- Email: [email protected]
OR6,33 Hyperglycaemia reduces the efficacy of chemotherapeutic agents in DU145 prostate cancer (CaP) cells: Involvement of IGFBP-2
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Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
in GH-induced activation of many genes including IGF-I, Socs2, Cis, IGFALS, and Spi2.1. Among these genes, unique features of IGF-I include that its transcribed region of >70 kb is substantially larger than the others, that it uses two independent promoters to direct transcription, and that its promoters lack Stat5 binding domains, which are instead scattered throughout its gene locus. We have recently characterized chromatin events at the two IGF-I promoters following acute GH stimulation in a hypophysectomized rat model, and observed that the mechanisms of activation of the two promoters are distinct. Whereas RNA polymerase II is recruited to promoter 2 upon GH treatment, both RNA polymerase II and the co-activator p300 occupy promoter 1 even in the absence of GH, indicative of a poised state. Here we sought to characterize the acute epigenetic actions of GH at a cohort of hormone-stimulated, Stat5b-dependent genes. We first found that the transcription of all five genes tested was rapidly stimulated by GH treatment, but as measured by quantitative transcription assay, the magnitude of gene activation for IGF-I, Socs2, and Cis was over 10-times greater than for ALS and Spi2.1. Histone modifications at the promoters were similar in the genes with acute GH-stimulated increases is H3 and H4 acetylation and significant enrichment in H3 K4 trimethylation, with the exception of Spi2.1 which did not possess these marks typical of activated genes. As expected, Stat5b was recruited to the promoters of the set of genes in a GH-dependent way, but interestingly we found the transcriptional coactivators p300 and TRAP220/Med1 were present at IGF-I promoter 1 but not at any of these four genes. RNA polymerase II was detected at transcribed segments following GH in all the genes, but was most abundant upstream of the transcription start site in the basal state at IGF-I promoter 1. Finally we detected enrichment of the transcriptional repressor Bcl6 at the promoters of the Socs2 and Cis genes in the absence of hormone signaling, with acute loss of this signal upon GH treatment coincident with the recruitment of Stat5 to the same regions. In contrast, the levels of Bcl6 before and after GH at IGF-I promoter 1 were similar to an unrelated control segment. In summary, we conclude that gene activation by GH via Stat5b involves distinct promoter-specific mechanisms utilizing different transcriptional co-regulatory complexes.
Oral session 6. IGFBPs: from evolution to cancer OR6,31 Identification and functional characterization of an ancient IGFBP from Amphioxus: Evolution of the IGFBP family J. Zhou1 , J. Xiang2 , C. Duan3 . 1 School of Medicine and Pharmacy, Ocean University of China, Qingdao, China; 2 Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; 3 MCDB, University of Michigan, Ann Arbor, MI, USA Insulin-like growth factor binding proteins (IGFBPs) are high affinity binding partners for IGFs and play important roles in regulating animal growth, reproduction, and longevity by controlling IGF availability and bioactivity. All known vertebrate IGFBPs are secreted proteins but some can also be found in the nucleus and possess IGF-independent actions. To understand the evolution of this gene family, we cloned and characterized an IGFBP gene from Amphioxus, the most basal lineage of chordates. In contrast to the presence of 6 IGFBP genes in human and other mammal genomes and about a dozen IGFBP genes in teleost genomes, only a single IGFBP gene is found in the Amphioxus genome. The genomic and protein structure of Amphioxus IGFBP (AmphiBP) is similar to its vertebrate counterparts. It is consisted of 4 exons and 3 introns and encodes a mature protein with a N-, L-, and C-domain. There is an IGFBP motif (GCGCCXXC) in the N-domain. To test whether AmphiBP is capable of IGF binding and to determine its cellular location, AmphiBP cDNA was cloned into an expression vector and introduced to culture mammalian cells by transient transfection.
AmphiBP was secreted into the cultured media. Ligand blot analysis indicated that secreted AmphiBP was capable of IGF binding. Like IGFBP-3, -5, and -6, AmphiBP is also found in the nucleus and its N-domain had transactivation activity when tested in a onehybrid assay. To examine its biological activity, AmphiBP mRNA was injected into zebrafish embryos along with vertebrate IGFBP-5 and -2 mRNA. Forced expression of IGFBP-5 in developing zebrafish embryos resulted in reduced trunk and tail, while forced expression IGFBP-2 caused no morphological abnormality but significantly decreased the growth and developmental rates. Forced expression of AmphiBP resulted in a phenotype very similar that of IGFBP-5. These results suggest that IGFBPs are ancient molecules present in all chordates. The AmphiBP shares many similar structural and biological properties with vertebrate IGFBP-3 and -5. These results provide novel insights into the structural and functional evolution of the IGFBP family. OR6,32 A novel role of insulin-like growth factor binding protein (IGFBP)-5 in calcium homeostasis in zebrafish embryos W. Dai1 , P.-P. Huang2 , C. Duan1 . 1 MCDB, University of Michigan, Ann Arbor, MI, USA; 2 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Insulin-like growth factor binding proteins (IGFBPs) are high affinity binding partners for IGFs and play important roles in regulating IGF availability and actions. IGFBP-5 is the most conserved member of this gene family. Recently, we have shown that zebrafish have two distinct IGFBP-5 genes, likely resulted from a gene duplication event during teleost evolution. These two genes exhibited distinct spatial and temporal expression patterns. Interestingly, one of the two genes, igfbp-5a, is specifically expressed in epidermal ionocytes surrounding the yolk sac and gill arches. Fluorescent double-labeled in situ hybridization analysis revealed that igfbp5a mRNA is co-localized with trpv6 mRNA but not with the H+-ATPase atp6v1al mRNA. In fish, the epithelial Ca2+ channel Trpv6-expressing ionocytes are known to be important for calcium uptake. Acclimation of zebrafish embryos to artificial water with altered ion concentrations showed that igfbp-5a mRNA levels are increased in response to reduced calcium concentrations. Low Ca2+ freshwater also increased the number and size of the igfbp-5amRNA expressing ionocytes, while it had no effect on the atp6v1almRNA expressing ionocytes. To test the role of IGFBP-5a in these cells and in calcium metabolism, antisense morpholinos were used to knockdown IGFBP-5a in zebrafish embryos. Compared with the control embryos, IGFBP-5a knocked down embryos had increased calcium content and calcium influx. Knocking down of IGFBP-5a also resulted in an increase in the number of Trpv6-expressing ionocytes. These findings suggest that IGFBP-5a is specifically expressed in Trpv6-expressing ionocytes and its expression is regulated by environmental calcium concentrations. Our study in zebrafish has also unraveled a novel role of IGFBP-5 in calcium homeostasis. OR6,33 Hyperglycaemia reduces the efficacy of chemotherapeutic agents in DU145 prostate cancer (CaP) cells: Involvement of IGFBP-2 C.M. Perks1 , C. Uzoh1 , L. Zeng1 , A. Bahl2 , R. Persad3 , J.M.P. Holly1 . 1 Clinical Science North Bristol, University of Bristol, Bristol, United Kingdom; 2 UBHT, Bristol Haematology & Oncology Centre, Bristol, United Kingdom; 3 Bristol Royal Infirmary, Department of Urology, Bristol, United Kingdom Background: Although prostate cancer is among the most prevalent cancers in Westernised societies; clinically evident disease is relatively rare in Eastern societies. Westernised lifestyle and consequent changes in metabolic status are the main causes of these geographical differences. In Western societies metabolic
Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
disturbances and type2 diabetes are very prevalent. Epidemiology studies have indicated that pre-existing diabetes adversely affects outcomes for patients with prostate cancer. Androgen deprivation therapy (ADT) remains the mainstay for managing recurrent or metastatic CaP. However when androgen-independence (AI) develops, chemotherapeutic agents (docetaxel) are favoured. Over 50% of men on ADT for at least 12 months develop insulin resistance and frank hyperglycaemia. Alterations in the insulin-like growth factor (IGF) axis also provide a link between insulin resistance, hyperglycaemia and the biology of CaP. Supra-physiological levels of glucose can induce production of IGFBP-2, which is increased 2–3 fold in patients with CaP and correlates with tumour grade, prostate specific antigen and poor prognosis. Aim: To assess the effects of supra-physiologic glucose concentrations on the response of DU145 prostate cancer cells to docetaxel and investigate the involvement of IGFBP-2. Methods: DU145 human CaP cells were seeded in 6 well plates (2×105 cells/well) in normal (5 mM) glucose-containing growth media for 24 hours and then placed into serum-free media containing either high (25 mM) or normal (5 mM) levels of glucose for a further 24 hours. They were then dosed with docetaxel (0–55 nM) +/− IGFBP-2 siRNA and a non-silencing control for 24 hours. Cell death was assessed using Trypan blue cell counting and apoptosis confirmed by measuring PARP cleavage. Abundance of IGFBP-2 was assessed using Western immunoblotting and ELISA. DNA methylation and acetylation status of the IGFBP-2 gene were assessed using combined bisulfite restriction analysis (COBRA) and chromatin immunoprecipitation assays (CHIP) respectively. Results: Docetaxel caused a dose-dependent increase in cell death under normal glucose conditions with a maximal increase of 32% at 45 nM docetaxel. However, under high glucose conditions, the cells were less sensitive to docetaxel-induced apoptosis, with 45 nM docetaxel only producing an increase of 21%. We confirmed apoptotic cell death by showing increased PARP cleavage. This reduced sensitivity to docetaxel observed under hyperglycaemic conditions was associated with a significant increase in IGFBP-2 production (p < 0.001). We found that the survival effect against docetaxel-induced apoptosis observed under hyperglycaemic conditions was mediated by IGFBP-2, since it was negated upon silencing IGFBP-2 using siRNA. We next determined that hyperglycaemic conditions epigenetically upregulated IGFBP-2, not via DNA methylation but through increased histone acetylation and that this involved histones 3 and 4. Summary: DU145 human CaP cells are more resistant to apoptosisinducing agents when grown in high glucose environments and this is via an epigenetic up-regulation of IGFBP-2. Conslusions: This may have important implications in the management of androgen independent prostate cancer especially in the poorly controlled diabetic. OR6,34 IGFBP-3 is a metastasis suppression gene for prostate cancer H. Mehta1 , L. Cobb1 , J. Said2 , A. Lee3 , D. Dong3 , K.-W. Lee1 , P. Cohen4 . 1 Pediatric Endocrinology, UCLA, Los Angeles, CA, USA; 2 Pathology, UCLA, Los Angeles, CA, USA; 3 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA; 4 Pediatric Endocrinolgy, UCLA, Los Angeles, CA, USA In addition to its role as the major serum carrier of IGFs, Insulinlike growth factor binding protein (IGFBP)-3 is a potent proapoptotic and anti-angiogenic protein in prostate cancer (CaP) in vitro and in vivo. Epidemiological studies suggest that low IGFBP-3 is associated with more aggressive CaP. In collaboration with Lexicon, we generated IGFBP3KO mice to study the in vivo actions of IGFBP-3. Initial characterization of the BP3KO mice revealed that they have 60% reduced serum IGF-1, and no detectable circulating IGFBP-3 and are 20% larger than wild-type by 3 weeks of age. No
S15
spontaneous prostate tumor development was observed in BP3KO mice; however, we did observe splenic lymphomas in 30 of BP3KO mice, but not WT, at 80 weeks of age. To investigate the significance of endogenous IGFBP-3 in the development of prostate cancer in vivo we crossed BP3KO mice with the Myc model of prostate cancer (expressing human c-Myc driven by a prostate specific promoter). Myc mice develop slow growing, non-metastatic tumors. Surprisingly, at 17 weeks of age, the prostates of BP3KO/Myc mice did not develop cancer and only displayed Prostate Intraepithelial Neoplasia while control Myc mice had well differentiated CaP (p < 0.05), which we attribute to the lower circulating IGF-I levels. By 24 weeks of age, well differentiated cancer was observed in all mice regardless of IGFBP3 status. However, when BP3KO/Myc mice were necropsied at 80 weeks of age, we identified lung metastases in 55% of them, while no WT/Myc mice exhibited any metastases. This is a remarkable finding, as metastases in the Myc model of CaP have never been described previously, and suggests that a major anti-tumorigenic role of endogenous IGFBP-3 is as a metastasis suppressor. To identify a mechanism for these effects, we assessed the expression of GRP78 by immunohistochemistry in the prostates of WT/Myc and BP3KO/Myc mice. GRP78 is a molecular chaperone, previously reported to interact with IGFBP-3, whose increased expression has been implicated with cancer metastasis and poor prognosis. GRP78 was barely detectable in WT/Myc mice but was 3- to 5-fold up-regulated in mice lacking IGFBP-3, suggesting that the anti-metastatic actions of IGFBP-3 may occur, in part, by downregulation of GRP78. These important findings reveal a novel role for IGFBP-3 (previously considered to be a tumor suppressor) as an anti-metastatic agent, opening new therapeutic possibilities for IGFBP-3 in cancer patients. OR6,35 Heparin binding properties of the IGF-II/IGFBP-2 complex J. Lund1 , M.T. Søndergaard1 , S.H. Jensen1 , F. Aasted1 , E.K. Andresen1 , C.A. Conover2 , M.T. Overgaard1 . 1 Section for Biotechnology, Aalborg University, Aalborg, Denmark; 2 Endocrine Research, Mayo Clinic Foundation, Rochester, MN, USA Insulin-like growth factor-I and -II (IGF-I and -II) are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF binding proteins (IGFBP-1–6) are primary regulators of the IGF bioavailability through mechanisms of transport in the circulation, regulation of metabolic clearance, delivery to target tissues and direct modulation of the cellular response. Generally, interaction with an IGFBP inhibits IGF-receptor activation, but some IGFBPs have been demonstrated to have a potentiating effect, presumably through targeting the IGF to or near the receptor located on the cell surface. Activation of an IGF from the binary complex can be achieved through specific proteolytic processing, post-translational modifications and extra-cellular matrix (ECM) binding. IGFs have been considered promising anabolic agents for the treatment of osteoporosis, and in particular the IGF-II/IGFBP-2 complex has been hypothesized to be able to specifically stimulate osteoblast function and increase skeletal mass, through targeted binding to bone ECM. IGF-II binding to IGFBP-2 greatly increases the affinity for 2- or 3-carbon O-sulphated glycosaminoglycans (GAGs) e.g. heparin and heparan sulphate, abundantly found in bone tissue ECM. However, GAGs and specifically heparan sulphate are ubiquitously found in proteoglycans throughout the human body. Hence, a detailed knowledge of the interactions between the IGFII/IGFBP-2 complex and GAGs is a pivotal point in understanding the extent of bone specific targeting and the risk of unintended side effects following administration of the growth factor complex. The goal of this study was to investigate GAG binding properties and mechanism of IGFBP-2 and IGF-II/IGFBP-2 complex. We have generated mutations within each of the three proposed heparin binding sites in the IGF-II/IGFBP-2 complex, two in IGFBP-2 (linker-
Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
in GH-induced activation of many genes including IGF-I, Socs2, Cis, IGFALS, and Spi2.1. Among these genes, unique features of IGF-I include that its transcribed region of >70 kb is substantially larger than the others, that it uses two independent promoters to direct transcription, and that its promoters lack Stat5 binding domains, which are instead scattered throughout its gene locus. We have recently characterized chromatin events at the two IGF-I promoters following acute GH stimulation in a hypophysectomized rat model, and observed that the mechanisms of activation of the two promoters are distinct. Whereas RNA polymerase II is recruited to promoter 2 upon GH treatment, both RNA polymerase II and the co-activator p300 occupy promoter 1 even in the absence of GH, indicative of a poised state. Here we sought to characterize the acute epigenetic actions of GH at a cohort of hormone-stimulated, Stat5b-dependent genes. We first found that the transcription of all five genes tested was rapidly stimulated by GH treatment, but as measured by quantitative transcription assay, the magnitude of gene activation for IGF-I, Socs2, and Cis was over 10-times greater than for ALS and Spi2.1. Histone modifications at the promoters were similar in the genes with acute GH-stimulated increases is H3 and H4 acetylation and significant enrichment in H3 K4 trimethylation, with the exception of Spi2.1 which did not possess these marks typical of activated genes. As expected, Stat5b was recruited to the promoters of the set of genes in a GH-dependent way, but interestingly we found the transcriptional coactivators p300 and TRAP220/Med1 were present at IGF-I promoter 1 but not at any of these four genes. RNA polymerase II was detected at transcribed segments following GH in all the genes, but was most abundant upstream of the transcription start site in the basal state at IGF-I promoter 1. Finally we detected enrichment of the transcriptional repressor Bcl6 at the promoters of the Socs2 and Cis genes in the absence of hormone signaling, with acute loss of this signal upon GH treatment coincident with the recruitment of Stat5 to the same regions. In contrast, the levels of Bcl6 before and after GH at IGF-I promoter 1 were similar to an unrelated control segment. In summary, we conclude that gene activation by GH via Stat5b involves distinct promoter-specific mechanisms utilizing different transcriptional co-regulatory complexes.
Oral session 6. IGFBPs: from evolution to cancer OR6,31 Identification and functional characterization of an ancient IGFBP from Amphioxus: Evolution of the IGFBP family J. Zhou1 , J. Xiang2 , C. Duan3 . 1 School of Medicine and Pharmacy, Ocean University of China, Qingdao, China; 2 Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; 3 MCDB, University of Michigan, Ann Arbor, MI, USA Insulin-like growth factor binding proteins (IGFBPs) are high affinity binding partners for IGFs and play important roles in regulating animal growth, reproduction, and longevity by controlling IGF availability and bioactivity. All known vertebrate IGFBPs are secreted proteins but some can also be found in the nucleus and possess IGF-independent actions. To understand the evolution of this gene family, we cloned and characterized an IGFBP gene from Amphioxus, the most basal lineage of chordates. In contrast to the presence of 6 IGFBP genes in human and other mammal genomes and about a dozen IGFBP genes in teleost genomes, only a single IGFBP gene is found in the Amphioxus genome. The genomic and protein structure of Amphioxus IGFBP (AmphiBP) is similar to its vertebrate counterparts. It is consisted of 4 exons and 3 introns and encodes a mature protein with a N-, L-, and C-domain. There is an IGFBP motif (GCGCCXXC) in the N-domain. To test whether AmphiBP is capable of IGF binding and to determine its cellular location, AmphiBP cDNA was cloned into an expression vector and introduced to culture mammalian cells by transient transfection.
AmphiBP was secreted into the cultured media. Ligand blot analysis indicated that secreted AmphiBP was capable of IGF binding. Like IGFBP-3, -5, and -6, AmphiBP is also found in the nucleus and its N-domain had transactivation activity when tested in a onehybrid assay. To examine its biological activity, AmphiBP mRNA was injected into zebrafish embryos along with vertebrate IGFBP-5 and -2 mRNA. Forced expression of IGFBP-5 in developing zebrafish embryos resulted in reduced trunk and tail, while forced expression IGFBP-2 caused no morphological abnormality but significantly decreased the growth and developmental rates. Forced expression of AmphiBP resulted in a phenotype very similar that of IGFBP-5. These results suggest that IGFBPs are ancient molecules present in all chordates. The AmphiBP shares many similar structural and biological properties with vertebrate IGFBP-3 and -5. These results provide novel insights into the structural and functional evolution of the IGFBP family. OR6,32 A novel role of insulin-like growth factor binding protein (IGFBP)-5 in calcium homeostasis in zebrafish embryos W. Dai1 , P.-P. Huang2 , C. Duan1 . 1 MCDB, University of Michigan, Ann Arbor, MI, USA; 2 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Insulin-like growth factor binding proteins (IGFBPs) are high affinity binding partners for IGFs and play important roles in regulating IGF availability and actions. IGFBP-5 is the most conserved member of this gene family. Recently, we have shown that zebrafish have two distinct IGFBP-5 genes, likely resulted from a gene duplication event during teleost evolution. These two genes exhibited distinct spatial and temporal expression patterns. Interestingly, one of the two genes, igfbp-5a, is specifically expressed in epidermal ionocytes surrounding the yolk sac and gill arches. Fluorescent double-labeled in situ hybridization analysis revealed that igfbp5a mRNA is co-localized with trpv6 mRNA but not with the H+-ATPase atp6v1al mRNA. In fish, the epithelial Ca2+ channel Trpv6-expressing ionocytes are known to be important for calcium uptake. Acclimation of zebrafish embryos to artificial water with altered ion concentrations showed that igfbp-5a mRNA levels are increased in response to reduced calcium concentrations. Low Ca2+ freshwater also increased the number and size of the igfbp-5amRNA expressing ionocytes, while it had no effect on the atp6v1almRNA expressing ionocytes. To test the role of IGFBP-5a in these cells and in calcium metabolism, antisense morpholinos were used to knockdown IGFBP-5a in zebrafish embryos. Compared with the control embryos, IGFBP-5a knocked down embryos had increased calcium content and calcium influx. Knocking down of IGFBP-5a also resulted in an increase in the number of Trpv6-expressing ionocytes. These findings suggest that IGFBP-5a is specifically expressed in Trpv6-expressing ionocytes and its expression is regulated by environmental calcium concentrations. Our study in zebrafish has also unraveled a novel role of IGFBP-5 in calcium homeostasis. OR6,33 Hyperglycaemia reduces the efficacy of chemotherapeutic agents in DU145 prostate cancer (CaP) cells: Involvement of IGFBP-2 C.M. Perks1 , C. Uzoh1 , L. Zeng1 , A. Bahl2 , R. Persad3 , J.M.P. Holly1 . 1 Clinical Science North Bristol, University of Bristol, Bristol, United Kingdom; 2 UBHT, Bristol Haematology & Oncology Centre, Bristol, United Kingdom; 3 Bristol Royal Infirmary, Department of Urology, Bristol, United Kingdom Background: Although prostate cancer is among the most prevalent cancers in Westernised societies; clinically evident disease is relatively rare in Eastern societies. Westernised lifestyle and consequent changes in metabolic status are the main causes of these geographical differences. In Western societies metabolic
Oral Presentations / Growth Hormone & IGF Research 20 (2010) S1–S38
disturbances and type2 diabetes are very prevalent. Epidemiology studies have indicated that pre-existing diabetes adversely affects outcomes for patients with prostate cancer. Androgen deprivation therapy (ADT) remains the mainstay for managing recurrent or metastatic CaP. However when androgen-independence (AI) develops, chemotherapeutic agents (docetaxel) are favoured. Over 50% of men on ADT for at least 12 months develop insulin resistance and frank hyperglycaemia. Alterations in the insulin-like growth factor (IGF) axis also provide a link between insulin resistance, hyperglycaemia and the biology of CaP. Supra-physiological levels of glucose can induce production of IGFBP-2, which is increased 2–3 fold in patients with CaP and correlates with tumour grade, prostate specific antigen and poor prognosis. Aim: To assess the effects of supra-physiologic glucose concentrations on the response of DU145 prostate cancer cells to docetaxel and investigate the involvement of IGFBP-2. Methods: DU145 human CaP cells were seeded in 6 well plates (2×105 cells/well) in normal (5 mM) glucose-containing growth media for 24 hours and then placed into serum-free media containing either high (25 mM) or normal (5 mM) levels of glucose for a further 24 hours. They were then dosed with docetaxel (0–55 nM) +/− IGFBP-2 siRNA and a non-silencing control for 24 hours. Cell death was assessed using Trypan blue cell counting and apoptosis confirmed by measuring PARP cleavage. Abundance of IGFBP-2 was assessed using Western immunoblotting and ELISA. DNA methylation and acetylation status of the IGFBP-2 gene were assessed using combined bisulfite restriction analysis (COBRA) and chromatin immunoprecipitation assays (CHIP) respectively. Results: Docetaxel caused a dose-dependent increase in cell death under normal glucose conditions with a maximal increase of 32% at 45 nM docetaxel. However, under high glucose conditions, the cells were less sensitive to docetaxel-induced apoptosis, with 45 nM docetaxel only producing an increase of 21%. We confirmed apoptotic cell death by showing increased PARP cleavage. This reduced sensitivity to docetaxel observed under hyperglycaemic conditions was associated with a significant increase in IGFBP-2 production (p < 0.001). We found that the survival effect against docetaxel-induced apoptosis observed under hyperglycaemic conditions was mediated by IGFBP-2, since it was negated upon silencing IGFBP-2 using siRNA. We next determined that hyperglycaemic conditions epigenetically upregulated IGFBP-2, not via DNA methylation but through increased histone acetylation and that this involved histones 3 and 4. Summary: DU145 human CaP cells are more resistant to apoptosisinducing agents when grown in high glucose environments and this is via an epigenetic up-regulation of IGFBP-2. Conslusions: This may have important implications in the management of androgen independent prostate cancer especially in the poorly controlled diabetic. OR6,34 IGFBP-3 is a metastasis suppression gene for prostate cancer H. Mehta1 , L. Cobb1 , J. Said2 , A. Lee3 , D. Dong3 , K.-W. Lee1 , P. Cohen4 . 1 Pediatric Endocrinology, UCLA, Los Angeles, CA, USA; 2 Pathology, UCLA, Los Angeles, CA, USA; 3 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA, USA; 4 Pediatric Endocrinolgy, UCLA, Los Angeles, CA, USA In addition to its role as the major serum carrier of IGFs, Insulinlike growth factor binding protein (IGFBP)-3 is a potent proapoptotic and anti-angiogenic protein in prostate cancer (CaP) in vitro and in vivo. Epidemiological studies suggest that low IGFBP-3 is associated with more aggressive CaP. In collaboration with Lexicon, we generated IGFBP3KO mice to study the in vivo actions of IGFBP-3. Initial characterization of the BP3KO mice revealed that they have 60% reduced serum IGF-1, and no detectable circulating IGFBP-3 and are 20% larger than wild-type by 3 weeks of age. No
S15
spontaneous prostate tumor development was observed in BP3KO mice; however, we did observe splenic lymphomas in 30 of BP3KO mice, but not WT, at 80 weeks of age. To investigate the significance of endogenous IGFBP-3 in the development of prostate cancer in vivo we crossed BP3KO mice with the Myc model of prostate cancer (expressing human c-Myc driven by a prostate specific promoter). Myc mice develop slow growing, non-metastatic tumors. Surprisingly, at 17 weeks of age, the prostates of BP3KO/Myc mice did not develop cancer and only displayed Prostate Intraepithelial Neoplasia while control Myc mice had well differentiated CaP (p < 0.05), which we attribute to the lower circulating IGF-I levels. By 24 weeks of age, well differentiated cancer was observed in all mice regardless of IGFBP3 status. However, when BP3KO/Myc mice were necropsied at 80 weeks of age, we identified lung metastases in 55% of them, while no WT/Myc mice exhibited any metastases. This is a remarkable finding, as metastases in the Myc model of CaP have never been described previously, and suggests that a major anti-tumorigenic role of endogenous IGFBP-3 is as a metastasis suppressor. To identify a mechanism for these effects, we assessed the expression of GRP78 by immunohistochemistry in the prostates of WT/Myc and BP3KO/Myc mice. GRP78 is a molecular chaperone, previously reported to interact with IGFBP-3, whose increased expression has been implicated with cancer metastasis and poor prognosis. GRP78 was barely detectable in WT/Myc mice but was 3- to 5-fold up-regulated in mice lacking IGFBP-3, suggesting that the anti-metastatic actions of IGFBP-3 may occur, in part, by downregulation of GRP78. These important findings reveal a novel role for IGFBP-3 (previously considered to be a tumor suppressor) as an anti-metastatic agent, opening new therapeutic possibilities for IGFBP-3 in cancer patients. OR6,35 Heparin binding properties of the IGF-II/IGFBP-2 complex J. Lund1 , M.T. Søndergaard1 , S.H. Jensen1 , F. Aasted1 , E.K. Andresen1 , C.A. Conover2 , M.T. Overgaard1 . 1 Section for Biotechnology, Aalborg University, Aalborg, Denmark; 2 Endocrine Research, Mayo Clinic Foundation, Rochester, MN, USA Insulin-like growth factor-I and -II (IGF-I and -II) are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF binding proteins (IGFBP-1–6) are primary regulators of the IGF bioavailability through mechanisms of transport in the circulation, regulation of metabolic clearance, delivery to target tissues and direct modulation of the cellular response. Generally, interaction with an IGFBP inhibits IGF-receptor activation, but some IGFBPs have been demonstrated to have a potentiating effect, presumably through targeting the IGF to or near the receptor located on the cell surface. Activation of an IGF from the binary complex can be achieved through specific proteolytic processing, post-translational modifications and extra-cellular matrix (ECM) binding. IGFs have been considered promising anabolic agents for the treatment of osteoporosis, and in particular the IGF-II/IGFBP-2 complex has been hypothesized to be able to specifically stimulate osteoblast function and increase skeletal mass, through targeted binding to bone ECM. IGF-II binding to IGFBP-2 greatly increases the affinity for 2- or 3-carbon O-sulphated glycosaminoglycans (GAGs) e.g. heparin and heparan sulphate, abundantly found in bone tissue ECM. However, GAGs and specifically heparan sulphate are ubiquitously found in proteoglycans throughout the human body. Hence, a detailed knowledge of the interactions between the IGFII/IGFBP-2 complex and GAGs is a pivotal point in understanding the extent of bone specific targeting and the risk of unintended side effects following administration of the growth factor complex. The goal of this study was to investigate GAG binding properties and mechanism of IGFBP-2 and IGF-II/IGFBP-2 complex. We have generated mutations within each of the three proposed heparin binding sites in the IGF-II/IGFBP-2 complex, two in IGFBP-2 (linker-