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P03-7 Hyperglycaemia, IGFs and insulin affect alternative splicing of the insulin receptor in DU145 prostate cancer cells
Posters / Growth Hormone & IGF Research 22 (2012) S33–S88
of human amylin/IAPP is also being measured. This data will be informative for developing pilot trials of vesiculin as a therapeutic agent in our proprietary human amylin/IAPP transgenic mouse model which is an excellent model for type 2 diabetes. In-vivo data has shown that murine synthetic vesiculin, like IGF-II has blood glucose lowering effects in mice with approximately 100x more peptide to elicit the same response. Ex-vivo experiments have also revealed that both human synthetic Insulin and synthetic murine vesiculin stimulate glucose conversion to muscle glycogen in isolated rat muscle with similar potencies. Findings to be presented will also include data from on-going in-vitro assessment on the proliferative and cytoprotective roles of both IGF-II and vesiculin as well as receptor specificities. P03-6 Structural studies of insulin-like growth factor I interacting with the type 1 Insulin-like growth factor receptor in the presence of inhibiting or enhancing monoclonal anti-bodies D. Thomas1 , G.K.W. Kong2 , J.G. Menting2 , M. Lawrence2 . 1 Max Planck Institute for Biochemistry, Molecular Structural Biology, Munich, Germany; 2 Walter+Eliza Hall Institute of Medical Research, Division of Structural Biology, Parkville, Australia The Type 1 insulin-like growth factor receptor (IGF-1R) is important for cell growth in both the normal physiological setting and many types of cancer. IGF-1R has attracted considerable pharmaceutical interest as a target for cancer therapy. Signaling via IGF-1R is initiated by the binding of insulin-like growth factor (IGF-I or IGF-II) to the receptor ectodomain, which, in the context of cancer, activates pathways leading to cellular proliferation, malignant transformation and/or inhibition of apotosis. Therapeutic monoclonal antibodies that block ligand binding or cause IGF-1R down-regulation have been developed and are in clinical trial. These complement an extensive suite of laboratory antibodies that modulate, to varying degrees, the affinity of IGF-1R for IGFs. It is thus of interest to elucidate the underlying mechanisms, such as conformational change, that are responsible for enhancement or inhibition of the ligand-receptor interaction. We have used cryo-electron microscopy to image the ectodomain of IGF-1R interacting with the mFabs alone or in combination with IGF-1 to determine what conformational change blocks ligand binding. We also have attempted to determine whether ligand binding interferes with mFab binding. There is also a specific monoclonal antibody which enhances the binding of the IGF-1. We are investigating the structural changes which lead to enhancement of binding of IGF-1. To date we can report that complete binding of the inhibiting mFab (2 mFabs per dimer) when the ligand is added first results in a pronounced asymmetry in the complex as compared to the complex with mFab alone. Monoclonal Fab binding is not complete, resulting in mixtures of complexes with one, two or no mFab bound. The complexes with only one mFab bound appear to have a common structure regardless of whether ligand added before or after the mFab, including when no ligand is present. These results and the technology that we have developed provide a platform for similar structural analyses of Fabs derived from monoclonal antibodies currently in clinical trial.
S73
P03-7 Hyperglycaemia, IGFs and insulin affect alternative splicing of the insulin receptor in DU145 prostate cancer cells J. Wang1 , C. Perks1 , E. Foulstone1 , M.L. Ladomery2 , J. Holly1 , IGFs and Metabolic Endocrinology Group (IMEG). 1 University of Bristol, School of Clincal Sciences, Bristol, United Kingdom; 2 University of the West of England, Faculty of Health and Life Sciences, Bristol, United Kingdom The insulin-like growth factor (IGF) family has been strongly implicated to play a role in prostate cancer and increasing evidence suggests that insulin may also be involved. Alternative splicing of the insulin receptor (IR) results in two isoforms: the full length IR-B and an IR-A isoform that lacks exon 11 and binds IGF-II in addition to insulin. IR-A has a 10 fold higher affinity for IGF-II than IR-B, and IGF-II is much more abundant in the body than insulin implying that IR-A may be a physiological receptor for IGF-II. We compared the abundance of the IR and the ratio of the IR-A: IR-B isoforms in different prostate cancer (CaP) cell lines (DU145, PC3, and LNCaP) and assessed if these were altered by the presence of the receptor ligands or altered glycaemic conditions. Total abundance of the IR was determined using immuno-blotting and total levels of mRNA for the IR together with specific assessment of the IR-A, IR-B isoforms were determined using Q-PCR. Immunoblotting showed that LNCaP and PC3 cells had comparable levels of IR that were higher than that found in DU145 cells. With DU145 cells the ratio of IR-A to IR-B significantly changed depending on whether the cells were grown in euglycaemic (5mM glucose) or hyperglycaemic (25mM glucose) conditions and following exposure to IGF-I, IGF-II or insulin. Hyperglycaemia increased the relative abundance of IR-B (From 40 to 70%) whereas exposure to IGF-I, IGF-II or insulin reduced the relative abundance of IR-B; even in hyperglycaemic conditions exposure to insulin and especially IGF-II resulted in predominant expression of IR-A. In euglycaemic conditions around 57% of insulin receptor expression was IR-A, but after exposure to IGF-I, IGF-II or insulin this increased to around 85%, 80% or 87% IR-A respectively. This means that in conditions with chronic hyperinsulinemia or IGFs the vast majority of the insulin receptors would be the isoform that responds to IGF-II. These data suggest that metabolic conditions that occur in diabetes may result in changes in the alternative splicing of the IR on epithelial cells resulting in an increase in the isoform that is activated by IGF-II. This could favour a more mitogenic response and potentially enhance the growth and survival of malignant cells promoting carcinogenesis. P03-8 Tumorigenicity of Ewing’s sarcoma cells is dependent on the level of beta-arrestin 1 I.I. Oprea1 , H. Zheng1 , C. Worrall1 , D. Nedelcu2 , A. Girnita1 , L. Girnita1 . 1 Karolinska Institute, Oncology-Pathology, Stockholm, Sweden; 2 ‘Iuliu Hatieganu’ University of Medicine and Pharmacy, Cluj-Napoca, Romania Background: The insulin-like growth factor receptor (IGF-1R) signaling plays essential roles in malignant transformation and treatment. External activation by the ligand, IGF-1, initiates a phosphorylation signaling cascade especially through the IRS/PI3K/AKT and Ras/Raf/MEK/ERK pathways. Recently, we have shown that beta-arrestin 1 (b-arr1) contribute to the IGF-1R signaling by controlling receptor internalization and scaffolding the components of the MAPK pathway. The Ewing’s sarcoma (ES) fusion protein fail to transform mouse embryonic fibroblasts in the absence of IGF-1R indicating a signal originating from the IGF-1R that facilitates and is "quasi-necessary" for transformation and maintenance of the ES malignant phenotype. Therefore present study’s aim was to investigate whether the b-arr1 mediated
of human amylin/IAPP is also being measured. This data will be informative for developing pilot trials of vesiculin as a therapeutic agent in our proprietary human amylin/IAPP transgenic mouse model which is an excellent model for type 2 diabetes. In-vivo data has shown that murine synthetic vesiculin, like IGF-II has blood glucose lowering effects in mice with approximately 100x more peptide to elicit the same response. Ex-vivo experiments have also revealed that both human synthetic Insulin and synthetic murine vesiculin stimulate glucose conversion to muscle glycogen in isolated rat muscle with similar potencies. Findings to be presented will also include data from on-going in-vitro assessment on the proliferative and cytoprotective roles of both IGF-II and vesiculin as well as receptor specificities. P03-6 Structural studies of insulin-like growth factor I interacting with the type 1 Insulin-like growth factor receptor in the presence of inhibiting or enhancing monoclonal anti-bodies D. Thomas1 , G.K.W. Kong2 , J.G. Menting2 , M. Lawrence2 . 1 Max Planck Institute for Biochemistry, Molecular Structural Biology, Munich, Germany; 2 Walter+Eliza Hall Institute of Medical Research, Division of Structural Biology, Parkville, Australia The Type 1 insulin-like growth factor receptor (IGF-1R) is important for cell growth in both the normal physiological setting and many types of cancer. IGF-1R has attracted considerable pharmaceutical interest as a target for cancer therapy. Signaling via IGF-1R is initiated by the binding of insulin-like growth factor (IGF-I or IGF-II) to the receptor ectodomain, which, in the context of cancer, activates pathways leading to cellular proliferation, malignant transformation and/or inhibition of apotosis. Therapeutic monoclonal antibodies that block ligand binding or cause IGF-1R down-regulation have been developed and are in clinical trial. These complement an extensive suite of laboratory antibodies that modulate, to varying degrees, the affinity of IGF-1R for IGFs. It is thus of interest to elucidate the underlying mechanisms, such as conformational change, that are responsible for enhancement or inhibition of the ligand-receptor interaction. We have used cryo-electron microscopy to image the ectodomain of IGF-1R interacting with the mFabs alone or in combination with IGF-1 to determine what conformational change blocks ligand binding. We also have attempted to determine whether ligand binding interferes with mFab binding. There is also a specific monoclonal antibody which enhances the binding of the IGF-1. We are investigating the structural changes which lead to enhancement of binding of IGF-1. To date we can report that complete binding of the inhibiting mFab (2 mFabs per dimer) when the ligand is added first results in a pronounced asymmetry in the complex as compared to the complex with mFab alone. Monoclonal Fab binding is not complete, resulting in mixtures of complexes with one, two or no mFab bound. The complexes with only one mFab bound appear to have a common structure regardless of whether ligand added before or after the mFab, including when no ligand is present. These results and the technology that we have developed provide a platform for similar structural analyses of Fabs derived from monoclonal antibodies currently in clinical trial.
S73
P03-7 Hyperglycaemia, IGFs and insulin affect alternative splicing of the insulin receptor in DU145 prostate cancer cells J. Wang1 , C. Perks1 , E. Foulstone1 , M.L. Ladomery2 , J. Holly1 , IGFs and Metabolic Endocrinology Group (IMEG). 1 University of Bristol, School of Clincal Sciences, Bristol, United Kingdom; 2 University of the West of England, Faculty of Health and Life Sciences, Bristol, United Kingdom The insulin-like growth factor (IGF) family has been strongly implicated to play a role in prostate cancer and increasing evidence suggests that insulin may also be involved. Alternative splicing of the insulin receptor (IR) results in two isoforms: the full length IR-B and an IR-A isoform that lacks exon 11 and binds IGF-II in addition to insulin. IR-A has a 10 fold higher affinity for IGF-II than IR-B, and IGF-II is much more abundant in the body than insulin implying that IR-A may be a physiological receptor for IGF-II. We compared the abundance of the IR and the ratio of the IR-A: IR-B isoforms in different prostate cancer (CaP) cell lines (DU145, PC3, and LNCaP) and assessed if these were altered by the presence of the receptor ligands or altered glycaemic conditions. Total abundance of the IR was determined using immuno-blotting and total levels of mRNA for the IR together with specific assessment of the IR-A, IR-B isoforms were determined using Q-PCR. Immunoblotting showed that LNCaP and PC3 cells had comparable levels of IR that were higher than that found in DU145 cells. With DU145 cells the ratio of IR-A to IR-B significantly changed depending on whether the cells were grown in euglycaemic (5mM glucose) or hyperglycaemic (25mM glucose) conditions and following exposure to IGF-I, IGF-II or insulin. Hyperglycaemia increased the relative abundance of IR-B (From 40 to 70%) whereas exposure to IGF-I, IGF-II or insulin reduced the relative abundance of IR-B; even in hyperglycaemic conditions exposure to insulin and especially IGF-II resulted in predominant expression of IR-A. In euglycaemic conditions around 57% of insulin receptor expression was IR-A, but after exposure to IGF-I, IGF-II or insulin this increased to around 85%, 80% or 87% IR-A respectively. This means that in conditions with chronic hyperinsulinemia or IGFs the vast majority of the insulin receptors would be the isoform that responds to IGF-II. These data suggest that metabolic conditions that occur in diabetes may result in changes in the alternative splicing of the IR on epithelial cells resulting in an increase in the isoform that is activated by IGF-II. This could favour a more mitogenic response and potentially enhance the growth and survival of malignant cells promoting carcinogenesis. P03-8 Tumorigenicity of Ewing’s sarcoma cells is dependent on the level of beta-arrestin 1 I.I. Oprea1 , H. Zheng1 , C. Worrall1 , D. Nedelcu2 , A. Girnita1 , L. Girnita1 . 1 Karolinska Institute, Oncology-Pathology, Stockholm, Sweden; 2 ‘Iuliu Hatieganu’ University of Medicine and Pharmacy, Cluj-Napoca, Romania Background: The insulin-like growth factor receptor (IGF-1R) signaling plays essential roles in malignant transformation and treatment. External activation by the ligand, IGF-1, initiates a phosphorylation signaling cascade especially through the IRS/PI3K/AKT and Ras/Raf/MEK/ERK pathways. Recently, we have shown that beta-arrestin 1 (b-arr1) contribute to the IGF-1R signaling by controlling receptor internalization and scaffolding the components of the MAPK pathway. The Ewing’s sarcoma (ES) fusion protein fail to transform mouse embryonic fibroblasts in the absence of IGF-1R indicating a signal originating from the IGF-1R that facilitates and is "quasi-necessary" for transformation and maintenance of the ES malignant phenotype. Therefore present study’s aim was to investigate whether the b-arr1 mediated