Monday, 7 November 2016

S1 Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

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Growth Hormone & IGF Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r . c o m / l o c a t e / g h i r

Abstracts of the Eighth International Congress of the GRS and the IGF Society 6–9 November 2016, Tel Aviv, Israel

Monday, 7 November 2016 Plenary: Plenary Session 1 602 Genetic diagnosis of growth hormone deficiency in children I.J. Prado Arnhold University of Sao Paulo, Brazil Genetic diagnosis of GHD is important to establish the etiology, predict prognosis including persistence of hormone deficiency as well as development of additional hormone deficiencies, allow genetic counseling and expand the knowledge of mechanisms involved in pituitary organogenesis and GH secretion. Congenital GHD can be isolated (IGHD), combined with deficiency of other pituitary hormones (CPHD), associated to developmental midline facial and/or brain defects, or with syndromes affecting different organs. The study of naturally occurring mutations in transcription factors and signaling molecules in mice and of transgenic animals improved our understanding of pituitary development and led to the diagnosis of several genetic causes of hypopituitarism by the candidate gene approach. The frequency of genetic diagnosis in congenital GHD has been higher in patients with a positive family history and/or offspring of consanguineous parents, usually related to founder effects. In sporadic cases the incidence has been lower and interaction of gene variants with environmental risk factors may explain incomplete penetrance. Hormonal studies and imaging of the hypothalamic-pituitary area and of other brain structures have been useful to select candidate genes for genetic studies. More recently, the advent of massive parallel sequencing has expanded the phenotypes related to known genes and indicated variants in potential novel players and modulating factors whose interpretation has been challenging. 603 Current ideas on the biology of IGFBP-6: more than an IGF-II inhibitor? L. Bach Department of Medicine, Monash University, Melbourne, Australia IGFBP-6 is unique among the IGFBPs for its ~50-fold preferential binding of IGF-II over IGF-I. It is a relatively selective inhibitor of IGF-II actions, including proliferation, survival and differentiation of a wide range of cells. However, IGFBP-6 has also recently been shown to have a number of IGF-independent actions; for example, IGFBP-6 inhibits angiogenesis in vitro and this was confirmed in vivo during zebrafish development and in rhabdomyosarcoma xenografts. IGFBP-6 expression is decreased in a number of cancer cells and it has been postulated to act as a tumor suppressor, which may be facilitated by these complementary inhibitory effects on IGF-II actions and angiogenesis. 1096-6374/© 2016 Elsevier Ltd. All rights reserved.

However, IGFBP-6 also induces migration of tumor cells including rhabdomyosarcomas by an IGF-independent mechanism. In different cell lines, this chemotactic effect is mediated by p38, ERK and JNK MAP kinases and cross-talk between them. Prohibitin-2 is a ubiquitously expressed protein that modulates a range of cellular functions by its effects in mitochondria and other cellular compartments. IGFBP-6 binds to prohibitin-2 on the cell surface and the latter is required for IGFBP-6-induced migration by a mechanism that is independent of MAP kinases. IGFBP-6 expression is increased in a small number of cancers, which may reflect either IGF-independent actions or a compensatory mechanism to control IGF-II actions. The relative balance of IGF-dependent and IGF-independent actions of IGFBP-6 in vivo together with the related question regarding the roles of IGFBP-6 binding to IGF and nonIGF ligands are keys to understanding the physiological role of this protein.

Parallel Oral Session: Parallel I Oral Presentations 1 – GH Clinical Correlations 154 Results of somavaratan (VRS-317) dose increase in the first two years of treatment in pre-pubertal children with growth hormone deficiency (GHD) R.W. Charlton1, K. Di Trapani1, B. Bakker1, D. Ng2, N.M. Wright3 1 Versartis, Inc., Menlo Park, CA, USA; 2ResearchPoint Global, Inc., Austin, TX, USA; 3 Florida State University College of Medicine, Tallahassee, FL, USA Background: Somavaratan demonstrated height velocity (HV) and IGF-1 improvements in Phase 2a (n=64), and an ongoing extension study. Dosing frequency varied in Year 1, but all subjects initially received 5 mg/kg monthly (M) in divided doses. By Year 2, 58 subjects increased to 7 mg/kg divided twice-monthly (TM). IGF1-SDS, treatment-related adverse events (AEs), and growth are compared at both doses. Methods: 23 subjects initially received 5 mg/kg/M, 22 increased to 3.5 mg/kg/ TM in Year 2. 20 started on 2.5 mg/kg/TM, 17 increased in Year 2. 21 received 1.15 mg/kg/weekly for 6 months; 1/21 discontinued, 5/21 increased to 3.5 mg/ kg/TM at M6, 15/21 increased at M9. Results: Baseline mean IGF1-SDS for the total cohort was –1.71 (0.78). Dose increase to 3.5 mg/kg/TM increased mean IGF1-SDS peak from 0.09 (1.35) to 0.60 (1.42) and trough from –1.39 (1.03) to –0.67 (1.25). On the lower dose, 7 subjects in the M cohort had IGF1 excursions >2SDS, one of which was >3SDS (range 2.05, 3.75). After dose increase, 12 IGF1-SDS excursions >2 occurred in n=9, 2 of which were >3SDS (range 2.01, 3.67). All peaks >2SDS with subsequent trough values were transient, none were associated with AEs. Fewer subjects reported related AEs after dose increase than before (8 v. 35). No related SAEs were reported. Dose increase maintained annualized HV into the second year of treatment (8.04±2.59 cm/year vs. 7.96±2.32), and HT-SDS continued to improve (–2.3±0.63 vs. –1.7±0.76).

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Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

Table 1 (abstract 72) Clinical characteristics of children and young adults who had a cerebrovascular event based on risk group No of patients (sex)

Age median (range)

Age at AE median (range)

GH dose at AE in mg/kg/wk median (range)

Gp1 (low): IGHD, ISS and SGA (low)

5 (1F, 4M)

8.6 (3.9–11.8)

9.8 (5.2–17.4)

Gp2 (intermediate): Organic GHD, severe chronic pediatric diseases, TS and PWS

8 (3F, 5M)

6.6 (1.0–12.7)

Gp3 (high): Organic GHD (malignant tumor), craniopha-ryngioma and CRI

14 (6F, 8M)

9.3 (2.6-15.6)

Risk group category

Crude incidence rates per 100,000 p/years (95% CI)

SAE

Causality

Outcome

0.27 (0.21–0.39)

5 Yes

4 No 1 Missing

2 recovered 2 recovered with a sequelae 1 unknown

2.7 (1.2–6.4)

9.9 (7.9–17.8)

0.21 (0.14–0.32)

7 Yes, 1 No

6 No 2 Missing

2 recovered 5 deaths 1 unknown

10.1 (5.1–19.9)

13.1 (6.4–17.5)

0.21 (0.12–0.31)

14 Yes 2 Yes 1 Missing

11 No

4 recovered 1 recovering 6 recovered with a sequelae 3 deaths

52.8 (31.4–88.5)

Conclusions: Increasing somavaratan dose to 3.5 mg/kg/TM safely improved IGF1-SDS and HV. Transient IGF1 excursions occurred in a small number of subjects, without apparent clinical significance. 72 Cerebrovascular morbidity and mortality in GH-treated children and adults: experience from KIGS (Pfizer International Growth Database) and KIMS (Pfizer International Metabolic Database) C.C. Hubner1, A. Lindberg2, A.F. Mattsson2, A.C. Ianos3, J. Heissler4, J. Cara1 1 Endocrine Care, Pfizer Inc. New York, USA; 2Endocrine Care, Pfizer Health AB, Sollentuna, Sweden; 3Safety Surveillance and Risk Management, Pfizer Inc., Sandwich, UK; 4Safety Surveillance and Risk Management, Pfizer Inc., New York, USA Background: GH treatment (GHT) of children with growth disorders and adults with GH Deficiency (GHD) is considered to be safe and efficacious. A recent study evaluated the incidence of stroke morbidity and mortality in GHT French patients prompting a thorough review of all cases of cerebrovascular (CV) hemorrhagic and ischemic strokes reported under GHT in children and adults enrolled in KIGS and KIMS. Methods: In KIGS, 82,658 (males 58 %) patients up to 18 years of age, the crude Incidence/100,000 patient-yrs of treatment was calculated in 3 risk categories. In addition 771 KIMS patients (males 64%) with childhood onset GHD were followed from entry into KIMS to age 40 or last visit, if it occurred earlier. Results: 27 CV events, 10 hemorrhagic and 17 ischemic strokes were reported in KIGS. GHT mean (SD) duration: 3.2 (3.0) years; dose (mg/kg/w): 0.23 (0.06); follow-up time until CV event: 3.4 (3.0) years. Crude incidence rates/100000 p-years (95% CI) were in GHD, ISS and SGA (n=56,380) 2.7 (1.2-6.4), in Organic GHD, TS and PWS (n=18,969) 10.1 (5.1–19.9) and in organic GHD secondary to a malignant tumor, craniopharyngioma and CRI (n=7,309), 52.8 (31.4–88.5). Eight patients died, none in a low- risk category (IGHD, ISS or SGA). One case, who recovered, was reported in KIMS during 4178 p-years (TIA; GH dose 0.38 mg/day). Conclusions: The risk of ischemic or hemorrhagic stroke in GH treated children, without known risk factors, is within the reported range in population based studies. Study limitations include lack of appropriate comparator groups and short-term follow-up.

159 GH actions during fasting in obese human subjects: impact of GH blockade M. Høgild Pedersen1, N. Jessen2, J.O. Lunde Jørgensen1 1 Medical Research Laboratory, Department of Clinical Medicine, Aarhus University, Denmark; Department of Endocrinology and Internal Medicine (MEA), Aarhus University Hospital, Denmark; 2Research Laboratory for Biochemical Pathology, Department of Clinical Medicine, Faculty of Health, Aarhus University & Department of Clinical pharmacology, Aarhus University Hospital, Aarhus C, Denmark Background: Obesity is associated with insulin resistance and metabolic inflexibility. Prolonged fasting is a useful model to investigate the shift in metabolism from glucose to lipid oxidation and the induction of insulin resistance. Since GH promotes lipolysis we aimed to study the impact of GH blockade by means of pegvisomant administration in fasting obese subjects. Methods: Nine obese males were studied on three occasions in a randomized, single-blinded, cross-over trial: After an overnight fast, after 72 hours of fasting and concomitant saline injections, and after 72 hours of fasting and concomitant pegvisomant injections (20 mg × 3). Results: Pegvisomant reduced the glucose infusion rate during the clamp owing to a lower hepatic glucose production without significantly increased peripheral glucose uptake (P=0.18). GHR blockade also enhanced the fastinginduced IGF-I reduction (P<0.05) without enhancing the fasting-induced increase in GH levels (P>0.05). Fasting alone induced a marked increase in FFA levels and lipid oxidation, which was not influenced by pegvisomant, but pegvisomant as compared with saline increased glycerol levels during fasting (P<0.05). Fasting furthermore decreased basal glucose oxidation independent of pegvisomant (P<0.05). Conclusions: 1) Pegvisomant-induced suppression of GH activity during fasting in obese subjects reverses hepatic—but not peripheral—insulin resistance and lowers hepatic IGF-I production, 2) Pegvisomant also resulted in elevated circulating glycerol levels without affecting serum FFA levels or lipid oxidation 3) Our data support a role for GH in the regulation of glucose and fat metabolism during fasting in obese subjects. 71 Candidate selection of a long-acting growth hormone antagonist to treat acromegaly I.R. Wilkinson1, S.L. Pradhananga1, R. Speak1, J.R. Sayers1, R.J. Ross2 1 University of Sheffield; 2Academic Unit of Diabetes, Endocrinology and Reproduction University of Sheffield E Floor The Medical School Beech Hill Road Sheffield UK Background: Pegvisomant, a growth hormone antagonist (GHA) controls disease in >95% cases, but requires high dose daily injections and was not considered cost-effective in the UK (2). We developed a technology for a longacting GH through fusion to its binding protein (GHBP) (2). We adapted this technology to generate a GHA.

Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

Methods: 3 GHA candidates were generated: site 2 mutation (GHA1), site 1 and 2 mutations (GHA2), site 1 and 2 + W104A mutation in GHBP (GHA3). The site 2 mutation (G120R) produces a GHA and site 1 mutations enhance receptor binding. The GHBP W104A mutation was introduced as we hypothesised the site 1 mutations would increase intra- and inter-molecular binding and reduce bioactivity. In vitro bioactivity was measured using a GHspecific bioassay. For in vivo studies, rats were given 1 nM/kg (i.v) and rabbits 2 mg/kg (s.c). Results: In vitro bioactivity gave IC50s of 44nM (GHA1); 198nM (GHA2); and 17nM (GHA3). GHA1-3 had terminal half-lives of ~ 20hrs in rat. In rabbit GHA3 had a terminal half-life of 40 hours and induced a 14% reduction in IGF-I. Allometric scaling predicted terminal half-life of GHA3 in humans as ~168 h. Conclusions: A fusion of GH antagonist to its binding protein generates a long acting GH antagonist and introducing the W104A mutation in GHBP enhanced antagonist activity. GHA3 has the potential to be a second generation longacting potent GHA with potential for weekly dosing. 122 Multi-center phase Iv study on long-acting pegylated recombinant human growth hormone for the treatment of growth hormone children in china: a preliminary report L. Xiaoping1, G. Chunxiu2, F. Junfen3, L. Feihong4 1 Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; 2Beijing Children’s Hospital, Capital Medical University, Beijing, China; 3Children’s Hospital of Zhejiang University School of Medicine, Zhejiang, China; 4Children’s Hospital of Fudan University, Shanghai, China Background: Long-acting pegylated recombinant human growth hormone (PEG-rhGH) has been approved by the China Food and Drug Administration in January 2014. After the approval, a multicenter phase IV study was carried out to investgate the long-term safety and efficacy of the PEG-rhGH with GHD children in China. Methods: The inclusion criteria was prepubertal children aged 3 or above with GH peak concentration of <10 ng/mL in two different GH provocation tests. The study has 4 investigation groups with different protocals, included patient were treated with PEG-rhGH or daily rhGH for 26 weeks. The detail study design was showed in Figure 1. All participants were assessed at baseline and 4, 13, 25 weeks after treatment initiation. At each assessment, height and weight were monitored and blood samples were tested for serum IGF-1. In addition, safety was monitored to assess possible GH treatment side effects.

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Results: 770 patients from 4 different groups were completed the 26 weeks study. The growth velocity of GHD children treated with PEG-rhGH increased significantly when compaired to the baseline. There were no serious adverse events consider to be PEG-rhGH related. All the patient whom finished the 26 weeks study will be treated with suitable PEG-rhGH dosage and follow-up untill final height. Patient will be accessed every 3 month for height, weight, IGF-1 level and adverse events, and adjusted the dosage accordingly. Conclusions: PEG-rhGH showed great efficacy and safety in GHD children within the dosage from 0.01 to 0.20 mg/kg/week. 144 Body composition and muscle function in patients with acromegaly before treatment and during 10–20 years of follow-up L.M. Füchtbauer1, D.S. Olsson1, L.L. Norrman3, K.S. Sunnerhagen4, G. Johannsson2 1 Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg and Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden; 2 Department of Endocrinology, Sahlgrenska University Hospital, Göteborg, Sweden; 3Departement of Internal Medicine, Södra Älvsborgs Sjukhus, Borås, Sweden; 4Institute of Neuroscience and Physiology Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Background: Patients with acromegaly have decreased body fat (BF), increased extracellular water (ECW) and body cell mass (BCM) i.e. muscle mass. Muscle strength is less well studied, but has been found to be reduced. Methods: Muscle strength and body composition were investigated in 48 acromegalics at diagnosis and at one and 10–20 years after surgery. Isometric and concentric knee flexion and extension, fatigue-index and hand grip strength were measured with dynamometers. Body composition was assessed by a 4-compartment model using potassium-40 and tritiated water. Results were compared with an age- and gender-matched healthy cohort (relative to predicted). Results: At diagnosis we found IGF-I 2.89 (2.47–3.31) [mean (95% confidence interval)] times the upper limit of normal (ULN), increased BCM 1.13 (1.10– 1.16) and ECW 1.10 (1.04–1.16), and reduced BF 0.67 (0.59–0.75). These changes normalized one year after treatment [(IGF-I 0.78 ULN (0.34–1.22), BCM 1.06 (1.03–1.09), ECW 1.01 (0.94–1.08), BF 0.84 (0.77–0.91)]. Strength and fatigue-index of knee flexors and extensors were comparable to the reference population at diagnosis. In contrast, grip strength 0.88 (0.81–0.95) and endurance 0.86 (0.78–0.94) were reduced, but normalized one year after treatment. At 10–20 years of follow-up isometric knee flexion had increased to 1.54 (1.37–1.71) of predicted and fatigue-index had increased to 1.39 (1.28– 1.50), corresponding to a decreased endurance. Conclusions: Despite the increase in muscle mass, muscle strength is not increased in patients with untreated acromegaly. With long-term remission, muscle strength is either unchanged or increased.

Parallel Oral Session: Parallel II Oral Presentations 1 – IGF Binding Proteins 125 Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 is a predictive biomarker of Ewing sarcoma progression at diagnosis: relationship with ABCG2 and ABCF1 K. Scotlandi, C. Mancarella, L. Calzolari, S. Ferrari, P. Picci Laboratory of Experimental Oncology, Rizzoli Institute, Bologna, Italy

Fig. 1 (abstract 122). Study design.

Background: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF

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Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker. Methods: Gene expression microarray and quantitative PCR were used to analyze expression of principal components of IGF system in a cohort of 30 (training set) and 109 primary localized Ewing sarcoma samples (validation set). Association with prognosis was obtained with log-rank and survival curves. Mechanistic studies were performed in EWS cell lines overexpressing or silenced for IGF2BP3. Tests for in vitro parameter of malignancy were applied (MTT assay, soft-agar growth, migration, chemosensitivy to conventional chemotherapeutic drugs). Results: IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed considering either univariate and multivariate analysis (p=0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions. Oncogenic potential of IGF2BP3 was found to be mediated by ABCF1, an ABC family member with a key role in translation initiation.Direct correlation between IGF2BP3 and ABCF1 was also found in 109 EWS patients (p-value 0.0001). Conclusions: IGF2BP3 represents an indicator of prognosis in EWS. ABCF1 is novel putative mediator of IGF2BP3 effects. (Grants: FIRB RBAP11884M_005; AIRC_IG2013_14049) 64 IGFBP-4 fragments provide incremental prognostic information on cardiovascular events and mortality in patients with ST-elevation myocardial infarction R. Hjortebjerg1, S. Lindberg3, S. Pedersen3, R. Mogelvang3, J.S. Jensen3,4, C. Oxvig5, J. Frystyk1,2, M. Bjerre1 1 Medical Research Laboratory, Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus C, Denmark; 2Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus C, Denmark; 3 Department of Cardiology P, Gentofte University Hospital, Copenhagen, Denmark; 4Institute of Clinical Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 5Department of Molecular Biology and Genetics, Faculty of Science and Technology, Aarhus University, Aarhus C, Denmark Background: Fragments of insulin-like growth factor binding protein-4 (IGFBP-4) are potential new biomarkers for cardiac risk assessment. The fragments are generated upon specific cleavage by pregnancy-associated plasma protein-A (PAPP-A), which exerts proatherogenic activity. This study investigated the prognostic value of IGFBP-4 fragments in patients with STelevation myocardial infarction (STEMI). Methods: We prospectively included 656 patients with STEMI treated with percutaneous coronary intervention (PCI) from September 2006 to December 2008. Blood samples were drawn before PCI, and levels of intact IGFBP-4, N-terminal (NT)-IGFBP-4 and C-terminal (CT)-IGFBP-4 fragments were measured by specific assays. End-points were 5-year all-cause mortality, cardiovascular mortality and the combined end-point of major adverse cardiac events (MACE). Prognostic potential was evaluated relative to a clinical model in terms of discrimination, calibration, and reclassification analysis. Results: During follow-up, 136 patients died, of whom 69 died from cardiovascular causes. IGFBP-4 fragments were associated with all end-points (p<0.001). After multivariable adjustments, NT- and CT-IGFBP-4 fragment levels remained associated with all end-points, including cardiovascular mortality with hazard ratios (HR) (95% confidence interval (CI)) per doubling in protein concentration of 2.54 (1.59–4.07) (p<0.001) and 2.07 (1.41–3.04) (p<0.001), respectively. Incorporation of NT- or CT-IGFBP-4 into a clinical model with 15 risk factors improved discrimination (cardiovascular mortality C-statistic (95% CI) increase: 2.9% (0.38–5.5%) (p=0.025) and 2.7% (0.15–5.3%) (p=0.038), respectively) and calibration, and provided incremental prognostic contribution as assessed by net reclassification improvement and integrated discrimination improvement. Conclusions: Circulating IGFBP-4 fragments are associated with increased risk of all-cause mortality, cardiovascular mortality, and MACE in patients with STEMI.

66 Recombinant human (rh)IGF-1/rhIGFBP-3 improves alveolar growth and lung structure and prevents pulmonary hypertension in rodent models of bronchopulmonary dysplasia D. Keefe1, T. Nowlin2, G. Seedorf2, B. Wallace2, A. Peisl2, J. Martineau Bosco1, S. Abman2 1 Shire, Lexington, MA, USA; 2Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Denver Anschutz Medical Center, Aurora CO, USA Background: Antenatal factors such as chorioamnionitis and preeclampsia are strongly associated with increased risk for bronchopulmonary dysplasia (BPD), which in turn has been associated with decreased serum IGF-1 in preterm infants. We evaluated whether increasing IGF-1 through postnatal treatment of newborn rats with rhIGF-1/rhIGFBP-3 would improve lung growth and prevent pulmonary hypertension in models of BPD induced by chorioamnionitis or preeclampsia. Methods: Models of chorioamnionitis and preeclampsia were as previously established in our laboratory. Chorioamnionitis model: Endotoxin was administered intra-amniotically at 20 days gestation (E20). Preeclampsia model: sFlt-1 was administered intra-amniotically at E20. For both models, rat pups were delivered by c-section at E22, and treated with rhIGF-1/ rhIGFBP-3 (0.2–20 mg/kg/day, intraperitoneal injection) or formulation buffer for 2 weeks. Alveolarisation was measured by radial alveolar counts (RAC); right ventricular hypertrophy (RVH), a marker of pulmonary hypertension, was assessed with the weight ratio (right ventricle)/(left ventricle+septum). Results: Chorioamnionitis model: Compared with untreated controls, antenatal endotoxin reduced RAC and increased RVH by 42% and 72% in 2-week rats, respectively (p<0.01 for each). Preeclampsia model: Compared with controls, antenatal sFlt-1 reduced RAC and increased RVH by 32% and 46% in 2-week rats, respectively (p<0.01 for each). For both models, postnatal rhIGF-1/rhIGFBP-3 treatment enhanced RAC to normal values and prevented RVH at all study doses vs formulation buffer. Conclusions: Postnatal rhIGF-1/rhIGFBP-3 treatment restores lung structure and prevents RVH in two rodent models of BPD. We speculate that rhIGF-1/ rhIGFBP-3 treatment may provide a novel strategy for the prevention of BPD associated with chorioamnionitis or preeclampsia. 89 Decidual IGFBP-1 expression and phosphorylation are increased in human IUGR S.B. Singal2, M.S. Abu Shehab3, K. Nygard3, T. Jansson2, M.B. Gupta1 1 Departments of Pediatrics and Biochemistry, University of Western Ontario, London, ON Canada and CHRI, London, Ontario, Canada; 2Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; 3Biotron Laboratory, University of Western Ontario, London, Ontario, Canada Background: Maternal insulin-like growth factor (IGF)-I is a positive regulator of placental function and fetal growth. IGF-I bioavailability is determined by IGF binding proteins, in particular IGFBP-1. Phosphorylation of IGFBP-1 markedly increases the affinity for binding IGF-I, thereby inhibiting IGF-I action. The decidua is the major source for maternal circulating IGFBP-1 and changes in the expression and phosphorylation of decidual IGFBP-1 may therefore have a major impact on placental function and fetal growth. We hypothesize that IUGR is associated with increased IGFBP-1 expression/ phosphorylation in decidua. Our aim was to determine decidual IGFBP-1 levels and phosphorylation in IUGR. Methods: Basal plate decidua dissected from IUGR (<3rd percentile) (n=13) or gestational age matched control placenta (n=11) were used to determine total IGFBP-1 (Mab 6303) and IGFBP-1 phosphorylation (pSer101, 119 and 169-specific-antibodies) using immunobloting. Placenta (IUGR/ control (n=5 each)) were subjected to quantitative immunofluorescenceimmunohistochemistry (IHC) using vimentin (Alexa 660) and Mab6303 and/ or phospho-site-specific (Ser101/119/169) IGFBP-1 antibodies (Alexa 568). The significance was determined by simple regression and correlation with birthweight.

Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

Results: Immunobloting demonstrated an increase in IGFBP-1 (1.5-fold, P=0.0364) and IGFBP-1 phosphorylation (pSer101/2.2-fold (P=0.036), pSer119/1.8-fold (P=0.003) and pSer169/1.8-fold (P=0.008) in IUGR. Quantitative image analysis (Image Pro Premier software) demonstrated higher IGFBP-1 (1.8-fold (P=0.0001); negative correlation with birthweight), and increased IGFBP-1 phosphorylation (pSer169/ 2-fold (p=0.0030); pSer101/ 2-fold (P=0.05) in vimentin-positive-mesenchymal decidua cells in IUGR. Conclusions: This study offers first-clear evidence for site-specific hyperphosphorylation of decidual IGFBP-1 in IUGR and strong-basis to elucidate the mechanisms that regulate decidual (maternal) IGFBP-1 phosphorylation and contribute to the development of a growth-restricted human fetus. 112 The role of IGFBP-6 in the differentiation of placental mesenchymal stem cells into skeletal muscle V. Han, D. Aboalola Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada Background: Insulin-like growth factors are major components of the stem cell niche, as they regulate proliferation and differentiation of many tissues including skeletal muscle. Insulin-like growth factor binding protein-6 (IGFBP-6) is expressed in developing muscle cells and is the main regulator of IGF-II (fetal IGF). To date, no previous studies have been reported relating placental mesenchymal stem cells (PMSCs), muscle differentiation, and IGFBP-6. We hypothesized that IGFBP-6 regulates the maintenance of pluripotency of PMSCs and promotes their differentiation into muscle. Objectives: 1) to investigate the capacity of PMSCs to differentiate into muscle; and 2) to evaluate the intracellular and extracellular actions of IGFBP-6 on PMSCs muscle differentiation. Methods: Chorionic villi were collected from preterm placenta and stem cell identity was verified. Cells were then differentiated into muscle for 14 days and the impact of IGFBP-6 was investigated by adding IGFBP-6 or silencing it using siRNA. Results: Isolated cells differentiated into muscle cells, forming multinucleated fibers expressing muscle markers with increased levels of IGFBP-6 and decreasing levels of pluripotency markers. Additionally, extracellular addition of IGFBP-6 significantly increased protein levels of muscle commitment maker Pax3/7, with an increase at the earlier time points for muscle markers that goes down by time. Interestingly, both SOX2 and OCT4 levels correlate with IGFBP-6. On the other hand, silencing IGFBP-6, significantly decreased both pluripotency and differentiation markers at the earlier time points. Conclusions: IGFBP-6 regulates PMSC differentiation into muscle, with more prominent effects at the beginning of the differentiation process when PMSCs commit to muscle formation.

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97 Saturation of IGFBP-2 and levels of IGF carried by IGFBP-2 in human serum J. Ring Agerholm1, M. Bjerre4, W.A. Hassan1, J. Frystyk2,3 1 Department of Clinical Medicine, Medical Research Laboratories, Aarhus University, Aarhus, Denmark; 2Medical Research Laboratory, Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus C, Denmark; 3 Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus C, Denmark; 4Medical Research Laboratory, Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus C, Denmark Background: Serum IGFBP-2 is elevated in numerous diseases. IGFBP-2 is considered to bind the IGF-hormones, but the saturation of IGFBP-2 is unknown. Therefore, we aimed to measure IGFBP-2 saturation and IGFbinding in human serum. Methods: Serum IGFBP-2 was affinity purified with Dynabeads (Figure 1). Bound IGFBP-2 was eluted by acid extraction. After neutralization, eluates were assayed for IGFBP-2, IGF-I and -II concentrations by immunoassays and compared to serum levels prior to purification. This allowed estimation of the amount of IGF-I and -II bound to IGFBP-2 and calculation of IGFBP-2 saturation. To validate the method clinically, 52 serum-samples, 26 from patients with lung cancer and 26 from age- and sex-matched controls with other lung diseases were analyzed. Results: We were able to deplete IGFBP-2 from undiluted serum with high specificity and minimal cross-reactivity (Table 1). Clinical evaluation showed that IGF-binding to IGFBP-2 was unaltered in cancer patients compared to controls (Table 2). Furthermore, saturated IGFBP-2 correlated to total IGFBP-2 with an incline of 0.37 (r^2= 0.69, P<0.0001, Figure 2). Finally, IGFBP-2 bound approximately twice as much IGF-II than IGF-I (Table 2). Table 1 (abstract 97) Results are presented as means (95% confidence interval) Validation parameter How?

Results

IGFBP-2 depletion

Compare serum IGFBP-2 before and after depletion

97.1% (96.2-98.0)

IGFBP-2 recovery

Compare IGFBP-2 in eluate with serum before minus serum after

73.5% (69.5-77.8)

Specificity

Depletion of IGFBP-2 with non-sense rat IgG coated beads.

9.8% (7.2-12-4)

Cross-reactivity to IGFBP-3

Measure IGFBP-3 in the eluate

Undetectable

Table 2 (abstract 97) IGF-binding and IGFBP-2 saturation in patients with no-cancer and cancer Measure

No-cancer

Cancer

P value

IGF-I bound to IGFBP-2/serum total IGF-I *

6.7% (4.6–9.6)

5.5% (4.1–7.4)

0.4

IGF-II bound to IGFBP-2/serum total IGF-II

9.0% (6.8–11.3)

9.8% (7.3–12.3)

0.7

Saturated IGFBP-2/serum total IGFBP-2

38.5% (32.2–44.7)

40.3% (36.2–44.4)

0.6

Unsaturated IGFBP-2/serum total IGFBP-2

61.5% (55.3–67.8)

59.7% (55.6–63.8)

0.6

Results are presented as means (95% confidence interval). *IGF-I data are presented as geometric means (95% confidence interval).

Fig. 1 (abstract 97). Dynabead affinity purification of IGFBP-2 from serum. Principle sketch of the method.

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Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

124 The contribution of hepatic growth hormone receptor to osteodystrophy S. Yakar, Z. Liu David B. Kriser Dental Center, Department of Basic Science and Craniofacial Biology New York University College of Dentistry New York, NY, USA

Fig. 2 (abstract 97). Saturated IGFBP-2 plotted as a function of total IGFBP-2.

Conclusions: We have developed a method to determine the concentration of the IGF-hormones bound to IGFBP-2 in human serum. We show that approximately 40% of IGFBP-2 is saturated, and that the saturation is unchanged by the presence of lung cancer. We propose that this method may be helpful in elucidating the pathophysiological role of IGFBP-2 in humans.

Background: Hepatic osteodystrophy (HO) refers to metabolic bone disease (MBD) seen in patients with chronic liver disease. The duration and severity of the liver disease relates directly to MBD, such that patients with less sever liver disease manifest osteoporosis mainly loosing trabecular bone, and as liver disease progresses patients develop osteomalacia (softening of the bone tissue). The pathogenesis of HO is multifactorial and includes deficiency in IGF-1, subnormal vitamin D levels, and reduced osteoprotegrin (OPG) levels. The main goal of the present study was to define the contribution of growth hormone (GH) receptor in liver to HO. Methods: Liver specific GH receptor inactivation (Li-GHRKO) results in nonalcoholic fatty liver disease (NFLD) and dyslipidemia, reductions in serum insulin-like growth factor-1 (IGF-1), and severe osteopenia. To rule out the contribution of serum IGF-1 to osteopenia in the Li-GHRKO, we restored liver IGF-1 gene expression via hepatic IGF-1 transgene (HIT). Results: Li-GHRKO-HIT mice show normal levels of IGF-1 in serum, but still display NFLD and osteopenia (Figure 1). Using osteocyte-like cell line, primary osteocytes and primary osteoblast cultures we show that the dyslipidemia in the Li-GHRKO mice contributed to reduced GHR signaling in bone and increased osteocyte apoptosis, likely leading to increased bone resorption.

Parallel Poster Discussion Session: Parallel I GH Short Basic Science Posters 81 Quantification of UCP1 and oxygen consumption rate in adipose tissue depots of bGH mice K.M. Troike1, B.E. Henry3, G.C. Gase4, J.A. Young4, E.O. List4, J.J. Kopchick2, D.E. Berryman3 1 College of Health Sciences and Professions, Ohio University, Athens, OH, USA; 2 Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA; 3The Diabetes Institute, Ohio University, Athens, OH, USA; 4The Edison Biotechnology Institute, Ohio University, Athens, OH, USA Background: Studies investigating the effect of growth hormone (GH) on brown adipose tissue (BAT) activity and white adipose tissue (WAT) beiging have provided contradictory evidence for the role GH in these processes. The purpose of the current study was to quantify uncoupling protein 1 (UCP1) in BAT and WAT of bovine growth hormone transgenic (bGH) mice. Methods: Male bGH and littermate control mice were used for all studies. Quantitative PCR and RNA-Seq data were used for gene expression analysis while immunohistochemical staining and western blots were used for protein analysis. A Seahorse Bioscience XFe24 was used to quantify basal, whole tissue respiration. Results: UCP1 RNA was undetectable in the WAT depots of bGH and WT mice after qPCR and RNA-Seq analysis. Additionally, immunohistochemistry and western blot analysis revealed no UCP1 staining in WAT. UCP1 RNA and protein were present in BAT, though no significant genotype differences were observed. Oxygen consumption rate (OCR) in the WAT of WT mice was significantly higher than that of bGH at 4 months of age, a trend which reversed at 11 months of age. Conclusions: Under standard housing conditions, we observed no detectable amounts of UCP1 in WAT at the level of RNA or protein. Future studies will attempt to utilize cold exposure in order to activate BAT and induce beiging of WAT, thereby enabling detection of UCP1. This study revealed genotype differences in OCR, which suggests a potential age-dependent effect of GH on WAT respiration. This observation will be further investigated using isolated mitochondria from WAT.

Fig. 1 (abstract 124). Bone morphology. Micro computed tomography of femurs at 16, 52, and 104 weeks of age from control, HIT, Li-GHRKO, Li-GHRKO-HIT mice (n>8 in each group at each age). Scans were taken with a Skyscan 1172 instrument at 9.7 microns resolution.

Conclusions: We conclude that liver GHR leads to HO via its effects on overall lipid metabolism.

Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

106 Somapacitan binds reversibly to human albumin at two distinct binding sites E. Johansson, H. Demuth, A. Dybdal Nielsen, P. Thygesen Novo Nordisk A/S, Novo Nordisk Park, Maaloev, Denmark Background: Somapacitan is a human growth hormone (hGH) derivative. Somapacitan binds reversibly to albumin via a fatty acid attached to a single mutated amino acid on hGH. Somapacitan is currently in development for once-weekly treatment of growth hormone disorders. Methods: X-ray crystallography, small angle X-ray scattering (SAXS) and size exclusion chromatography (SEC) were used to identify somapacitan binding sites on human albumin. A ternary complex of somapacitan, albumin and the neonatal Fc receptor (FcRn) was crystallised. FcRn binds domain I and III of albumin, potentially blocking binding sites on these domains. The stoichiometry of somapacitan binding to albumin subunit preparations was examined. The albumin subunits used were domains I+II and III alone. Surface plasmon resonance (SPR) biosensor technology and Isothermal Titration Calorimetry (ITC) were used to investigate the binding of somapacitan and human albumin. Results: One somapacitan binding site on domain II was identified by X-ray crystallography. SEC suggested a possible binding site on domain III, while domain I+II only has one binding site for human albumin, corresponding to the site localised in the crystal structure (Figure 1). SPR and ITC analyses suggest that somapacitan binds to high and a low affinity binding sites on human albumin.

Fig. 1 (abstract 106). Somapacitan (grey) binding to human albumin (dark blue). Two binding sites identified at sites 5 and 6.

Conclusions: A model of human albumin with somapacitan binding at two sites: one at site 6 on domain II, as determined from crystal structures, and one at site 5 on domain III, deduced from SEC studies on full length and subunits of human albumin, have been identified. 43 Multiple Ca2+ channel-dependent components in growth-hormone secretion from rat anterior-pituitary somatotrophs I. Nussinovitch, A. Tzour, E. Sosial Department of Medical Neurobiology, Hebrew University Faculty of Medicine, IMRIC, Jerusalem, Israel Background: The involvement of L-type Ca2+ channels in growth-hormone (GH) secretion is well established, yet knowledge about the involvement of non-L-type Ca2+ channels is lacking. We recently demonstrated the existence

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on non-L-type Ca2+ channels in somatotrophs. In this study, we investigated whether these non-L-type Ca2+ channels participate in regulating GH secretion. Methods: GH secretion was monitored from dissociated male rat somatotrophs which were incubated for 15 minutes with 2 mM K+ (‘basal’ secretion), or 60 mM K+ (‘stimulated’ secretion). The role of non-L-type Ca2+ influx was investigated using specific Ca2+ channel blockers, including w-agatoxin-IVA, w-conotoxin GVIA or SNX-482, to block P/Q-, N- or R-type Ca2+ channels, respectively. Results: Our results demonstrate that P/Q-, N- and R-type Ca2+ channels contributed ~21%, ~20% and ~11%, respectively, to ‘basal’ GH secretion and ~18%, ~24% and ~14%, respectively, to ‘stimulated’ GH secretion. Following treatment with a ‘cocktail’ that comprised the previously described non-Ltype blockers, non-L-type Ca2+ channels contributed 50.9±0.4% and 45.5±2.0% to ‘basal’ and ‘stimulated’ GH secretion, respectively (Figure 1). Lipid raft disruption abrogated the cell surface compartmentalisation of Ca2+ channels and substantially reduced both ‘basal’ and ‘stimulated’ GH secretion, indicating the role of Ca2+ channels compartmentalisation in GH secretion.

Fig. 1 (abstract 43). Multiple Ca2+ channel dependent components in GH secretion: the relative contribution of Ca2+ channels to GH secretion was determined using specific Ca2+ channels blockers as described in the text. P/Q-, N- and R-type Ca2+ channels were blocked using the specific channels blockers; w-agatoxin-IVA (250 nM), w-conotoxin GVIA (2 M) and SNX-482(30 nM), respectively. The ‘cocktail’ was comprised of the same three blockers at the same concentrations. L-type Ca2+ channels were blocked using Nifedipine (10 M). Cadmium (Cd, 300 M) was used for complete block of voltage-gated Ca2+ influx. Adapted from: Sosial and Nussinovitch (2015) J Neuroendocrinology 27, 166–176.

Conclusions: This study demonstrates that GH secretion depends on Ca2+ influx through both L-type and non-L-type Ca2+ channels. The existence of multiple Ca2+ channels in somatotrophs provides a subcellular mechanism for selective regulation, or fine-tuning, of GH secretion. Moreover, these Ca2+ channels may serve as therapeutic targets for treatments of GH secretion disorders.

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Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

65 Production and characterization of adult onset growth hormone receptor knockout mice S. Duran Ortiz1, R.K. Junnila3, O. Suer3, E.O. List3, J.J. Kopchick2 1 Department of Biological Sciences, College of Arts and Sciences, Ohio University, Athens, Ohio, USA; 2Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio, USA; 3Edison Biotechnology Institute, Ohio University, Athens, OH, USA Background: Reduction in the growth hormone/insulin like growth factor-I (GH/IGF-I) axis increases lifespan in most animals. For example, the GH receptor (R) knockout (GHR–/–) mouse, which possesses a germline mutation that disrupts the Ghr gene, holds the record for the longest lived laboratory mouse. These mice are dwarf and have low rates of cancer, improved insulin sensitivity, and are resistant to diabetes. Because of the beneficial healthand lifespan effects seen in germline GHR–/– mice, we hypothesized that disruption of GH action in adult life would also increase longevity and health parameters. Methods: An adult-onset GHR knockout (aGHRKO) mouse was generated by global disruption of the Ghr gene at 6 weeks of age by using a Tamoxifen inducible ROSA26 Cre-Lox system. We characterized the aGHRKO mice by evaluating: (1) disruption of Ghr in 9 tissues; (2) growth; (3) body composition; and (4) metabolic parameters. Results: The aGHRKO mice had a decreased level of Ghr mRNA in all tissues tested except the heart. They were smaller than controls and showed lower levels of IGF-I and higher levels of GH than control mice. Additionally, aGHRKO mice exhibited increased relative fat mass, and decreased lean mass, as well as improved insulin sensitivity and impaired glucose tolerance relative to control mice. Conclusions: We generated a new mouse line with the Ghr gene disrupted at 6 weeks of age. These mice share characteristics with germline GHR–/– mice, in that they are dwarf, fat and insulin sensitive. Aging studies of the aGHRKO mice are ongoing. 147 Role of SOCS2 silencing on chronic kidney disease (CKD) related somatic growth and remnant kidney fibrosis D. Landau1,2, M. Hani Assadi3, R. Abu Hilal3, Y. Chen4,5, J. Medrano6, R. Rabkin4,5, Y. Segev3 1 Department of Pediatrics, Faculty of Health Sciences, Ben Gurion University, Beer Sheva, Israel; 2Department of Pediatrics B, Schneider Children’s Medical Center of Israel, Petach Tikva, Israel; 3Shraga Segal Dept. of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University, Beer Sheva, Israel; 4Research Service, Veterans Affairs Health Care System, Palo Alto, CA, USA; 5 Department of Medicine, Stanford University, Stanford, CA, USA; 6Department Animal Science, University of California, Davis, CA, USA Background: Short stature related GH resistance in CKD is partly due to a postreceptor defect in STAT5a/b phosphorylation (p-). Since SOCS2, a negative regulator of GH signaling is overexpressed in CKD and GH overexpression in mice may cause glomerulosclerosis, we tested whether lack of SOCS2 improves body growth, but accelerates kidney fibrosis in CKD. Methods: HG and normal wild-type mice (N) underwent 5/6 nephrectomy (CKD) or sham operation (SO), sacrificed after 12 weeks, generating 4 groups: SO-N, SO-HG, CKD-N, CKD-HG. Results: Weight and length gain, reduced in CKD-N, increased significantly in SO-HG and CKD-HG. Liver and kidney SOCS2 mRNA, totally absent in HG mice, was increased in CKD-N. Liver GHR protein levels were unchanged. Liver p-STAT5 was decreased in CKD-N but not in CKD-HG. Renal insufficiency was similar in CKD-N and CKD-HG. Kidney TGF-β and collagen type IV mRNA levels were increased in SO-HG, CKD-HG and CKD-N Vs SO-N. Renal GHR mRNA was decreased in SO-HG, CKD-HG and CKD-N Vs SO-N. Kidney p-STAT5 was decreased in CKD-N and further decreased in CKD-HG. IL6 and SOCS3 mRNA and p-STAT3 were increased similarly in C-HG and both CKD groups Vs SO-N.

Conclusions: CKD related growth retardation is prevented by spontaneous SOCS2 silencing, in association with increased p-STAT5. Renal insufficiency and fibrosis are not worsened by this manipulation, in association with decreased p-STAT5. This may be explained by the elevation of kidney proinflammatory cytokines and their mediators (IL6, phospho-STAT3 and SOCS3). These findings explain the renal safety of long term GH therapy for short stature in CKD. 62 Effects of modulation of the growth hormone axis in human melanoma cells R. Basu1, S. Wu3, J.J. Kopchick2 1 Edison Biotechnology Institute, Ohio University, Athens, Ohio, USA; 2Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio, USA; 3Molecular and Cellular Biology Program, Ohio University, Athens, Ohio, USA Background: Human melanoma has remained the most aggressive and therapy resistant form of cancer worldwide since 1950. Intracellular signaling networks used by the human melanoma cells for active proliferation, migration, invasion, therapy resistance and metastases strongly overlap with those regulated by human growth hormone (hGH). Consistently maximum levels of GHR expression has been observed in most melanoma cell lines in the NCI60 human cancer panel. Methods: Effects of exogenously added hGH or of siRNA-mediated GHR knock-down (GHR-KD) on the RNA and protein levels of intracellular signaling intermediates, relevant cell-surface ligand-receptor pairs and downstream phenotypes in four different human melanoma cell lines (SK-MEL-5, SK-MEL-28, MALME-3M and MDA-MB-435) were studied. Results: Phosphorylation states of the JAK2, SRC, STATs1, 3, and 5, ERK1/2, AKT and mTOR increased in a dose-dependent manner with hGH stimulation and were significantly attenuated by GHR-KD. Differential and significant changes were observed in the relative mRNA levels of endogenous GH, prolactin, and IGF1, and their receptors (GHR, PRLR, IGF1R, IGF2R, IR) as well as endogenous HGF and MET, following modulations of the GH-axis. Following GHR-KD, we observed attenuated tumor cell migration, proliferation, invasion and colony formation in soft agar. Additionally, we found an inhibitory effect of GHR-KD on markers of epithelial-mesenchymal transition and of the melanogenesis pathway, both of which are critical in metastasis and drug-resistance. Conclusions: Our findings provide multiple mechanistic details of GHdependent regulation of melanoma physiology and identify GHR as a unique point of intervention in melanoma therapy. 61 Intestinal fibrosis in response to GH J.A. Young1 , A. Stevens3, J.J. Kopchick2 1 Department of Biological Sciences, Ohio University, Athens, OH, USA; 2 Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA; 3Edison Biotechnology Institute, Ohio University, Athens, Ohio, USA Background: Fibrosis, or excess production of extracellular matrix components including collagen, is associated with intestinal disorders such as Crohn’s disease. Growth hormone (GH) is known to induce collagen production throughout the body, including in muscle, kidney and adipose tissue and is associated with the development of fibrosis. Conversely, GH has also been used somewhat successfully to treat Crohn’s disease in adults and children. Thus, it would be interesting to understand GH’s effects on the development of intestinal fibrosis to better understand its two seemingly opposed effects on intestinal health. Methods: Mice with altered levels of GH action were developed and studied. The mice included bovine GH (bGH) transgenic mice (with excess GH action), GH antagonist transgenic mice (with decreased GH action), GH receptor (GHR) gene disrupted mice (with no GH action), and intestine-specific GHR gene disrupted mice (with no GH action in the intestinal epithelium). Fibrosis

Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

in these mice was measured using a hydroxyproline assay, which quantifies tissue collagen content. Results: bGH transgenic mice displayed a significant increase in intestinal fibrosis, while mice with decreased GH action showed a trend towards reduced fibrosis that did not reach statistical significance. RNA expression of collagen I, III, and V (the predominant intestinal collagens) will be assessed using qPCR to confirm the results. Conclusions: The results show that excess GH does induce fibrosis in the intestine, thus, the mechanism of its anti-Crohn’s activity remains to be elucidated. 85 Mild increase in GH secretion but no gigantism or pituitary adenoma development in 3- and 12-month-old Aip-deficient male mice A. Lise Lecoq1, P. Zizzari2, M. Hage3, C. Adam4, S. Viengchareun1, J.D. Veldhuis5, V. Geoffroy6, M. Lombès1, A. Karhu7, L. Kappeler8, P. Chanson9, P. Kamenicky9 1 Institut National de la Santé et de la Recherche Médicale (Inserm) Le Kremlin-Bicêtre, France - Université Paris-Sud, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France; 2Inserm U894, Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 3Institut National de la Santé et de la Recherche Médicale (Inserm), Le Kremlin-Bicêtre, France Université Paris-Sud, Faculté de Médecine Paris-Sud, Université Paris Saclay, Le Kremlin-Bicêtre, France; 4Assistance Publique-Hôpitaux de Paris, Service d’Anatomie et Cytologie Pathologiques, Hôpital Bicêtre, Le Kremlin Bicêtre, France; 5Department of Medicine, Endocrine Research Unit, Mayo School of Graduate Medical Education, Clinical Translational Science Center, Mayo Clinic, Rochester, USA; 6Inserm U1132, Hôpital Lariboisière, Université Paris Diderot, Sorbonne Paris Cité, Paris, France; 7Department of Medical Genetics, Genome-Scale Biology Research Program Biomedicum, University of Helsinki, Helsinki, Finland; 8Sorbonne Universités, Univ Paris 06 UMRS 938, Inserm U938, CDR Saint-Antoine, Paris, France; 9Assistance Publique-Hôpitaux de Paris, Service d’Endocrinologie et des Maladies de la Reproduction, Hôpital Bicêtre, Le Kremlin Bicêtre, France Background: Mutations in the Aryl hydrocarbon receptor Interacting Protein (AIP) gene lead, in humans, to pituitary tumorigenesis, affecting particularly the somatothroph cell line. Mice with global heterozygous inactivation of Aip (Aip+/–) also develop pituitary adenomas but differ from AIP– mutated patients by the high penetrance of pituitary disease, as reported by previous studies. The endocrine phenotype of this mouse model has not been investigated. Methods: We compared the somatic growth, body composition, ultradian pattern of GH secretion and IGF-I concentrations of longitudinally followed wild-type (WT) and Aip+/– male mice at 3 and 12 months of age. To determine the early stages of AIP-related pituitary tumorigenesis, we also studied the pituitary histology in these mice. Results: Aip+/– mice did not develop gigantism but exhibited changes in body composition with a leaner phenotype compared to wild-type mice. Analysis of GH pulsatily by deconvolution showed a mild increase in total GH secretion in 12-month-old Aip+/– mice with conserved GH pulsatility pattern, without differences in IGF-I concentrations. No pituitary adenomas were detected up to 12 months of age. The increased ex vivo response of pituitary explants to GHRH observed in 3-month-old Aip+/– mice and the areas of enlarged acini identified on pituitary histology of some Aip+/– mice were suggestive of somatotroph hyperplasia. Conclusions: Global heterozygous Aip deficiency in mice is accompanied by subtle increase in GH secretion which does not result in gigantism. The absence of pituitary adenomas in 12-month-old Aip+/– mice demonstrates the important phenotypic variability of this congenic mouse model.

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47 Increasing fibrosis: a novel means by which growth hormone may limit white adipose tissue (WAT) expansion D.E. Berryman1, L.A. Householder1, R. Comisford1, K. Troike2, C. Wilson3, B. Henry1, E.O. List3, J.J. Kopchick4 1 The Diabetes Institute, Ohio University, Athens, OH, USA; 2School of Applied Health Sciences and Wellness, College of Health Sciences and Professions, Athens, OH, USA; 3Edison Biotechnology Institute, Ohio University, Athens, OH, USA; 4 Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA Background: WAT fibrosis—buildup of extracellular matrix (ECM) proteins, primarily collagen—is now a recognized hallmark of obesity. GH is well known to increase collagen in other tissues, but no previous research has addressed its effect on collagen in WAT. This study examined collagen content, adipocyte size, and fibrosis-associated gene expression in GH-altered mice. Methods: Six month old male bovine GH (bGH) transgenic, GH antagonist (GHA) transgenic, adipose specific GH receptor (GHR) gene disrupted (FaGHRKO) and liver specific GHR gene disrupted (LiGHRKO) mice along with littermate controls for each line (n=6–9) were studied. Body composition, depot size and adipocyte size were assessed for all mice. In the epididymal and subcutaneous depots, collagen content was assessed by picrosirius red staining of paraffin embedded tissue, hydroxyproline content and RNA expression studies. Results: The bGH mice had increased collagen content, decreased adipocyte size and significantly different RNA expression for some fibrosis-associated genes although RNA expression results did not always correlate with the level of fibrosis. Evaluation of GHA males, which have decreased GH action, showed significantly decreased collagen in the subcutaneous depot. Lastly, FaGHRKO mice had a decrease in WAT collagen content while LiGHRKO mice had an increase in collagen content, with significant changes in both lines only in the subcutaneous depot. Conclusions: In conclusion, our results show that GH causes a striking increase in the collagen content of WAT, which is depot dependent and which alters adipocyte size. These data reveal a novel means by which GH may affect WAT mass.

Parallel Poster Discussion Session: Parallel II IGF Short Basic Science Posters 167 Characterisation and quantification of IGF-1 receptors in equine lamellae: a step towards uncovering the disease mechanism for insulin-induced laminitis S.N. Nanayakkara1, P.A. Harris2, S.T. Anderson3, M.A. De Laat1, M.N. Sillence1 1 Earth, Environmental and Biological Sciences School, Queensland University of Technology, Queensland, Australia; 2WALTHAM Centre for Pet Nutrition, Mars Horsecare UK Ltd., Waltham-on-the-Wolds, Leicestershire, UK; 3School of Biomedical Sciences, University of Queensland, Australia Background: Hyperinsulinaemia has been well established as a main cause of equine laminitis, a debilitating condition that affects the hooves. It has been suggested that uncontrolled cell proliferation in the lamellae, triggered by hyperinsulinaemia, causes separation of the weight-bearing structure from the hoof wall. The soft tissue at the base of the hoof may then be crushed by the failing weight-bearing apparatus, causing intense pain; and consequently, these animals have to be euthanized. Insulin receptors are sparse in the lamellae and the mechanism by which insulin causes cell proliferation is unknown. Suggestions include insulin acting via the IGF-1 receptor (IGF-1R), and gene expression and histology studies have confirmed the presence of IGF-1R in the lamellae. However, the ability of insulin to bind to IGF-1R has not been demonstrated in horses.

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Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

Methods: Cell membrane fragments containing IGF-1R were extracted from the lamellae harvested from >100 randomly selected hoofs from horses not euthanized for research purposes. Results: Radioligand-binding studies using 125I-IGF-1 revealed 92% of the binding sites were IGF-1R, with a receptor density (Bmax) of 243 fmol/mg of membrane protein and a binding affinity (Kd) of 0.16 nM, similar to that found in other mammals. However, the affinity of insulin for binding to these receptors was >5,600 fold lower (Ki 905nM) than that of IGF-1, suggesting that equine IGF-1R are unlikely to bind to insulin at physiological concentrations. There was no evidence for InsR/IGF-1R hybrid receptors. Conclusions: These findings suggest that an alternative theory is required to explain the mechanism of insulin action in laminitis. 84 Cancer protection in Laron syndrome patients is associated with IGF1-dependent increase in thioredoxin interacting protein (TXNIP) gene expression K. Nagaraj1, L. Lapkina1, R. Sarfstein1, Z. Laron2,3, H. Werner1 1 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; 2Endocrinology and Diabetes Research Unit, Schneider Children’s Medical Center, Petach Tikva, Israel; 3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Background: Laron syndrome (LS) is a congenital autosomal recessive disorder caused by molecular defects of the growth hormone receptor (GHR) gene, leading to congenital IGF1 deficiency. Consistent with the prosurvival, antiapoptotic role of IGF1, recent epidemiological studies reported that patients with congenital IGF1 deficiency have a reduced risk of cancer development. The thioredoxin-interacting protein (TXNIP) plays an important role in redox homeostasis, and glucose metabolism. In addition, TXNIP has been identified as a candidate tumor suppressor gene. The aim of our study was to investigate the involvement of the TXNIP gene product in IGF1R signaling pathways associated with protection of LS patients from cancer. Methods: HEK293 and prostate cancer-derived cell lines P69 and M12 were used in this study. Quantitative Real-time PCR (qRT-PCR) was carried out following hormonal treatment at different time points. Expression levels of receptors, proteins, and activation of signaling cascades were measured by Western immunoblotting. Results: Genomic analyses conducted on lymphoblastoid cell lines revealed that TXNIP mRNA levels in LS patients were several-fold higher than in controls. In addition, qRT-PCR and Western blot revealed that IGF1 and insulin significantly downregulated TXNIP gene expression in a time-dependent manner. Furthermore, Western blot analysis revealed that stress induced TXNIP expression was significantly downregulated by IGF1. Conclusions: Our analyses have identified an important novel link between the TXNIP gene and the IGF1R signaling pathway, with potential implications in cell homeostasis during oxidative and glucose stresses. Further studies will dissect the TXNIP-mediated mechanisms at the molecular and cellular levels. 156 Insulin-like growth factor-I (IGF-I) as a novel regulator of prolactin secretion A. Leko1, M. Cservenak1,2, H. Öllös2, J. Hanics3, A. Alpar3,4, A. Dobolyi1,2 1 Laboratory of Neuromorphology, Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary; 2MTA-ELTE NAP B Laboratory of Molecular and Systems Neurobiology, Institute of Biology, Hungarian Academy of Sciences and Eötvös Loránd University, Budapest, Hungary; 3MTA-SE NAP B Research Group of Experimental Neuroanatomy and Developmental Biology, Hungarian Academy of Sciences, Budapest, Hungary; 4 Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary Background: IGF-I expressed in the central nervous system may influence some neuroendocrine functions. However, its effects on prolactin secretion have not been examined even though it is conceivable due to major

rearrangement of the prolactin regulatory system in the maternal hypothalamus. Methods: First, we administered IGF-I intracerebroventricularly (icv.) to rat mothers via osmotic minipumps for a 2-week period starting 2 days after parturition. On the 14th postpartum day, blood was obtained via jugular canuls and serum prolactin levels measured. Second, we tested the effects of IGF-I on primary mediobasal hypothalamic cell culture in vitro. Results: We observed significantly decreased suckling-induced prolactin levels in IGF-I-treated mothers, compared to saline-treated mothers. 4 days IGF-I treatment caused increase in tyrosine-hydroxylase (TH) expression in vitro. Subsequently, we demonstrated the presence of endogenous IGF-I in the mediobasal hypothalamus by immunohistochemistry without any change during lactation. In contrast, a markedly elevated IGFBP-3 expression level was found in the arcuate nucleus of lactating mothers using newly developed in-situ hybridization probes. We also discovered co-localization of IGFBP-3 with TH positive neurons using a combination of in situ hybridization and immunohistochemistry. Conclusions: In conclusion, we showed that exogenous IGF-I decreased serum prolactin levels in suckled rat dams possibly by increasing TH expression in the arcuate nucleus leading to elevated dopamine levels in the pituitary. We also showed the presence of endogenous IGF-I in the arcuate nucleus whose extracellular level may be reduced by elevated IGFBP-3 during lactation. 74 The role of insulin-like growth factors (IGFs) and low oxygen tension (PO2) in placental mesenchymal stem cell differentiation towards endothelial cells A. Youssef, H. Chakma, V. Km Han Children’s Health Research Institute, Lawson Health Research Institute, Departments of Pediatrics and Biochemistry, The University of Western Ontario, London, ON, Canada Background: Differentiation of stem cells towards endothelial cells provides potential new strategy for vascular regeneration therapy in coronary or peripheral arterial diseases. Human placenta is a readily available and noncontroversial source of mesenchymal stem cells (PMSCs). Low PO2 and IGFs are two niche factors that are present in the vascular microenvironment and participate in vascular remodeling and regulate endothelial cell proliferation, growth and survival. In this study, we hypothesized that PMSCs can differentiate into vascular endothelial cells and the process can be enhanced by IGF-1 and -2, and oxygen tension. Methods: Preterm PMSCs were differentiated towards endothelial cells under low PO2 (1% O2) in presence/absence of 100 ng/mL of IGF-1 or -2. Differentiation was determined by the expression of endothelial markers using western-blotting, RT-PCR and immunocytochemistry. Results: Under differentiation conditions, low PO2 decreased OCT4 and SOX2 suggesting a reduced multipotency. Low PO2, in the presence of IGF-1 more than IGF-2, increased sum tube length in angiogenesis assay. In static/ shear-stress conditions, differentiated PMSCs aligned with the direction of force more so in low PO2 vs. room air, and enhanced by the presence of IGFs. von-Willebrand factor (vWF) was increased in low PO2, while IGFs increased propeptide processing. Immunocytochemistry confirmed that low PO2 enhanced vWF, PECAM1 and eNOS. Conclusions: Alteration of the oxygen tension in the microenvironment allows PMSCs to differentiate into vascular endothelial cells and the inclusion of IGFs improves the differentiation efficiency, especially in low PO2. These maneuvers may be utilized in the regenerative therapy for vascular disorders using PMSCs or other stem cells.

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90 Targeting insulin receptor in breast cancer J. Ying Chan2, B.J. Hackel3, D. Yee1 1 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA; 2 Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA; 3 College of Science and Engineering, University of Minnesota, Minneapolis, MN, USA Background: The insulin-like growth factor (IGF) signaling system has been implicated in breast cancer. Multiple trials have tested anti-IGF regimens but not have shown clinical benefit. Our preclinical data implicate insulin receptor (InsR) signaling as an equally important pathway in regulating cancer cell growth. Specifically, InsR is an active signaling receptor in tamoxifen resistant breast cancer (Chan; Oncogene, 2016 PMID: 26876199) and the fetal InsR-A subtype is more commonly expressed in breast cancer (Zhang; Oncogene, 2010 PMID: 20154728). Our goal was to develop novel InsR targeting agents for the detection and treatment of breast cancer. Methods: Using yeast surface display and directed evolution, a gene 2 protein (Gp2) scaffold library was sorted against recombinant InsR and cell lysates with magnetic beads and flow cytometry, and evolved via error-prone PCR, to isolate InsR-specific Gp2 binders. Results: Our screen identified multiple Gp2 variants with high affinity nanomolar binding to the insulin receptor. Estrogen receptor-a (ER) positive tamoxifen and resistant (TamR) breast cancer cell lines were used to test binding and biological effect. Identified Gp2s did not bind IGF1R and inhibited insulin stimulated cell growth in TamR cell lines. Unlike monoclonal antibodies, the scaffolds did not downregulate InsR expression suggesting that the scaffolds disrupted insulin binding to its receptor. Conclusions: Yeast surface display libraries can yield high affinity binding scaffolds to key cell surface targets in breast cancer. While our study used a bait sequence that detects both isoforms of InsR, this technology might be used to detect InsR-A specific binders. 59 Gold nanoparticles conjugated quercetin inhibits IGFR mediated epithelial-mesenchymal transition and tumor growth of breast cancer B. Solaimuthu1, F. Ahmad Bhat1, S. Mukherjee2, C. Ranjan Patra2, A. Jagadeesan1,2 1 Department of Endocrinology, Dr.ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, 600113, India; 2 Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad, 500007, India Background: Insulin like growth factor (IGF) plays a critical role in tumor angiogenesis and metastasis of breast cancer. Our aim is to study the effect of gold nanoparticles conjugated quercetin (AuNPs-Qu-5) on IGFR mediated epithelial mesenchymal transition (EMT) using breast cancer (MCF-7 and MDA-MB-231) cell lines as well as on in vivo using 7, 12-dimethylbenz-(a)anthracene (DMBA) induced breast cancer rat model. Methods: Gold nanoparticles conjugated quercetin (AuNPs-Qu-5) was synthesized and characterized by physicochemical techniques. Cell viability, protein expression, anti-angiogenesis and therapeutic efficacy of AuNPsQu-5 were assessed by various in vitro and in vivo assays. Results: AuNPs-Qu-5 shows stronger inhibitory effects on EMT markers like Vimentin, N-cadherin and transcriptional factors including snail, slug and twist as compared to free Qu. Cell survival, proliferation, migration and invasion of both breast cancer cell lines were regressed on treatment with AuNPs-Qu-5 as compared to free Qu. Further, we tested the anti-angiogenesis effect of AuNPs-Qu-5 in HUVEC cells and results showed that AuNPs-Qu-5 inhibited the cell viability, capillary-like tube formation, invasion, migration and VEGFR-2 protein expression in HUVEC cells. In vivo anti-angiogenesis assay using Chick embryo angiogenesis (CEA) showed that AuNPs-Qu-5 inhibited the new blood vessel formation. Treatment with AuNPs-Qu-5 resulted stronger in tumor growth regression compared to free Qu in DMBA induced mammary carcinoma in Sprague Dawley rats.

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Conclusions: AuNPs-Qu-5 effectively enhanced the therapeutic efficacy of Qu by blocking IGFR mediated EMT in breast cancer and could be exploited as a potential candidate for cancer therapy. 28 CD24 cell surface expression in MVT1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF-1R therapy R. Rostoker, S. Ben Shmuel, R. Rashed, Z. Shen Orr, D. Leroith Diabetes and Metabolism Clinical Research Center of Excellence Clinical Research Institute at Rambam Rambam-Health Care Campus, Haifa, Israel Background: The pro-tumorigenic effects of the insulin-like growth factor receptor (IGF-1R) are well described. However, phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies. Methods: shRNA vectors were used in order to determine IGF-1R’s role mammary tumorigenesis. Control and IGF-1R-knock down (IGF1R-KD) cells were FACS sorted into CD24– and CD24+ subsets and further characterized invitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF-1R’s effect on the plasticity of this cell’s phenotype. Metastatic capacity was assessed using the tail vein assay. Results: IGF-1R-KD in cancer cells expressing CD24 affects both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF-1R-KD abolished both CD24+ cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG (tumor suppressor) and a significant reduction of SLP1 (tumor promoting) gene expression in the CD24+/IGF-1R-KD cells. Moreover, we demonstrate that IGF-1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF-1R-KD induces in-vivo differentiation of the CD24+ cells toward the CD24– phenotype. Conclusions: These findings suggest that CD24 cell surface expression may serve as a valuable biomarker in order to identify mammary tumors that will positively respond to targeted IGF-1R therapies. 171 IGF-1R inhibition sensitizes breast cancer cells to ATM-related kinase (ATR) inhibitor and cisplatin C.H.O. Flanagan1, S.O. Shea1, A. Lyons1, F.M. Fogarty1, N. McCabe2, R.D. Kennedy2, R. O’Connor1 1 University College Cork, Ireland; 2Queens University Belfast, Ireland Background: The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. Methods: A targeted siRNA library was used as well as siRNA, kinase inhibitors and clonogenic assays to assess additive or synergistic cytotoxic responses. Results: By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included the ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI 906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines.

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Conclusions: Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs. 139 Targeting IGF-1R in breast cancer: combined treatment with conventional chemotherapeutic drugs T. Sinai1, Z. Cohen2, H. Werner1, R. Berger2 1 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 2Division of Medical Oncology, Sheba Medical Center, Tel-Hashomer Hospital, Ramat-Gan, Israel Background: Most tumors, including breast cancers, express high levels of IGF-1R mRNA and protein. Cancer cells activate survival pathways to overcome death signals induced by cytotoxic chemotherapy. IGF-1R protects cancer cells from apoptosis through activation of survival pathways. Our study was aimed at evaluating the hypothesis that targeting IGF-1R reduces the IGF1-R-mediated activation of survival pathways and, combined with chemotherapy, may lead to synergistic growth inhibition. Accordingly, IGF-1R levels might predict responsiveness to combined treatment. Methods: Malignant breast cancer cell lines with high (MCF7) or low (HCC 1937) IGF-1R levels, were treated with a panel of standard chemotherapeutic drugs as a single agent or in combination with AEW541, a selective IGF-1R tyrosine kinase inhibitor. Cell proliferation was measured by XTT assays. Results: The combined treatment of AEW541 and chemotherapy synergistically enhanced growth inhibition in MCF7 cells. On the other hand AEW541 had a minor effect on HCC 1937 viability. Conclusions: The effect of IGF-1R inhibitor on cancer cells proliferation was correlated with IGF-1R expression and activity levels. Specifically, combination of IGF-1R inhibitor and chemotherapy was highly synergistic in cells expressing high level of IGF-1R, but not in cells with low IGF-1R level. Thus, IGF-1R targeted therapy should adopt IGF-1R values as a key biomarker for patient evaluation. In conclusion, our results strongly suggest that combined treatment with IGF-1R inhibitor and chemotherapeutic drugs can greatly improve the treatment efficiency in breast cancer patients whose tumor cells express high IGF-1R level.

Plenary: Plenary Session 2 604 GH/IGF1 genetics and aging N. Barzilai Departments of Medicine and Genetics, Albert Einstein College of Medicine, Bronx NY USA The link between altered insulin-IGF pathway and longevity was shown in lower organisms and variety of mammalian models. However, paradoxically low GH/IGF-1 levels are associated with increased prevalence of many agerelated diseases in humans including diabetes, cardiac diseases and cognitive decline. Therefore it is of the utmost importance to ling this pathway to aging/longevity in humans. We studied individuals with exceptional longevity (>600 genetically and socially homogeneous Ashkenazi Jews ages 95-112YO) and their offspring and age- and sex-matched controls without a family history of unusual longevity. We have implicated the following genetic components in functional alterations in GH/IGF1 pathways: 1) Functional variants in the IGF1 receptor in 2% of our centenarians. Phenotype includes short stature; 2) A relatively rare exon 3 deletion variant homozygosity in the GH receptor frequency in AJs increased monotonically to a prevalence of ~12% in centenarians, validated in other populations; 3) Markedly increased levels of micro RNA (miRNA) in ~30% of centenarians, some of them modulating GH/IGF1 signaling pathway; 4) From a phenotypic perspective, in 95-year old subjects, low IGF1 levels have significantly longer survival that is greatest in females and with a history of cancer. Overall, these data suggest that diminished GH/IGF-1 signaling through several genetic mechanisms may provide protection against variety of age-related diseases and allow for

exceptional longevity also in humans. Future questions and directions will be discuss in this talk and this session. 605 Gigantism: X-linked acrogigantism-GPR101 mutations M. Korbonits Queen Mary University of London, UK Early in 20th-century acromegaly was suggested to be an acquired disease, while gigantism congenital. Indeed this suggestion seems to be more and more true. While gigantism due to McCune-Albright or MEN1 syndromes is known to have genetic origin, familial and simplex AIP gene mutationinduced, usually adolescent-onset gigantism also has genetic origin. But notably, few of the AIP-related giants were diagnosed under 10 years, while the world tallest patients usually presented before the age of 5 years. In December 2014 X-linked acrogigantism (XLAG) has been identified as a cause of very early-onset GH excess, in most patients with concomitant hyperprolactinaemia, resulting from Xq26.3 microduplication. While some patients present with a pituitary tumour showing peculiar histopathological features, others have pituitary hyperplasia. Females have de novo germline mutations, while sporadic male patients reported so far are somatic mosaics. Two familial cases have also been described. The causative gene is the orphan G protein-coupled receptor, GPR101, normally expressed in the CNS and at particularly high levels in the hypothalamus and nucleus accumbens. While the physiological function and the endogenous ligand of GPR101 are unknown, the high expression of GPR101 in the arcuate nucleus and the occurrence of increased circulating GHRH levels in some patients with XLAG, suggest that increased hypothalamic GHRH secretion could play a role in the pathogenesis of this condition. No GPR101 mutations have been identified in GH deficient children so far. GPR101 therefore may be an important regulator of GH release and future physiological studies must be performed to understand the pathogenesis of this unique from of gigantism.

Parallel Oral Session: Parallel I Pituitary Society Symposium 606 Screening for genetic causes of GH hypersecretion A. Beckers Domaine University, du Sart-Tilman, Belgium The majority of cases of GH excess arise from pituitary tumors. This can produce gigantism when GH/IGF-1 excess occurs before the epiphyseal closure and acromegaly thereafter. Most cases occur as a result of sporadic GH-secreting pituitary adenomas, however, in about 5% of cases, they can have genetic or familial predisposition. There are now several known genetic causes of acromegaly-gigantism, including multiple endocrine neoplasia syndromes (MEN1 and MEN4), Carney complex, McCune Albright syndrome, familial isolated pituitary adenoma (FIPA), pituitary adenomas with paraganglioma/pheochromocytoma due to SDHx mutations, and the recently identified X-linked acrogigantism syndrome (XLAG) due to microduplication involving GPR101. Screening for genetic causes of pituitary adenomas is necessary in familial cases and in patients with a suggestive syndromic presentation. Germline mutations in genes involved in these genetic syndromes are very rare in unselected sporadic pituitary adenomas. MEN1 and AIP variants are more frequent among specific subgroups of patients, such as children and young adults, especially those with large and aggressive adenomas. About 20% of FIPA kindreds have an AIP mutation and tend to have a distinctive clinical profile (younger age at diagnosis and larger tumor size with relative resistance to medical therapy). XLAG syndrome is characterized by very specific phenotype of infant-onset gigantism that can present either sporadically or in the setting of FIPA. Somatic mosaicism underlies XLAG in sporadic male cases. Early detection of genetic alterations and diagnosis of associated conditions are essential for timely management.

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607 Detection of GH abuse in athletes C.J. Strasburger Department of Clinical Endocrinology, Campus Charité Mitte, Universitaetsmedizin Berlin, Sauerbruchweg, Berlin, Germany Doping with growth hormone (GH) has been considered undetectable in the past. Athletes apparently fancy the protein-anabolic and lipolytic effects of GH. Two strategies have been pursued to detect GH doping: Pharmacological endpoints and GH isoform composition. For the former approach the consortium GH 2000/2004 has identified markers of GH action and found a combination of parameters from the IGFsystem and collagen markers to provide a suitable combination to detect GH abuse. The latter approach is based on the molecular nature of isoform composition in which GH is released from the pituitary. Monomeric 22 kD hGH in its unbound form accounts only for approximately one quarter of all GH isoforms in circulation, while the vast majority of recombinant hGH is in the monomeric 22 kD form. We have therefore developed the differential immunoassay strategy in which every sample is analyzed by two independent immunoassays: The first assay preferentially recognizes 22 kDa monomeric hGH while the second assay recognizes the bulk of isoforms as released from the pituitary but has only moderate affinity for the 22 kDa monomeric form of hGH. The 22 kD preferential antibody as well as the “permissive” antibody are immobilized and allowed to react with the sample before incubation with a common labeled anti-hGH antibody. This test has been introduced and certified at WADA accredited laboratories since the Summer Olympic Games in Athens and meanwhile has detected several cases of hGH abuse in Olympic as well as in professional athletes. The decision limit for the ratio between the recombinant and the pituitary-preferential assays has been established as a consequence of the meanwhile thousands of samples analyzed from athletes of different disciplines and ethnic background, neither of which affect the observed ratio significantly. The pharmacological marker test in its development was hampered by assay discontinuations and the need for restandardization. This test should be capable of detecting GH abuse in athletes for a longer period of time than the isoform approach, but may fail after initial GH injections. It can therefore be speculated, that both strategies will be applied side by side in the future. 609 New medical therapies of acromegaly F. Maffezzoni, S. Frara, G. Mazziotti, A. Giustina Endocrinology, University of Brescia Italy Acromegaly is a rare, chronic, progressive disease caused by a pituitary adenoma in the vast majority of cases and characterized by an excess secretion of growth hormone (GH) and increased circulating insulin-like growth factor 1 (IGF-1) concentrations. Therapy for acromegaly aims at decreasing GH and IGF-1 levels, ameliorating patients’ symptoms, decreasing any local compressive effects of the pituitary adenoma, controlling comorbidities and normalizing mortality. Somatostatin receptor ligands (SRLs) currently in use such as octreotide LAR and lanreotide Autogel (with high affinity for the subtype 2 of the somatostatin receptor) remain the cornerstone of medical treatment, whereas cabergoline and pegvisomant are used in selected often resistant patients. However, despite all these treatment options, approximately 30% of patients are not adequately controlled in the long term with relevant impact on quality of life and outcome of systemic complications. Management of patients partial responders or with poor compliance to conventional treatment are currently the most important challenges that clinicians are facing. In this scenario newer formulation of octreotide (oral, implant and needle-free injections) have been developed in order to overcome patient discomfort and compliance whereas SRLs with a wider spectrum affinity such as pasireotide, ITF2984 and somatoprim can increase response to treatment in patients with total or partial resistance to conventional SRLs, determined by several factors inherent to the biology of the tumor and somatostatin receptors expression on cell surface. Moreover, the current attempt is to obtain with the same SRL an increased affinity

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for subtype 5 of the somatostatin receptor without increasing the risk of hyperglycemia (as observed with pasireotide). Furthermore, somatostatin receptors are only one of the different targets involved in acromegaly treatment: from this point of view new therapies such as antisense drugs (ATL1103) and botulin neurotoxin (SXN101959), targeting GH (potential alternative to pegvisomant) or GH-releasing hormone signal respectively, could prospectively be important additional medical tools in acromegaly treatment.

Parallel Oral Session: Parallel II Oral Presentations 2 – IGF Basic aspects 158 The interplay between BRCA1 and IGF-I actions in estrogen-receptor (ER) positive breast cancer cell lines M.O. Koobotse, J.M. Holly, C.M. Perks School of Clinical Sciences, Faculty of Health Sciences, University of Bristol, Bristol, UK Background: We have shown previously that in ER-positive breast cancer cell lines, IGF-I induces growth in a fatty acid synthase (FASN)-dependent manner. In these cells, BRCA1 binds the phosphorylated and inactive form of acetyl-CoA carboxylase (ACCA) to reduce fatty acid synthesis. Since FASN is downstream of ACCA, we hypothesised that when promoting cell growth, IGF-I may also reduce BRCA1-ACCA association. Methods: ER-positive breast cancer cells (MCF7 and T47D) and UBR60-bcl2 cell line with an inducible BRCA1 expression were used. Cells were treated with IGF-I and proliferation assessed using a 3-H thymidine incorporation assay. Changes in protein abundance were analysed using Western blotting. Protein localisation and protein-protein associations were studied using subcellular fractionation and immunoprecipitation respectively. Results: We found that BRCA1 was predominantly cytoplasmic in ER-positive cell lines and the addition of IGF-I dephosphorylated ACCA.IGF-I also reduced the interaction between BRCA1 and ACCA, accompanied by increased FASN abundance and growth of MCF7 and T47D cells. In UBR60 cells, BRCA1 also restricted fatty acid synthesis by regulating both ACCA phosphorylation and FASN abundance. All cells became desensitized to high doses of IGF-I but when BRCA1 was repressed sensitivity was restored, supporting the role of BRCA1 as a brake on proliferation, potentially via inhibiting fatty acid synthesis. Conclusions: Our novel findings suggest that IGF-I stimulates ACCA activity by reducing the interaction between BRCA1 and phosphorylated ACCA and this is associated with increased FASN and cell growth. The interplay between loss of BRCA1 and high IGF-I levels may result in worse outcome for ERpositive breast cancer patients. 94 TMPRSS2-ERG fusion protein regulates insulin-like growth factor-1 receptor (IGF1R) gene expression in prostate cancer S. Meisel Sharon, Y. Pozniak , T. Geiger, H. Werner Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Background: Prostate cancer is a major health issue in the Western world. The most common gene rearrangement in prostate cancer is the TMPRSS2ERG fusion, which results in aberrant expression of the transcription factor ERG. The insulin-like growth factor-1 receptor (IGF1R) plays a key role in tumorigenesis and is overexpressed in most malignancies, including prostate cancer. Our aim was to analyze the involvement of the TMPRSS2-ERG fusion protein in the regulation of IGF1R gene expression and IGF1 signaling pathway. Methods: T-ERG-expressing cells were transfected with a siRNA directed against the fusion protein. Protein levels of IGF1R and ligand-induced IGF1R phosphorylation were measured by Western blotting. To assess the T-ERG

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effect on IGF1R transcription, luciferase activity assays were performed. Physical interaction between T-ERG and upstream regulatory factor Sp1 was examined by co-immunoprecipitation assays. Mass spectrometry analysis was applied to reveal new T-ERG interactors. Results: Silencing of T-ERG resulted in downregulation of both IGF1R and Sp1. Co-immunoprecipitation assays revealed a physical interaction between transcription factors ERG and Sp1, with potential relevance in IGF1R gene regulation. Promoter assays indicated that transactivation of the IGF1R gene by ERG was mediated at the level of transcription. Mass spectrometry assays identified new interactors of the TMPRSS2-ERG fusion protein, which are known to play a mechanistic role in IGF1R internalization. Conclusions: Our analyses are consistent with a potential novel function of TMPRSS2-ERG as a major regulator of IGF1R gene expression. Results may impinge upon ongoing efforts to target the IGF1R in the clinics.

Results: We identified 300 PDLIM2-interacting proteins and classified them into functional clusters which were: 1) ribosome, translational elongation; 2) proteasome, regulation of ubiquitin E3 ligase activity; 3) RNA binding and processing; 4) macromolecular complex assembly; and 5) cell cycle. Importantly, the PDLIM2 interactome contains p53 and 13 E3 ubiquitin ligases, eight of which reportedly interact with and regulate p53 and/or Mdm2. This is consistent with an increase in protein ubiquitination upon re-constitution of PDLIM2 knockout cells with PDLIM2. Moreover, PDLIM2 interacts with RNA Polymerase II, several histone proteins and transcription factors. Conclusions: Overall, our findings indicate that PDLIM2 is active in protein complexes that promote transcription factor ubiquitination, likely at the sites of Polymerase II interaction with chromatin. Our results support a model for PDLIM2-dependent regulation of p53 transcriptional output that includes IGF-1 signalling pathways, and that determines cancer cell phenotype.

121 Functional selectivity of the insulin-like growth factor type 1 receptor (IGF-1R) signaling: therapeutic implications for cancer treatment

141 Homozygous deletion of Pregnancy-Associated Plasma Protein A (PAPPA) protects old long-lived mice from lipopolysaccharide endotoxicosis

L. Girnita, C. Crudden, D. Nedelcu, N. Suleymanova, E. Trocme, A. Girnita Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden

P. Griffin3, C.A. Conover2, L. Bale2, A. Snyder3, K. Gronborg3, J.J. Michel3, A.N. Vallejo1 1 Departments of Pediatrics and Immunology, Pittsburgh Claude Pepper Older Americans Independence Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 2Endocrine Research Unit, Department of Medicine, Mayo Clinic, Rochester, MN, USA; 3Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Background: The insulin like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer, however therapeutics targeting it have had disappointing results in clinic. Based on the main structural characteristic—the presence of an intracellular domain possessing tyrosine kinase activity—IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, or dissociation between signaling and receptor downregulation. During the last decade, we and others challenged the classical model by demonstrating IGF-1R as a RTK/GPCR functional hybrid, which integrates both canonical kinase signalling with many functions characteristic of a GPCR. In the present study we investigated the biological outcomes of selective IGF-1R signaling activation. Methods: By using various cancer models, we analyzed the functional effects of IGF-1R “biased-signaling” following site-specific mutation of the kinase domain with or without mutations within the “GPCR” domain. Results: Biased kinase signaling increase activation of the PI3K/Akt pathways and sustain cell proliferation whereas biased “GPCR” signaling caused hyperactivation of the MAPK/ERK pathway with increased effects on cell migration with parallel reduction in IGF-1R expression. Conclusions: This study highlight the complexity of the IGF-1R signaling, advocate its recognition as anti-cancer therapeutic target, but also points to the possibility of biased ligand therapeutics, which together may hold a very powerful key to unlocking the true potential of IGF-1R modulation. 169 PDLIM2 regulates ubiquitination, stability and activity of key transcription factors such as p53 J. Berghoff, M. Francisca Bustamante Garrido, B. Addario, H. Quinn, E. Tresse, R. O’Connor Department of Biochemistry and Cell Biology, University College Cork, Cork Background: Insulin-like growth factor 1 (IGF-1) regulates the expression of many genes associated with cancer progression. These include the PDZ-LIM protein PDLIM2, which is highly expressed in invasive cancer cells. PDLIM2 regulates stability of key transcription factors including NF-κB, cell cycle regulators p27KIP1 and p53, as well E cadherin and the IGF-1 receptor (IGF-1R) (1-3). However, it is still unknown how PDLIM2 controls the activity of so many key determinants of cellular transformation. Methods: To elucidate the PDLIM2 mechanism of action in controlling protein stability, we investigated the PDLIM2 protein interactome using GST-pulldown assays, mass spectroscopy and immunoprecipitation (IP). Annotation clustering was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID).

Background: Mice deficient in PAPPA–/– are long-lived. They have an overall reduced pathology, and are resistant to age-related atrophy of the thymus, the site of production of T cells that are required for normal pathogen-specific immunity. Thus, we have been using PAPPA–/– mice to examine mechanisms of immune homeostasis underlying longevity. PAPPA is the enzyme that degrades inhibitory proteins sequestering IGF, and thereby controls the bioavailability of IGF. Excess IGF has been linked to organ inflammation and tumorigenesis in old mice and humans. Independent of thymus preservation, we examined whether old PAPPA–/– mice have functionally competent innate non-specific immunity. Methods: Mice aged 18 months were injected IP with high dose LPS. Survival was monitored. Sera and spleens were harvested from moribund mice, and from survivors after 10 days. Luminex and flow cytometry were performed to examine humoral and cellular profiles. RNASeq was performed to examine splenic transcriptomes. Results: Old PAPPA–/– mice, even after two challenges, survived high dose of LPS that was lethal to old wildtype (WT) mice. Moribund WT mice showed classic LPS-induced cytokine storm. In contrast, LPS-treated old PAPPA–/– mice had significantly muted cytokine production. Furthermore, their innate cell populations expressed high levels of Bcl-xL with negligible cell death, and had low cellular expression of TNFα, IL-6, and RelA, molecules linked to inflammatory cascades. Transcriptomic signature of old PAPPA–/– mice included the induction of several antimicrobial genes, and negative regulators of LPS/TLR4 signaling. Conclusions: These data show robust innate immunity is critical to the promotion of favorable health outcomes during long-term survivorship.

Abstracts of the 8th International Congress of the GRS and IGF Society / Growth Hormone & IGF Research 30-31S1 (2016) S1–S53

95 Does IGF-1 have a role in the regulation of autophagy in the vertebrate inner ear? S. Pulido1, M. Aburto2, R. De Iriarte Rodríguez3, L. Rodríguez De La Rosa4, M. Magariños5, I. Varela Nieto1 1 Institute for Biomedical Research “Alberto Sols” (IIBm), CSIC-UAM, Madrid, Spain; 2Institute of Cell Biology and Neuroscience and BMLS, Goethe University Frankfurt, Frankfurt am Main, Germany; 3“Alberto Sols” Biomedical Research Institute (CSIC-UAM), Madrid, Spain; 4IdiPAZ, La Paz Hospital Institute for Health Research, Madrid, Spain; 5Department of Biology, Autonomous University of Madrid, Madrid, Spain Background: IGF-1 pathway regulates otic development and cochlear postnatal maturation and function. Deficiencies in IGF-1 pathway have been implicated in rare and severe human syndromes that include deafness. Autophagy is a catabolic process essential for development and adult homeostasis. Autophagy provides energy for the clearing of dying cells and neuronal differentiation during early otic development. IGF-1 pathway has been described to regulate autophagy in different contexts although its role is not well-defined yet. Here, we will discuss the role of IGF-1 in the regulation of autophagy during the early development of the otocysts (Gallus gallus) and its lifelong function in the inner ear (Mus musculus). Methods: The experiments were performed in ex vivo cultures of chicken otocysts treated with autophagy inhibitors and IGF-1, and in wild type and Igf1 null mice. Autophagy machinery was assessed by RT-qPCR and autophagy flux by Western blotting. Autophagosomes were evaluated by immunohistochemistry. Results: IGF-1 reduced the effects of autophagy inhibitors in chicken otocysts cultures. Autophagy machinery levels were up-regulated with age in the mouse inner ear. Autophagic flux increased with age. Autophagosomes were localized in spiral ganglion neurons. Although specific stages of Igf1 deficient inner ear showed a significantly different autophagic profile pattern, no definitive association with IGF-1 deficiency was observed in the adult mouse inner ear. Conclusions: Taken together, these results show that IGF-1 might play a role during early development and adult activity of the inner ear. IGF-1 effects were targeted to cell survival and therefore might be indirect on the modulation of autophagy. 179 An IGF-Trap inhibits spontaneous liver metastasis of pancreatic ductal adenocarcinoma M.C. Fernandez, S. Moffett, N. Wang, S. Perrino, P. Brodt Departments of Surgery and Medicine, McGill University, McGill University Health Centre, Montreal, QC, Canada Background: Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer related death with a 5 year survival rate of only 6%, due, in part, to the high incidence of liver metastases. The IGF-IR was identified as a marker of progression in PDAC and a target for cancer therapy. We previously reported on the production of an IGF-IR signaling inhibitor— the IGF-Trap—that markedly reduced experimental liver metastasis in preclinical models of colon and lung carcinoma. The objective of the present study was to to assess the effect of the IGF-Trap on spontaneous liver metastasis in a PDAC model that mimics progression of the human disease. Methods: Murine PDAC LMP cells were implanted intra-pancreatically in syngeneic B6/129 male mice. Mice were first treated with 5 mg/kg IGF-Trap 1 or 3 days later and received a total of 5 injections over 2 weeks. Mice were euthanized at 3 weeks, the primary tumours measured and metastases on the surface of the liver enumerated and sized. FFPE liver sections were H&E stained to quantify the surface area of hepatic metastases. Results: In the control mice, intra-pancreatic tumours progressed rapidly causing numerous liver and extra-hepatic metastases and accumulation of ascites. Mice were moribund within 3 weeks of tumor implantation. Treatment with the IGF-Trap markedly reduced the incidence, number and size of spontaneous metastases but did not affect the growth of local pancreatic tumours.

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Conclusions: The data identify the IGF-Trap as an inhibitor of PDAC dissemination and a potential drug for this fatal disease. 92 Loss of IGF-1R in the luminal lineage promotes basal and metastatic phenotypes in the wnt model of triple negative breast cancer T. Wood1, V. Ciliento1, A. Obr1, D. Leroith2 1 Department of Pharmacology, Physiology & Neuroscience & Cancer Center, New Jersey Medical School, Rutgers University, Newark, NJ, USA; 2Diabetes and Metabolism Clinical Research Center of Excellence Clinical Research Institute at Rambam Rambam- Health Care Campus, Haifa, Israel Background: A fundamental question in cancer biology is what causes tumor phenotypic diversity and, most critically, what leads to an aggressive, metastatic tumor phenotype? Here, we present studies designed to distinguish the function of the IGF-1R specifically in the luminal lineage in the Wnt model of TNBC. Methods: We have established a mouse line carrying floxed Igf1r alleles and a tamoxifen inducible Cre-recombinase transgene expressed from a luminalspecific (Keratin 8) promoter in the presence of the MMTV-Wnt1 transgene. Results: Loss of luminal-specific Igf1r results in an increase in percentage of basal cells compared to MMTV-Wnt1 tumors. Moreover, the K8-Cre/Igf1rflox/Wnt1 tumors have an increase in the luminal progenitor population. Lung tissues reveal increased lung metastases with luminal loss of Igf1r. Tumors resulting from loss of Igf1r show alterations in several pathways that mediate an aggressive tumor phenotype including Notch signaling and interleukin-6. Using expression of a reporter allele, we are currently determining whether the alterations in tumor phenotype and resulting metastases are due to cell-autonomous or paracrine effects from loss of Igf1r in the luminal cells. Analyses of TCGA human databases further support the relevance of our model in human TNBCs that have reduced IGF-1R expression and increased expression of Wnt1 and the Frizzled co-receptor Lrp6. Conclusions: Our results demonstrate that loss of IGF-1R specifically in the luminal epithelial cells is sufficient to enhance the basal phenotype of Wntmediated tumors and that this mouse model is relevant to specific subtypes of human TNBCs.

Tuesday, 8 November 2016 Plenary: Plenary Session 3 610 IGF-1R: its role in radioresistance and DNA homeostasis V. Macaulay University of Oxford, UK Type 1 insulin-like growth factor receptor (IGF-1R) mediates cell cycle progression, invasion and cell survival, and can also promote resistance to cancer therapy. In trials of IGF-1R inhibitory drugs some patients obtained very durable responses, suggesting that this is a viable target, but the majority did not benefit. It is essential to understand IGF biology in order to exploit the potential of these new drugs, and several avenues may prove fruitful. Firstly, genetic screens for predictive biomarkers may enable identification of patients likely to respond to IGF blockade. Secondly, response to IGF-1R inhibition has been linked with the recently-recognised ability of IGF-1R to undergo nuclear translocation and interaction with chromatin. Ongoing research is investigating the clinical/translational implications of this phenomenon. Finally, over-expression or activation of IGF axis components has been associated with chemo- and radio- resistance, and it may be possible to exploit this association clinically. In the context of combination treatment with cytotoxic drugs that cause phase-specific DNA damage, it is critical to optimise the sequencing of IGF-1R inhibition with chemotherapy. We have also shown that IGF-1R depletion or inhibition enhances radiosensitivity,