Targeting the insulin growth factor and the vascular endothelial growth factor pathways in ovarian cancer

Antiangiogenic therapy is emerging as a highly promising strategy for the treatment of ovarian cancer, but the clinical benefits are usually transitory. The purpose of this study was to identify and target alternative angiogenic pathways that are upregulated in ovarian xenografts during treatment with bevacizumab. For this, angiogenesis-focused gene expression arrays were used to measure gene expression levels in SKOV3 and A2780 serous ovarian xenografts treated with bevacizumab or control. Reverse transcription-PCR was used for results validation. The insulin growth factor 1 (IGF-1) was found upregulated in tumor and stromal cells in the two ovarian xenograft models treated with bevacizumab. Cixutumumab was used to block IGF-1 signaling in vivo. Dual anti-VEGF and IGF blockade with bevacizumab and cixutumumab resulted in increased inhibition of tumor growth. Immunohistochemistry measured multivessel density, Akt activation, and cell proliferation, whereas terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay measured apoptosis in ovarian cancer xenografts. Bevacizumab and cixutumumab combination increased tumor cell apoptosis in vivo compared with therapy targeting either individual pathway. The combination blocked angiogenesis and cell proliferation but not more significantly than each antibody alone. In summary, IGF-1 activation represents an important mechanism of adaptive escape during anti-VEGF therapy in ovarian cancer. This study provides the rationale for designing bevacizumab-based combination regimens to enhance antitumor activity

PDGF induced microRNA alterations in cancer cells

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells

Extracellular Tissue Transglutaminase Activates Noncanonical NF-κB Signaling and Promotes Metastasis in Ovarian Cancer

Tissue transglutaminase (TG2) is a multifunctional protein that binds to fibronectin and exerts protein transamidating activity in the presence of Ca2+. We previously reported that TG2 is upregulated in ovarian tumors and enhances intraperitoneal (i.p.) metastasis. TG2 is secreted abundantly in ovarian cancer (OC) ascites as an active enzyme, yet its function in the extracellular compartment remains unknown. To study the distinct functions of secreted TG2, we used recombinant His6-tagged TG2 and catalytically inactive enzyme in vitro and in vivo. By using i.p. and orthotopic ovarian xenografts, we show that extracellular transglutaminase promoted OC peritoneal metastasis. The main pathway activated by extracellular TG2 was noncanonical nuclear factor-kappa B (NF-κB) signaling, and the enzymatic function of the protein was required to induce phosphorylation of IκBkinaseá and processing of the precursor protein p100 into the active p52 subunit. A specific target of TG2-activated p52/RelB complex is the hyaluronan receptor, CD44. Noncanonical NF-κB activation by extracellular TG2 induced CD44 up-regulation and epithelial-to-mesenchymal transition, contributing to increased cancer cell invasiveness and OC peritoneal dissemination. Taken together, our data support that noncanonical NF-κB activation is the pathway through which extracellular TG2 promotes OC metastasis

Small Molecule Inhibitors Target the Tissue Transglutaminase and Fibronectin Interaction

<div><p>Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε−(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination.</p></div

The AlphaLISA assay is used to screen the ChemDiv library.

<p>A, The assay identifies SMIs that inhibit the TG2-FN42 interaction by >50% at 10 μM. B, Dose dependent inhibition of TG2-FN42 interaction by selected compounds. Bars represent means +/− SD of triplicate measurements. Asterisks denote <i>p <</i>0.05. C, Selected compounds are grouped in two categories based on chemical structure: diamino-pyrimidines and pyrolydinyl-pyrimidines.</p

Characteristics of the AlphaLISA^TM assay developed to measure the TG2-FN42 interaction.

<p>A, Design of the AlphaLISA assay that measures the TG2-FN interaction. Donor beads coated with streptavidin and acceptor nickel chelate beads were used to capture biotinylated FN42 and His tagged TG2 protein, respectively. After excitation at 680 nm singlet oxygen is transferred from donor to acceptor beads coming within a distance of 200 nm, resulting in a chemiluminescence signal. B, Cross-titration was performed to optimize detection of the TG2-FN interaction by the assay. Saturation isotherms of FN42 binding to TG2 were generated. The K_d was 2.43 nM. C, Titration curves represented with GraphPad Prism demonstrate reaching the hook point at 3 nM biotinylated FN42 and 10 nM TG2-His.</p

ELISA-based approach measures the TG2-FN interaction.

<p><b>A</b>, Design of the ELISA measuring the TG2-FN interaction. His tagged TG2 is captured by the anti-His antibody coating the wells. Biotinylated FN42 interacting with TG2 is recognized by streptavidin-HRP, which reacts with a TMB substrate. The signal is abrogated if the TG2-FN interaction is disrupted. <b>B</b>, Specificity of the assay is demonstrated by incubating His-tagged TG2 with increasing concentrations of biotinylated FN42 (from 0 nM to 16 nM) in the presence of unlabeled FN42 (from 0 nM to 16 nM). <b>C</b>, ELISA in the presence of the competitive inhibitor 4G3, an anti-TG2 antibody against the FN-binding domain of TG2 (5 μg/ml), and in the presence of unlabeled FN42 (3 nM). <b>D</b>, ELISA measures inhibition of the TG2-FN42 interaction by SMIs selected from the AlphaLISA HTS. Bars represent means +/− SD of triplicate measurements. Asterisks denote <i>p <</i>0.05. <b>E</b>, ELISA measures dose dependent inhibition of TG2-FN42 interaction by TG53. <b>F</b>, Saturation curves of FN42 in the presence of increasing concentrations of TG53 were used to calculate the Ki (4.15 μM) of TG53 for TG2. Inset corresponds to representative Lineweaver-Burk plots showing that TG53 competes for the same binding site in TG2 as FN42.</p