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SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, β-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival.

Glioblastomas (GBMs) are the most frequent malignant brain tumors with very limited treatment options and nearly all GBM patients dying within 1 year. Pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) is a secreted growth factor that shows mitogenic, chemotactic and transforming activity. While PTN expression is tightly regulated during embryogenesis and very limited in normal adult tissues, a marked PTN upregulation is seen in tumors including glioblastomas. Targeting of the PTN receptors, ALK and RPTP-zeta, indicates a contribution of PTN-activated signaling pathways in glioblastomas. However, the relevance of PTN expression itself is unknown especially since, besides PTN, at least one more growth factor, midkine (MK), signals through ALK and is expressed in glioblastoma. Here we demonstrate the biologic relevance of PTN in 2 glioblastoma cell lines in vitro and in vivo. We show that stable ribozyme-targeting leads to a robust reduction of PTN mRNA and protein levels. This results in decreased cell proliferation, cell migration and soft agar colony formation in vitro. Comparing clonal ribozyme-transfected cells with different residual PTN levels, we establish a PTN gene-dose effect of glioblastoma cell proliferation. In a subcutaneous tumor xenograft mouse model, in vivo growth is markedly reduced upon PTN depletion, which is paralleled by decreased PTN serum levels. Furthermore, the immunohistochemical analysis of the tumors shows reduced angiogenesis in PTN-depleted tumors. We conclude that PTN is a rate-limiting growth factor in glioblastoma. Since PTN is overexpressed in glioblastomas but rarely found in normal tissue, PTN may represent an attractive therapeutic target.

Over-expression of the ErbB-2 proto-oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. The ErbB-2 receptor is a member of the ErbB family of growth factor receptors, and within this complex signaling network, ErbB-2-containing heterodimers are preferentially formed. To assess whether ErbB-2 is a critical component in epidermal growth factor (EGF)–mediated stimulation of tumor cell proliferation, we used as a model SK-OV-3 ovarian cancer cells, which over-express EGF receptor (EGFR) and ErbB-2 receptors. In these cells, we reduced ErbB-2 mRNA and protein expression by transfection with ErbB-2-targeted hammerhead ribozymes and generated cell lines expressing different levels of ErbB-2. In SK-OV-3 cells, ErbB-2 expression conferred a growth advantage and soft agar experiments revealed that ErbB-2 was rate-limiting for anchorage-independent growth. The induction of colony formation by EGF was completely abrogated in ErbB-2-depleted cells, despite unchanged expression levels and tyrosine phosphorylation of the EGFR. The duration of EGF-mediated c-Fos mRNA up-regulation was decreased in parallel with loss of ErbB-2 expression. Furthermore, the rate of spontaneous apoptosis was increased in ErbB-2-depleted cells. Our results demonstrate that in human ovarian cancer cells the EGFR–ErbB-2 heterodimer, and not the EGFR homodimer, can be rate-limiting for EGF-mediated proliferation, thus suggesting that the oncogenic activity of ErbB-2 in human tumors is due in part to its ability to increase the growth response to stroma-derived EGF-like growth factors. Int. J. Cancer 86:644–651, 2000. © 2000 Wiley-Liss, Inc.

SATB1 (special AT-rich binding protein 1) is a global chromatin organizer regulating the expression of a large number of genes. Overexpression has been found in various solid tumors and positively correlated with prognostic and clinicopathological properties. In colorectal cancer (CRC), SATB1 overexpression and its correlation with poor differentiation, invasive depth, TNM (tumor, nodes, metastases) stage and prognosis have been demonstrated. However, more detailed studies on the SATB1 functions in CRC are warranted. In this article, we comprehensively analyze the cellular and molecular role of SATB1 in CRC cell lines with different SATB1 expression levels by using RNAi-mediated knockdown. Using siRNAs with different knockdown efficacies, we demonstrate antiproliferative, cell cycle-inhibitory and proapoptotic effects of SATB1 knockdown in a SATB1 gene dose-dependent manner. Tumor growth inhibition is confirmed in vivo in a subcutaneous tumor xenograft mouse model using stable knockdown cells. The in-depth analysis of cellular effects reveals increased activities of caspases-3, -7, -8, -9 and other mediators of apoptotic pathways. Similarly, the analysis of E- and N-cadherin, slug, twist, β-catenin and MMP7 indicates SATB1 effects on epithelial-mesenchymal transition (EMT) and matrix breakdown. Our results also establish SATB1 effects on receptor tyrosine kinases and (proto-)oncogenes such as HER receptors and Pim-1. Taken together, this suggests a more complex molecular interplay between tumor-promoting and possible inhibitory effects in CRC by affecting multiple pathways and molecules involved in proliferation, cell cycle, EMT, invasion and cell survival.

Ovarian cancer is one of the most lethal gynecological malignancies and the small success rate of routine therapeutic methods justifies efforts to develop new approaches. Evaluation of targets for effective inhibition of ovarian cancer cell growth should precipitate clinical application of gene silencing therapy. In our previous work, we showed upregulation of HMGA2 gene expression as a result of Ras-induced rat ovarian surface epithelial cell transformation. This gene codes the HMGA2 protein, a member of the high-mobility group AT-hook (HMGA) family of nonhistone chromatin proteins. Genome-wide studies revealed upregulation of the HMGA2 gene in human ovarian carcinomas. Herein we have evaluated over-expression of the HMGA2 gene, relevant to ovarian cancer, in subsets of human specimens and cell lines by in situ RNA hybridization and RT-PCR. Transient silencing of HMGA2 gene by means of siRNA inhibited proliferation of those ovarian cancer cells, which over-express this gene initially. Growth suppression was mediated by cell-cycle arrest. Stable silencing of highly expressed HMGA2 gene by shRNAi in A27/80, Ovcar-3 and OAW-42 ovarian cancer cell lines resulted in growth inhibition because of G1 arrest and increase of apoptosis as well. The tumor growth inhibition effect of HMGA2 silencing for Ovcar-3 cells was validated in vivo. Our findings revealed that the HMGA2 gene represents a promising target for gene silencing therapy in ovarian cancer.

Fibroblast growth factors FGF-1 (aFGF) and FGF-2 (bFGF) are found in most embryonic and adult normal and tumor tissues, where they are immobilized in the extracellular matrix (ECM). Mobilization of these FGFs is part of a tightly controlled process resulting in the activation of high-affinity FGF receptors. Recently, we have shown that a secreted FGF-binding protein (FGF-BP) binds non-covalently to FGF-2 and is able to release it from the ECM. This process of growth factor bioactivation seems to play a pivotal role in the growth of squamous cell carcinomas, especially through induction of tumor angiogenesis. Since previous studies provided only indirect evidence for the proposed mechanism of FGF-BP-mediated FGF-2 release, we decided to use recombinant purified FGF-BP to study further the underlying mechanism of FGF-BP action. Here we show that FGF-BP is able to bind directly to FGF-2 without additional cofactors and to exhibit bioactivity. The purified recombinant FGF-BP stimulates tumor cell growth as well as endothelial cell growth and chemotaxis, indicating a dual growth-supporting role of FGF-BP in tumors. We show that this paracrine FGF-BP effect is dependent on endogenously expressed FGF-2, since it can be completely blocked by anti-FGF-2 antibodies. In tumor xenografts and in tumor cells, we detected a pattern of specific FGF-BP-immunoreactive high molecular weight forms, which presumably represent stable covalent complexes of FGF-BP and show marked differences in their occurrence in different tumors and in their heparin binding affinity. By providing further insight into the mechanism of FGF-BP action, our results emphasize the relevance of FGF-BP and of FGF-2 in tumor growth.