[Show abstract][Hide abstract] ABSTRACT: 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.
Molecular Cancer Therapeutics 06/2012; 11(7):1576-86. DOI:10.1158/1535-7163.MCT-11-0961 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-β, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-β-activated kinase 1-mediated activation of the nuclear factor-κB complex. TGF-β-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-β-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44+/CD117+). Furthermore, CD44+/CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-β-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype.
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 05/2011; 39(10):4035-47. DOI:10.1093/nar/gkq1305 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Molecular Cancer Therapeutics 02/2011; 10(4):626-36. DOI:10.1158/1535-7163.MCT-10-0912 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Cancer Research 12/2009; 69(24):9192-201. DOI:10.1158/0008-5472.CAN-09-1257 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tissue transglutaminase 2 (TG2) is overexpressed in epithelial ovarian cancer (EOC) and promotes intraperitoneal metastasis. How TG2 facilitates the spread of EOC is unknown. Here, we show that TG2 regulates the expression and function of matrix metalloproteinase-2 (MMP-2), a critical mediator of tissue invasiveness. TG2 knockdown down-regulates MMP-2 protein and mRNA expression in SKOV3, IGROV-1, MDA-MB-436, and PC-3 cancer cells. TG2 knockdown or inhibition of TG2 activity using KCC009 decreases MMP-2 gelatinase activity in cancer cells. MMP-2 expression and function are regulated by TG2 at transcriptional level, as demonstrated by quantitative PCR and reporter assays. We used bioinformatics and chromatin immunoprecipitation to identify a CREB binding site in the MMP-2 promoter. Binding of CREB to the MMP-2 promoter was diminished in cells that expressed decreased TG2 levels. TG2 knockdown decreased CREB phosphorylation, and CREB knockdown decreased MMP-2 expression. The effect of TG2 on CREB activity and MMP-2 transcription is mediated by TG2-dependent degradation of protein phosphatase 2 (PP2A-alpha). We show that PP2A-alpha complexes with and is targeted for degradation by TG2. In addition to their related in vitro expression levels, TG2 and MMP-2 expression were significantly correlated in vivo, as shown by concordant immunostaining in peritoneal xenografts and in human ovarian tumors. The capacity of TG2 to regulate MMP-2 expression in vitro and in vivo identifies a mechanism that may facilitate tissue invasion and the spread of EOC. The demonstration that TG2 induced degradation of PP2A-alpha activates CREB, and thereby increases MMP-2 transcription, provides novel mechanistic insight into the pro- metastatic function of TG2.