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OR8,47 IGF-I:Vitronectin interactions modulate breast cancer cell survival, migration and epithelial to mesenchymal transition
Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARa) that can switch energy metabolism from glycolysis to fatty acid b-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal. Methods: The effects of fenofibrate on glioma cell motility, IGF-I receptor (IGF-IR) signaling, PPARa activity, reactive oxygen species (ROS) metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines. Results: Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARa-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC), restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated G9lioma cell lines. Conclusions: Our results indicate that although fenofibratemediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARa-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells. OR8,47 IGF-I:Vitronectin interactions modulate breast cancer cell survival, migration and epithelial to mesenchymal transition Z. Upton1 , A. Kashyap1 , B. Hollier2 , H. McCosker1 , K. Manton3 , D. Leavesley1 . 1 Cells and Tissues Domain, QUT Institute of Health and Biomedical Innovation, Brisbane, QLD, Australia; 2 Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3 Cells and Tissue Domain, QUT Institute of Health and Biomedical Innovation, Brisbane, QLD, Australia The biological effects of insulin-like growth factors (IGF) are determined by their diverse interactions with many molecules, including the extracellular matrix protein vitronectin (VN). Recent studies have demonstrated that IGFs associate with VN through IGFbinding proteins (IGFBP) and modulate IGF-stimulated biological functions such as cell proliferation, attachment and migration. Since IGFs play important roles in the transformation and progression of breast tumours, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function, to dissect mechanisms underlying these responses and to extend our findings into clinical samples from breast cancer patients. In this study, we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival over 72 hours; this being mediated by transient activation of the ERK/MAPK and sustained activation of the PI3-K/AKT pathways. Furthermore, use of pharmacological inhibitors of MAPK and PI3-K reveal that both pathways are pivotal to IGF-I:IGFBP:VN complex-mediated enhanced cell survival. IGF-I:IGFBP:VN complexes also stimulate an increase in breast cell migration coincident with a greater than 2-fold change in expression of genes involved in migration, invasion, survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1) and epithelial to mesenchymal transition (Snail, Slug, Twist, E-cadherin). This differential gene expression was detected by microarray analysis on breast cells which had migrated in response to the IGF-I:IGFBP:VN comples and was further confirmed using real-time PCR analysis. These changes were not detected when the IGF-I analogue ([L24 ][A31 ]-IGF-I), which fails to bind to the IGF-I receptor, was substituted. This supports the involvement of the IGF system in the differential expression of these genes associated with enhanced cell movement. These cell-based studies have established that IGF and VN synergistically act to enhance survival and epithelial to mesenchymal transition of breast cells,
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processes central to facilitating transformation towards the invasive and metastatic phenotype. Preliminary tissue microarray studies in clinical samples from breast cancer patients also suggest that differences in the expression and localisation of proteins within the IGF/VN signalling pathway are implicated in breast cancer progression. Taken together, this study highlights the interdependence of ECM and growth factor interactions in breast cancer metastasis and identifies novel targets that may hold potential for the development of therapies that prevent breast cancer progression. OR8,48 Differential impacts of IGFBP-3 in epithelial IGF-induced lung cancer development W.-Y. Kim1 , M.-J. Kim2 , H.-J. Moon3 , P. Yuan4 , J.-S. Kim1 , J.-K. Woo1 , G. Zhang1 , Y.-A. Suh5 , L. Feng6 , C. Behrens1 , C.S. Van Pelt7 , H. Kang8 , J.J. Lee6 , W.-K. Hong1 , I.I. Wistuba9 , H.-Y. Lee10 . 1 Thoracic/Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2 Pathology, University of Ulsan College of Medicine, Seoul, South Korea; 3 Department of Mathematics and Statistics, California State University – Long Beach, Long Beach, CA, USA; 4 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 5 Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 6 Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 7 Veterinary Medicine and Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 8 Roosevelt Hospital, Columbia Unversity College of Physicians and Surgeons, New York, NY, USA; 9 Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 10 Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA IGF axis has been implicated in the risk of various cancers. We previously reported a potential role of tissue-derived IGFs in lung tumor formation and progression. However, the role of IGF-binding protein (IGFBP)-3, a major IGFBP, on the activity of tissue-driven IGFs in lung cancer development is largely unknown. Here, we show that IGF-1, but not IGF-2, protein levels in non-small cell lung cancer (NSCLC) were significantly higher than those in normal and hyperplastic bronchial epithelium. We found that IGF-1 and IGFBP-3 levels in NSCLC tissue specimens were significantly correlated with phosphorylated IGF-1 receptor (pIGF-1R) expression. We investigated the impact of IGFBP3 expressions on the activity of tissue-driven IGF-1 in lung cancer development using mice carrying lung-specific human IGF-1 transgene (Tg), a germ linenull mutation of IGFBP-3, or both were analyzed. Compared to wild-type (BP3+/+ ) mice, mice carrying heterozygous (BP3+/− ) or homozygous (BP3−/− ) deletion of IGFBP-3 alleles exhibited decreases in circulating IGFBP-3 and IGF-1. Unexpectedly, IGFTg mice with 50% of physiological IGFBP-3 (BP3+/− ; IGFTg ) showed higher levels of pIGF1R/IR and a greater degree of spontaneous or tobacco carcinogen [4(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK]-induced lung tumor development and progression than did the IGFTg mice with normal (BP3+/+ ; IGFTg ) or homozygous deletion of IGFBP-3 (BP3−/− ; IGFTg) . These data show that IGF-1 is overexpressed in NSCLC, leading to activation of IGF-1R, and that IGFBP-3, depending on its expression level, either inhibit or potentiates IGF-1 actions in lung carcinogenesis. This work was supported by National Institutes of Health grants R01 CA109520 and CA100816–01A1 (to H.-Y. L.) and in part by grants from the National Foundation for Cancer Research, the Department of Defense, the VITAL program (W81XWH-04–1–0142), and the BATTLE program (W81XWH-06–1–0303) (to W.-K. H.).
anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARa) that can switch energy metabolism from glycolysis to fatty acid b-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal. Methods: The effects of fenofibrate on glioma cell motility, IGF-I receptor (IGF-IR) signaling, PPARa activity, reactive oxygen species (ROS) metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines. Results: Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARa-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC), restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated G9lioma cell lines. Conclusions: Our results indicate that although fenofibratemediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARa-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells. OR8,47 IGF-I:Vitronectin interactions modulate breast cancer cell survival, migration and epithelial to mesenchymal transition Z. Upton1 , A. Kashyap1 , B. Hollier2 , H. McCosker1 , K. Manton3 , D. Leavesley1 . 1 Cells and Tissues Domain, QUT Institute of Health and Biomedical Innovation, Brisbane, QLD, Australia; 2 Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3 Cells and Tissue Domain, QUT Institute of Health and Biomedical Innovation, Brisbane, QLD, Australia The biological effects of insulin-like growth factors (IGF) are determined by their diverse interactions with many molecules, including the extracellular matrix protein vitronectin (VN). Recent studies have demonstrated that IGFs associate with VN through IGFbinding proteins (IGFBP) and modulate IGF-stimulated biological functions such as cell proliferation, attachment and migration. Since IGFs play important roles in the transformation and progression of breast tumours, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function, to dissect mechanisms underlying these responses and to extend our findings into clinical samples from breast cancer patients. In this study, we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival over 72 hours; this being mediated by transient activation of the ERK/MAPK and sustained activation of the PI3-K/AKT pathways. Furthermore, use of pharmacological inhibitors of MAPK and PI3-K reveal that both pathways are pivotal to IGF-I:IGFBP:VN complex-mediated enhanced cell survival. IGF-I:IGFBP:VN complexes also stimulate an increase in breast cell migration coincident with a greater than 2-fold change in expression of genes involved in migration, invasion, survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1) and epithelial to mesenchymal transition (Snail, Slug, Twist, E-cadherin). This differential gene expression was detected by microarray analysis on breast cells which had migrated in response to the IGF-I:IGFBP:VN comples and was further confirmed using real-time PCR analysis. These changes were not detected when the IGF-I analogue ([L24 ][A31 ]-IGF-I), which fails to bind to the IGF-I receptor, was substituted. This supports the involvement of the IGF system in the differential expression of these genes associated with enhanced cell movement. These cell-based studies have established that IGF and VN synergistically act to enhance survival and epithelial to mesenchymal transition of breast cells,
S21
processes central to facilitating transformation towards the invasive and metastatic phenotype. Preliminary tissue microarray studies in clinical samples from breast cancer patients also suggest that differences in the expression and localisation of proteins within the IGF/VN signalling pathway are implicated in breast cancer progression. Taken together, this study highlights the interdependence of ECM and growth factor interactions in breast cancer metastasis and identifies novel targets that may hold potential for the development of therapies that prevent breast cancer progression. OR8,48 Differential impacts of IGFBP-3 in epithelial IGF-induced lung cancer development W.-Y. Kim1 , M.-J. Kim2 , H.-J. Moon3 , P. Yuan4 , J.-S. Kim1 , J.-K. Woo1 , G. Zhang1 , Y.-A. Suh5 , L. Feng6 , C. Behrens1 , C.S. Van Pelt7 , H. Kang8 , J.J. Lee6 , W.-K. Hong1 , I.I. Wistuba9 , H.-Y. Lee10 . 1 Thoracic/Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2 Pathology, University of Ulsan College of Medicine, Seoul, South Korea; 3 Department of Mathematics and Statistics, California State University – Long Beach, Long Beach, CA, USA; 4 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 5 Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 6 Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 7 Veterinary Medicine and Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 8 Roosevelt Hospital, Columbia Unversity College of Physicians and Surgeons, New York, NY, USA; 9 Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 10 Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA IGF axis has been implicated in the risk of various cancers. We previously reported a potential role of tissue-derived IGFs in lung tumor formation and progression. However, the role of IGF-binding protein (IGFBP)-3, a major IGFBP, on the activity of tissue-driven IGFs in lung cancer development is largely unknown. Here, we show that IGF-1, but not IGF-2, protein levels in non-small cell lung cancer (NSCLC) were significantly higher than those in normal and hyperplastic bronchial epithelium. We found that IGF-1 and IGFBP-3 levels in NSCLC tissue specimens were significantly correlated with phosphorylated IGF-1 receptor (pIGF-1R) expression. We investigated the impact of IGFBP3 expressions on the activity of tissue-driven IGF-1 in lung cancer development using mice carrying lung-specific human IGF-1 transgene (Tg), a germ linenull mutation of IGFBP-3, or both were analyzed. Compared to wild-type (BP3+/+ ) mice, mice carrying heterozygous (BP3+/− ) or homozygous (BP3−/− ) deletion of IGFBP-3 alleles exhibited decreases in circulating IGFBP-3 and IGF-1. Unexpectedly, IGFTg mice with 50% of physiological IGFBP-3 (BP3+/− ; IGFTg ) showed higher levels of pIGF1R/IR and a greater degree of spontaneous or tobacco carcinogen [4(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK]-induced lung tumor development and progression than did the IGFTg mice with normal (BP3+/+ ; IGFTg ) or homozygous deletion of IGFBP-3 (BP3−/− ; IGFTg) . These data show that IGF-1 is overexpressed in NSCLC, leading to activation of IGF-1R, and that IGFBP-3, depending on its expression level, either inhibit or potentiates IGF-1 actions in lung carcinogenesis. This work was supported by National Institutes of Health grants R01 CA109520 and CA100816–01A1 (to H.-Y. L.) and in part by grants from the National Foundation for Cancer Research, the Department of Defense, the VITAL program (W81XWH-04–1–0142), and the BATTLE program (W81XWH-06–1–0303) (to W.-K. H.).