19 research outputs found

    Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

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    Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (\u3e95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo

    Aspartate β-Hydroxylase Expression Promotes a Malignant Pancreatic Cellular Phenotype

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    Pancreatic cancer (PC) is one of the leading causes of cancer related deaths due to aggressive progression and metastatic spread. Aspartate β-hydroxylase (ASPH), a cell surface protein that catalyzes the hydroxylation of epidermal growth factor (EGF)-like repeats in Notch receptors and ligands, is highly overexpressed in PC. ASPH upregulation confers a malignant phenotype characterized by enhanced cell proliferation, migration, invasion and colony formation in vitro as well as PC tumor growth in vivo. The transforming properties of ASPH depend on enzymatic activity. ASPH links PC growth factor signaling cascades to Notch activation. A small molecule inhibitor of β-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway. These findings demonstrate the critical involvement of ASPH in PC growth and progression, provide new insight into the molecular mechanisms leading to tumor development and growth and have important therapeutic implications

    LRH1 as a driving factor in pancreatic cancer growth

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    Liver receptor homolog 1 (LRH1), directs the development and differentiation of embryonic pancreas, and is overexpressed in pancreatic cancer (PC). We hypothesized that LRH1 promotes PC growth. Cell proliferation and tumorigenicity in nude mice were compared between empty vector-transfected (control) and stable LRH1-overexpressed PC cell lines. The subsequent tumor burden, vasculature development, and histologic features were evaluated. LRH1 overexpression enhanced the expression of downstream target genes (cyclin D1/E1) and stimulated cell proliferation in PC cell lines. LRH1 upregulated cyclin E1 truncated T1/T2 isoforms expression which may occur through ERα–calpain1 signaling. Compared with the control, LRH1 overexpressing stable cells generated tumors with increased weight, proliferation index and enhanced angiogenesis. Cyclin D1/E1 and calpain1 were overexpressed in human PC tumors compared to adjacent normal pancreas. These observations demonstrate that LRH1 promotes PC growth and angiogenesis, suggesting that LRH1 is a driving factor in tumorigenesis and may serve as a potential therapeutic target

    Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

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    We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation

    Correlation of TFR1 and AF20 staining in colon cancer samples.

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    <p>Three pairs of adjacent tissue sections of normal colon (<b>A</b>) and colon cancer (<b>B</b>) were stained with TFR1 (upper panels, 1:50 dilution) and AF20 mAb (lower panels, 1:500 dilution), respectively. Images were taken at 1000x magnification. Note that overexpression of both TFR1 and AF20 antigen was only observed in colon cancer but not in normal colon. In addition, the expression pattern and localization as revealed by TFR1 and AF20 Ab are indistinguishable.</p

    AF20 mAb failed to recognize deglycosylated TFR1.

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    <p>Huh7 cell lysate was subject to immunoprecipitation with AF20 mAb and retained proteins on protein G beads were treated with PNGase F at 37°C for 1hr. Samples were directly loaded onto 10% SDS-PAGE (minigel) in duplicate followed by Western blot detection using AF20 (left) and TFR1 antibodies (right), respectively. Note that an additional protein band above the 75-kd size marker was detected by TFR1 antibody from PNGase F treated sample, most likely corresponding to deglycosylated form of TFR1 (79kDd).</p

    Comparison of AF20 antigen levels in different cell lines.

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    <p>Cell lysate containing 200μg of proteins was subject to immunoprecipitation with AF20 antibody followed by Western blot using the same antibody. The 50-kDd protein band in the blot corresponds to the heavy chain of AF20 antibody (IgG). Pro RG: proliferating HepaRG cells. Note that for the left panel, lane 1 and lanes 2/3 were cropped from different parts of the same blot.</p

    Holo but not apo transferrin could interfere with AF20 antigen-antibody interaction.

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    <p><b>A</b>. Lysate of LS180 cells was subjected to immunoprecipitation with AF20 mAb in the absence (w/o) or presence of 33μg/ml of apo or holo transferrin (TF), and retained proteins were detected by Western blot with AF20 mAb. <b>B</b>. Increasing concentrations (3.3, 16.5, and 66μg/ml) of holo transferrin were added during immunoprecipitation with AF20 mAb, followed by Western blot with the same antibody.</p

    Purification of AF20 antigen from Huh7 cells for proteomic analysis.

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    <p><b>A</b>. Cell pellet (2 grams) was lyzed by freeze/thaw and diluted with Tris buffer. The sample was loaded onto a DEAE-cellulose column. After flow through (FT), the column was washed with Tris buffer (wash). Proteins were eluted successively with 100 mM, 200 mM and 400 mM of NaCl, and protein concentration was determined by BCA assay using known concentrations of albumin as a standard. <b>(B & C)</b> AF20 antigen was imunoprecipitated from the three protein peaks by AF20 mAb and subjected to Western blot (minigel format) using the same antibody (B), or pooled and subject to SDS-PAGE (large gel format) followed by Coomassie blue staining (C). The area of three protein bands was cut out for proteomic analysis. MW: molecular size markers.</p
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