Most antiangiogenic therapies currently being evaluated in clinical trials target the vascular endothelial growth factor pathway; however, the tumor vasculature can acquire resistance to vascular endothelial growth factor-targeted therapy by shifting to other angiogenesis mechanisms. Insulin-like growth factor binding protein-3 (IGFBP-3) has been reported to suppress tumor growth and angiogenesis by both IGF-dependent and IGFindependent mechanisms; however, understanding of its IGF-independent mechanisms is limited. We observed that IGFBP-3 blocked tumor angiogenesis and growth in non-small cell lung cancer and head and neck squamous cell carcinoma. Conditioned media from an IGFBP-3-treated non-small cell lung cancer cell line displayed a significantly decreased capacity to induce HUVEC proliferation and aortic sprouting. In cancer cells, IGFBP-3 directly interacted with Erk1/2, leading to inactivation of Erk1/2 and Elk-1, and suppressed transcription of early growth response protein 1 and its target genes, basic fibroblast growth factor and platelet-derived growth factor. These data suggest that IGF-independent Erk1/2 inactivation and decreased IGFBP-3-induced Egr-1 expression block the autocrine and paracrine loops of angiogenic factors in vascular endothelial and cancer cells. Together, these findings provide a molecular framework of IGFBP-3's IGF-independent antiangiogenic antitumor activities. IntroductionAngiogenesis, the formation of new capillaries from existing blood vessels, is essential to carcinogenic processes, including solid tumor formation, growth, invasion, and metastasis. 1 Most tumors can stimulate angiogenesis by switching on the production of numerous cytokines and growth factors, including fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), and platelet-derived growth factors (PDGFs). 2 Several antiangiogenic agents are in various phases of clinical trials for human cancer; however, most of these agents target the VEGF signaling pathway. 3 Therefore, other potential therapeutic agents that block non-VEGF angiogenic pathways need to be evaluated.Insulin-like growth factor-binding protein-3 (IGFBP-3), a member of a family of 6 IGFBPs, has demonstrated antiproliferative, proapoptotic, antiangiogenic, and antimetastatic activity in a variety of cancer cells. [4][5][6][7][8] It may also have IGF-independent antitumor activities through cell-surface or intracellular protein interaction, its nuclear translocation, or its transcriptional regulation. 7,[9][10][11][12] However, the mechanisms that mediate IGFBP-3's IGF-independent antitumor activity have not been clearly defined.The 82-kDa phosphoprotein transcription factor early growth response protein 1 (Egr-1), an immediate early gene product, has been implicated in multiple cellular processes, including cell growth, apoptosis, wound healing, and angiogenesis. Mitogenic stimuli, including serum, PDGF, peptide growth factors, and B-Raf, and nonmitogenic stresses, including ␥-irradiation and hypoxia, activate Egr-1 expre...

BackgroundThe complexity of wnt signaling likely stems from two sources: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.Methodology/Principal FindingsHere we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of β-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-β-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of β-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.Conclusions/SignificanceThese data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling.

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In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies.

Ovarian cancer growth under hypoxic conditions results in hypoxia-inducible factor-1a (HIF1a) stabilization. HIF1a is an adverse prognostic factor that may contribute to worse outcomes via its capacity to bind to p53, potentially blocking p53-mediated apoptosis. We determined whether HIF1a-p53 binding occurred in hypoxic ovarian cancer cell lines, and if this blocked p53 transcriptional activity. Topotecan (TPT), used in the treatment of ovarian cancer, inhibits HIF1a translation via a topoisomerase-1 (TOPO1)-dependent mechanism. We examined if TPT knockdown of HIF1a restored p53 transcriptional function. TPT effects on HIF1a and p53related transcriptional targets were assessed by PCR. Associations between TPT effects and TOPO1 expression levels were examined by Western blots and knockdown by siRNA. RNAbinding protein immunoprecipitation was used to assess if TOPO1 was resident on HIF1a mRNA. We determined if sublethal doses of TPT, used to knockdown HIF1a, reversed hypoxia-related cisplatin and paclitaxel resistance (XTT assay). Flow cytometry was used to assess HIF1a-mediated upregulation of ABCB1 and ABCB5 efflux pump expression. We found that HIF1a binding to, and inhibition of, p53 transcriptional activity in hypoxic ovarian cancer cells was associated with drug resistance. TPT-mediated downregulation of HIF1a in hypoxic cells required TOPO1 resident on HIF1a mRNA, restored p53 transcriptional activity, downregulated ABCB1/ ABCB5 cell surface expression, and reversed hypoxia-related cisplatin and paclitaxel resistance.Implications: TPT-mediated reduction of HIF1a accumulation in hypoxic ovarian cancer cell lines restores p53 tumorsuppressor function, offering a novel approach to reverse chemoresistance. Further clinical investigation is warranted.