Small molecule inhibitors target the tissue transglutaminase and fibronectin interaction.

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.

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 Ca(2+). 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 IκB kinase α 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.

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.

Targeting ovarian tumor cell adhesion mediated by tissue transglutaminase.

Tissue transglutaminase (TG2) is a transpeptidase involved in protein cross-linking through generation of ε-(γ-glutamyl)lysine isopeptide bonds. It also promotes cell adhesion through interaction with fibronectin and facilitates formation of fibronectin-integrin complexes. This interaction is involved in tumor cell adhesion to the matrix and in the process of tumor dissemination. Its inhibition by small molecules may therefore be useful in blocking metastasis. To that end, we screened more than 800,000 compounds following an in silico docking approach targeting two distinct cavities in the vicinity of the fibronectin-binding site on TG2. A total of 120 compounds were acquired and tested in cell culture-based assays for inhibition of ovarian tumor cell adhesion and proliferation. Seven compounds showed more than 50% inhibition of cell adhesion at a concentration of 25 µmol/L. A follow-up fluorescence polarization study revealed that one compound in particular (ITP-79) inhibited binding of a TG2 peptide to a 42-kDa fragment of fibronectin in a dose-dependent manner. This inhibition was confirmed in cancer cells by coimmunoprecipitation. A competition assay with surface plasmon resonance showed that ITP-79 modulated binding of TG2 to fibronectin. Direct binding of compounds that inhibited adhesion to TG2 were examined with differential scanning fluorimetry, which measures the effect of the compound on the melting temperature of the target. Two compounds, including ITP-79, reduced TG2 stabilization, mimicking the effects of GTP, a known negative allosteric regulator of TG2 enzymatic function. This suggests a potential allosteric mechanism for the compound in light of its distal target site.

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.

Epithelial-to-mesenchymal transition and ovarian tumor progression induced by tissue transglutaminase.

Tissue transglutaminase (TG2), an enzyme that catalyzes Ca(2+)-dependent aggregation and polymerization of proteins, is overexpressed in ovarian cancer cells and tumors. We previously reported that TG2 facilitates tumor dissemination using an i.p. xenograft model. Here we show that TG2 modulates epithelial-to-mesenchymal transition (EMT), contributing to increased ovarian cancer cell invasiveness and tumor metastasis. By using stable knockdown and overexpression in epithelial ovarian cancer cells, we show that TG2 induces a mesenchymal phenotype, characterized by cadherin switch and invasive behavior in a Matrigel matrix. This is mediated at the transcriptional level by altering the expression levels and function of several transcriptional repressors, including Zeb1. One mechanism through which TG2 induces Zeb1 is by activating the nuclear factor-kappaB complex. The effects of TG2 on ovarian cancer cell phenotype and invasiveness translate into increased tumor formation and metastasis in vivo, as assessed by an orthotopic ovarian xenograft model. Highly expressed in ovarian tumors, TG2 promotes EMT and enhances ovarian tumor metastasis by activating oncogenic signaling.

Renal ischemia reperfusion inhibits VEGF expression and induces ADAMTS-1, a novel VEGF inhibitor.

Reductions in vascular density occur following acute ischemia-reperfusion (I/R) injury that may predispose the development of chronic kidney disease. The mechanisms mediating vascular loss are not clear but may relate to the lack of effective vascular repair responses. To determine the regulation of the VEGF/VEGFR pathway following I/R injury, male Sprague-Dawley rats were subjected to bilateral renal ischemia (45 min) and allowed to recover for 1, 3, 7, and 35 days. VEGF mRNA expression was repressed by greater than 50% of control values up to 3 days postischemia, while VEGF protein was repressed for up to 7 days postischemia. The renal mRNA expression of receptors was not altered postischemia; however, VEGFR1 (flt-1) protein was transiently reduced in kidney while soluble flt-1 was elevated in plasma at 7 days following injury. Microarray analysis of angiogenesis-related genes identified the enhanced expression of a number of genes, among these was ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif-1), a secreted VEGF inhibitor. The altered expression of ADAMTS-1 was confirmed using RT-PCR and Western blot analysis; immunofluorescence localized its expression to proximal tubules following I/R injury. Other genes identified using microarray included aminopeptidase N, Smad-1, and Id-3 and their localization was also examined using immunohistochemistry. In summary, the data indicate no clear pattern of anti-angiogenic gene expression following renal I/R injury. However, the studies do suggest an overall inhibition of the VEGF pathway during the early injury and repair phase of renal ischemia that may contribute to an overall reduction in renal microvascular density.