^xin the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLe^xpresent in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLe^xwas present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-<it>N</it>-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).</p> <p>Results</p> <p>We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.</p> <p>Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.</p> <p>Conclusion</p> <p>C2-O-sLe^x, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLe^xis a potentially useful early predictor of metastasis.</p

Status of Agents Targeting the HGF/c-Met Axis in Lung Cancer

Hepatocyte growth factor (HGF) is the ligand for the tyrosine kinase receptor c-Met (Mesenchymal Epithelial Transition Factor also known as Hepatocyte Growth Factor Receptor, HGFR), a receptor with expression throughout epithelial and endothelial cell types. Activation of c-Met enhances cell proliferation, invasion, survival, angiogenesis, and motility. The c-Met pathway also stimulates tissue repair in normal cells. A body of past research shows that increased levels of HGF and/or overexpression of c-Met are associated with poor prognosis in several solid tumors, including lung cancer, as well as cancers of the head and neck, gastro-intestinal tract, breast, ovary and cervix. The HGF/c-Met signaling network is complex; both ligand-dependent and ligand-independent signaling occur. This article will provide an update on signaling through the HGF/c-Met axis, the mechanism of action of HGF/c-Met inhibitors, the lung cancer patient populations most likely to benefit, and possible mechanisms of resistance to these inhibitors. Although c-Met as a target in non-small cell lung cancer (NSCLC) showed promise based on preclinical data, clinical responses in NSCLC patients have been disappointing in the absence of MET mutation or MET gene amplification. New therapeutics that selectively target c-Met or HGF, or that target c-Met and a wider spectrum of interacting tyrosine kinases, will be discussed

C2GnT1 mediates invasion.

<p>(A) LS174T cells transduced with C2GnT1 shRNA were significantly less invasive than cells transduced with scrambled shRNA, *p<0.0001, in Matrigel transwell invasion assays. (B) Similarly, decreased invasion was observed for HepG2 cells that were transfected with C2GnT1 shRNA compared to transfection with a control vector, *p = 0.0005. (C) Representative photomicrographs from two separate experiments of invasion of LS174T cells transduced with the scrambled shRNA vector compared to (D) cells transduced with C2GnT1 shRNA and (E) HepG2 cells transfected with the control vector compared to (F) HepG2 cells transfected with C2GnT1 shRNA. The invading cells were stained and counted in five separate fields of view at 100X magnification. Arrows indicate invasive cells.</p

C2GnT1 and FucT-III genes are endogenously expressed in human carcinoma cell lines.

<p>(A) Endogenous C2GnT1 and FucT-III mRNA transcripts in LS174T colorectal adenocarcinoma (lane 1) and HepG2 hepatic carcinoma (lane 2) cell lines were detected by RT-PCR. (B) Flow cytometric analysis of positive staining of LS174T and HepG2 cells labeled with CHO-131 mAb (anti-C2-O-sLe^X). A representative example of 3 experiments is shown for (A-B). (C) LS174T (*p = 0.008) and HepG2 cells (#p = 0.001) substantially bind to E-selectin in the presence of calcium ions (Ca²⁺). Binding is impeded in the presence of EDTA, a calcium ion chelator. The average of two experiments is shown.</p

C2GnT1 gene knock-down results in decreased binding of cells to E-selectin.

<p>(A) Significant differences in E-selectin binding were not observed among LS174T un-manipulated cells, cells transduced with scrambled shRNA, or with C2GnT1 shRNA by flow cytometry. (B) E-selectin binding was significantly decreased for HepG2 cells transfected with C2GnT1 shRNA compared to cells transfected with the control vector, *p = 0.02. (C) Shear flow assays were performed at an E-selectin/Fc chimera concentration of 1 µg/ml and shear stresses ranging from 0.5 – 1.5 dynes/cm² in the presence of 10 µg/ml of an IgG isotype control mAb. At a shear stress of 0.5 dynes/cm², significantly fewer C2GnT1 shRNA transduced cells accumulated on E-selectin than un-manipulated LS174T cells, *p = 0.04. (D) In the same run of experiments, at a shear stress of 0.5 dynes/cm², significantly fewer un-manipulated LS174T cells treated with a 10 µg/ml of a blocking anti-E-selectin mAb accumulated on E-selectin than those cells treated with an isotype control mAb, ^#p = 0.004. Significantly fewer LS174T cells transduced with C2GnT1 shRNA and treated with a blocking anti-E-selectin mAb accumulated on E-selectin than those cells treated with an isotype control mAb, ^&p = 0.01. The same assay as shown in (C) at 0.5 dynes/cm² is included for comparison. For both groups of cells treated with an isotype control mAb, significantly fewer un-manipulated LS174T cells accumulated on E-selectin than LS174T cells transduced with C2GnT1 shRNA, *p = 0.04. Each continuous shear flow assay was performed in duplicate for each shear stress and the bars represent the mean ± standard deviations. Representative data from three independent experiments are shown.</p