Insulin Receptor A Research Articles

Effect of insulin on IR and GLP1-R expressions in HT22 cells.

Insulin is a significant growth factor that specifically binds to the insulin receptor (IR) in the brain and then activates the PI3K-AKT pathway. Glucagon-like peptide 1 (GLP-1) has a variety of functions including neuroprotection, support for neurogenesis, and increasing insulin signal. This study aims to investigate the effect of insulin administered to immortalized clonal mouse hippocampal cell line (HT22) at different doses and intervals on IR, insulin receptor A (IRA), insulin receptor B (IRB), and Glucagon-like peptide 1 receptor (GLP1-R) mRNA expression and protein levels. The cells were planted in 6 well plates at a density of 3 × 105/4 × 105. Cells treated with insulin at different concentrations (5, 10, and 40nM) were collected at 0.5, 2, 8, 16, and 24h. RT-PCR and western blot analysis were used to measure mRNA expression and protein levels. Our results showed that insulin has short and long-term effects on IR and GLP1-R expression depending on dose and time. These findings may guide future studies targeting IR isoforms and GLP1-R in particular, as well as determining the optimal dose and duration of insulin stimulation in insulin signaling research.

Abstract 3560: Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development

Abstract Introduction: Ewing sarcoma (ES) is the second most common pediatric bone cancer, and patients with metastases have five-year survival rates of <30%. Both local and metastatic ES tumors are characterized by the EWS-FLI1 chromosomal translocation, resulting in an aberrant transcription factor that increases ES cell proliferation and metastasis. EWS-FLI1 activity and ES transformation are reliant upon increased IGF-1 signaling, so initial therapies have centered on IGF-1R inhibition. However, compensatory upregulation of insulin receptor A (IR-A), an oncogenic spliced isoform of IR, provides a viable resistance mechanism to support ES growth and progression. IR has two spliced isoforms formed by the inclusion or exclusion of exon 11 (IR-B and IR-A respectively), and IR-A is differentially expressed in cancers due to its high affinity binding to IGF-II. We hypothesize that the pathogenesis and metastatic potential of ES is driven in part by alternative splicing of IR and other key genes. Furthermore, modulation of this splicing via antisense oligonucleotides (ASOs) aided by adeno-associated viral (AAV) delivery may be a novel therapeutic avenue for ES. Results: We performed long-read mRNA sequencing to identify differentially spliced isoforms between the matched ES cell lines CHLA-9 and CHLA-10 (derived from a localized and metastatic ES tumor respectively). Our analysis revealed global differences in alternative splicing between CHLA-9 and CHLA-10 and identified gene isoforms in the insulin receptor/IGF signaling pathway (IGFBP3) that are unique to CHLA-10. Additionally, we detected both previously reported and novel isoforms for CD99, RHOA, RACK1, splicing factors (PTBP1), and eukaryotic initiation factors, among other genes. To further understand IR alternative splicing, our labs have developed an IR ASO targeting the CUG-BP1 regulatory site upstream of exon 11, and we have previously validated its efficacy in HeLa cells transfected with an IR minigene and in pediatric sarcoma cell lines. In ES cell lines, we observed significant IR-A upregulation in a panel of ES cell lines and patient-derived xenografts derived from local and metastatic tumors, highlighting the opportunity for intervention with our ASO. To facilitate delivery, we have explored the use of AAV vectors, a clinically approved option for gene therapy, and shown successful transduction of AAV-8 over a time-course in ES cells. Conclusions: Our results indicate that alternative splicing is differential between local and metastatic ES and produces isoforms unique to the metastatic setting. Additionally, the oncogenic IR-A isoform and other spliced isoforms are viable targets for ASO therapy aided by AAV delivery. By better understanding splicing in ES, we can identify targetable interactions at the transcriptional level and develop innovative therapeutics that can improve the clinical outcomes of ES patients. Citation Format: Akila S. Venkataramany, Safiya Khurshid, Anthony R. Miller, Saranga Wijeratne, Pin-Yi Wang, Timothy P. Cripe, Dawn S. Chandler. Understanding alternative splicing in Ewing sarcoma progression and metastasis to inform novel therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3560.

Effects of dietary berberine hydrochloride inclusion on growth, antioxidant capacity, glucose metabolism and intestinal microbiome of largemouth bass (Micropterus salmoides)

The present study was conducted to investigate the influence of berberine inclusion on growth, antioxidant capacity, glucose metabolism and intestinal microbiome of largemouth bass. Three experimental diets were formulated with 0, 100, and 400 mg/kg berberine hydrochloride, which were fed to triplicate groups of juvenile largemouth bass twice daily for 11 weeks. The results of feeding trial revealed that dietary berberine inclusion produced no significant influence on growth performance of largemouth bass. However, the inclusion of dietary berberine significantly reduced serum glucose content, and alleviated hepatic glycogen accumulation. Gene expression analysis indicated that dietary berberine inclusion significantly elevated the expression of insulin pathway related genes, such as insulin receptor a (IRa), phosphatidylinositol-3-kinase p85 alpha (PI3KR1) and serine/threonine kinase 1 (AKT1), which was partly associated with the repressed expression of glycolysis related genes, glucokinase (GK) and phosphofructokinase liver type (PFKL). Additionally, the inclusion of dietary berberine significantly decreased the content of lipid peroxidation products, MDA. Meanwhile, the antioxidant capacity was significantly elevated at both transcriptional level and enzymatic level, which was suggested to be mediated by Nrf2/Keap1. The analysis of bacterial 16S rRNA V3–4 region indicated that the intestinal microbiota profile was significantly altered at both phylum and genus level, with elevated relative abundance of Fusobacteria, and Cetobacterium, and deceased enrichment for potential pathogenic bacteria genera Plesiomonas with the inclusion of dietary berberine. In summary, the inclusion of dietary berberine improved glucose metabolism, antioxidant capacity and intestinal microbiome of largemouth bass.

Endocrine Dysfunction in Patients With Myotonic Dystrophy.

Myotonic dystrophy is a dominantly inherited multisystem disorder that results from increased CTG repeats in the 3' region of the myotonic dystrophy protein kinase gene (DMPK). The mutant DMPK mRNA remains in the nucleus and sequesters RNA-binding proteins, including regulators of mRNA splicing. Myotonic dystrophy is characterized by a highly variable phenotype that includes muscle weakness and myotonia, and the disorder may affect the function of many endocrine glands. DMPK mRNA is expressed in muscle, testis, liver, pituitary, thyroid, and bone; the mutated form leads to disruption of meiosis and an increase in fetal insulin receptor-A relative to adult insulin receptor-B, resulting in adult primary testicular failure and insulin resistance predisposing to diabetes, respectively. Patients with myotonic dystrophy are also at increased risk for hyperlipidemia, nonalcoholic fatty liver disease, erectile dysfunction, benign and malignant thyroid nodules, bone fractures, miscarriage, preterm delivery, and failed labor during delivery. Circulating parathyroid hormone and adrenocorticotropic hormone levels may be elevated, but the mechanisms for these associations are unclear. This review summarizes what is known about endocrine dysfunction in individuals with myotonic dystrophy.

Molecular investigation of hormonal alterations in Oreochromis niloticus as a bio-marker for long-term exposure to zinc oxide nanoparticles

This study aimed to investigate the potential effects of long-term zinc oxide nanoparticles’ (Zn-ONPs) exposure to the hormonal profile of O. niloticus and their application as a bio-marker for fresh water pollution by Zn-ONPs. Two hundred-forty female O. niloticus were used. Growth hormone (GH), thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), follicular stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), glucagon, insulin, Adrenocorticosteroid hormone (ACTH) serum concentrations and the expression levels of GH, insulin-like growth factor gene (IGF-I), insulin and insulin receptor A (IRA) genes were determined. The levels of GH, TSH, FSH, LH, T3, T4, Estradiol, E2, insulin and GH, IGF-I, insulin and IRA genes expression decreased significantly in Zn-ONPs’ exposed fish in a concentration-dependent manner; meanwhile, serum ACTH and cortisol increased. The alterations in hormone levels and their gene expressions are considered as good bio-markers for the detection of fresh water pollution with Zn-ONPs.

Unraveling the Resistance of IGF-Pathway Inhibition in Ewing Sarcoma.

Simple SummaryThe insulin-like growth factor-1 receptor (IGF1R) is a receptor commonly overexpressed and overactivated in a variety of cancers, including Ewing sarcoma, and promotes cell growth and survival. After promising results with targeting and inhibiting the receptor in vitro, multiple different IGF1R targeting compounds have been clinically tried but showed limited efficacy. Here we discuss several possible resistance mechanisms which could explain why IGF1R targeting fails in the clinic and discuss possible ways to overcome these resistances.Insulin-like growth factor-1 receptor (IGF1R) inhibitors are effective in preclinical studies, but so far, no convincing benefit in clinical studies has been observed, except in some rare cases of sustained response in Ewing sarcoma patients. The mechanism of resistance is unknown, but several hypotheses are proposed. In this review, multiple possible mechanisms of resistance to IGF-targeted therapies are discussed, including activated insulin signaling, pituitary-driven feedback loops through growth hormone (GH) secretion and autocrine loops. Additionally, the outcomes of clinical trials of IGF1-targeted therapies are discussed, as well as strategies to overcome the possible resistance mechanisms. In conclusion, lowering the plasma insulin levels or blocking its activity could provide an additional target in cancer therapy in combination with IGF1 inhibition. Furthermore, because Ewing sarcoma cells predominantly express the insulin receptor A (IRA) and healthy tissue insulin receptor B (IRB), it may be possible to synthesize a specific IRA inhibitor.

Nanocurcumin improved glucose metabolism in streptozotocin-induced diabetic rats: a comparison study with Gliclazide.

In the present study, the biochemical effect of nanocurcumin (nanoCUR) compared with Gliclazide (GLZ) on the diabetic rats was studied. Forty male albino rats (Sprague Dawley) weighted 110 ± 20g were used. Rats were randomly separated into two groups. Control, received no treatment. Streptozotocin (STZ)-induced diabetic groups take 5ml/kg of STZ in normal saline daily for 30days, further divided into diabetic non-treated group, did not receive any treatment: diabetic group treated by nanoCUR, received 15mg/kg/day of nanoCUR orally for 30days; diabetic group treated by GLZ, received 2mg/kg/day of GLZ for 30days. The mean body weights of all rats were registered and serum samples were collected for determination of fasting blood glucose (FBG), insulin concentration, liver glucokinase (GK), and glycogen synthase (GS) activities. Liver tissues were collected for determination of mRNA expression of insulin (INS), insulin receptor A (IRA), glucokinase (GK), and glucose transporter 2 (GLUT2). The results revealed a significant reduction of body weight in diabetic rats, with no significant differences in nanoCUR and GLZ groups. There was a decline in FBG levels and significant elevation of INS levels, GK, and GS activities in diabetic rats received nanoCUR and GLZ. mRNA expression of INS, IRA, GK, and GLUT2 significantly upregulated in diabetic rats received nanoCUR and GLZ. The amazing observation was a non-significant difference in all measured parameters between nanoCUR and GLZ groups. In conclusion, nanoCUR is able to improve cellular uptake of glucose, the hepatic insulin signaling, and insulin sensitivity in diabetic rats. Its effect was similar to standard hypoglycemic drug (GLZ).

Hormonal and molecular alterations induced by sub-lethal toxicity of zinc oxide nanoparticles on Oreochromis niloticus.

This study was carried out to determine the biochemical and molecular potential effects of Zn-ONPs sub-lethal toxicity on the hormonal profile of Oreochromis niloticus (O. niloticus). One hundred and fifty O. niloticus juvenile female were used in this experiment; Ninety for determination of LC50 and other 60 fish were divided into 3 groups with 20 fish each (two replicate in each group). Group I used as control group whereas other groups treated with 1/20 and 1/30 of LC50 respectively for 4 days. Serum, pituitary gland, hepatic, pancreatic and muscular tissues were obtained for hormonal and molecular evaluation. Serum growth hormone (GH), thyroid stimulating hormone (TSH), triiodothyronine (T3), tetraiodothyronine (T4), follicular stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone and insulin hormones were significantly decreased with a significant increase in both Adrenocorticosteroid hormone (ACTH) and cortisol levels with no change in serum glucagon levels. On molecular levels there were a significant down regulation in transcriptional levels of GH, Insulin like growth factor I (IGF-I), insulin and Insulin receptor-A (IRA genes. These results suggested that, hormonal and molecular alterations can be used as an early biomarkers for Zn-ONPs toxicity in fish.

Panax ginseng improves glucose metabolism in streptozotocin-induced diabetic rats through 5' adenosine monophosphate kinase up-regulation.

5′ AMP-activated protein kinase (AMPK), insulin receptors and transporters are distorted in diabetes mellitus. In this study, the effect of Panax ginseng was assessed on glucose manipulating enzymes activities and gene expression of AMPK, IRA and GLUT2 in streptozotocin-induced diabetic male rats. Forty male albino rats were randomly divided to four groups 10 rats of each, group I, normal control group (received saline orally); group II, normal rats received 200 mg/kg of Panax ginseng orally; group III, Streptozotocin (STZ) –induced diabetic rats and group IV, STZ-induced diabetic rats received 200 mg/kg of Panax ginseng orally. The duration of experiment was 30 days. Results showed the ability of Panax ginseng to induce a significant decrease in the blood glucose and increase in the serum insulin levels, hepatic glucokinase (GK), and glycogen synthase (GS) activities with a modulation of lipid profile besides high expression levels of AMPK, insulin receptor A (IRA), glucose transporting protein-2 (GLUT-2) in liver of diabetic rats. In conclusion, the obtained results point to the ability of Panax ginseng to improve the glucose metabolism in diabetic models.

Biochemical study on the potential therapeutic use of novel curcumin copper oxide nanocomposite in diabetic complications in rats

The aim of this study was to investigate the hypoglycemic and antioxidant effect of CurcuminCopper Oxide nano-composite in STZ- induced diabetic rats. Sixty male rats were divided into fourequal groups. Group 1 (control) comprised 15 rats received no drugs, Group 2 (Diabetic nontreatedgroup) included 15 rats received no drugs, serve as STZ induced diabetic group. Group 3(Diabetic nano-curcumin treated group) consists of 15 rats, received curcumin nanoparticle orally ata dose level of 2 mg/kg b.wt/day. Group 4 (Diabetic Curcumin Copper Oxide Nano-Compositetreated group): included 15 rats, received curcumin Copper Oxide Nano-Composite at a dose levelof 7 mg/kg b.wt/day orally. The obtained results revealed that, diabetic rats showed significantincrease in serum glucose and Malondialehyde (MDA) concentrations. However, serum Superoxidedismutase (SOD) activity, Reduced Glutathione (GSH) and insulin concentration in pancreatictissue were markedly decreased. Moreover, a significant down regulation in Insulin Receptor-A(IRA) and Insulin Receptors Substrate-2 (IRs-2) mRNA gene levels in hepatic tissue were observedin diabetic rats. Administration of Curcumin Copper Oxide Nano-Composite in diabetic rats causedsignificant improvement of all previous parameters. These results suggested that, Curcumin CopperOxide Nano-Composite treatment may decrease the diabetic complications as hyperglycemia andoxidative stress in rats through free radical scavenging as well as regulation of endogenousantioxidant defense system mechanisms.

Targeted gene expression panels and microbiota analysis provide insight into the effects of alternative production diet formulations on channel catfish nutritional physiology

The present research evaluated targeted gene expression panels and microbiota analysis to provide greater insight into the effects of alternatively-sourced dietary ingredients on production indices, gut health, changes in the gut microbiota and genes involved in the regulation of appetite, growth, metabolism, and intestinal inflammation. Four dietary formulations were based primarily on distinguishing protein sources: (D1-MFM) menhaden fishmeal (control), (D2-MBM) porcine meat and bone meal, (D3-SBM) soybean meal, and (D4-CSM/CGM) cottonseed meal/corn germ meal, respectively, and fed to channel catfish for 12 weeks. Differences in feed conversion ratio (FCR), specific growth rate, feed intake, body condition, weight gain, proximal intestine histology, intestinal microbiota composition, and quantitative gene expression were analyzed. FCR was significantly (P < 0.05) increased in D2–4 relative to D1-MFM; however, other production indices were unaffected by treatment. Dietary treatment also had no effect on intestinal histology (P < 0.05). Effects of alternative dietary formulations on the gut microbiota were minimal, although when using Chao1, a significant effect of dietary treatment was detected (P = 0.0497) on gut-associated microbiota richness estimates. D3-SBM caused diet-specific differences (P < 0.05) in the expression of neuropeptide Y, peptide YY, and D2-MBM, D3-SBM, and D4-CSM/CGM resulted in differences in α-amylase, insulin receptor-a, glucose-6-phosphate-dehydrogenase, glucocorticoide receptor 1, and glucocorticoide receptor 2, relative to D1-MFM. These changes likely relate to differences in diet-mediated regulation of appetite and glucose metabolism, and perhaps the modulation of gut passage rate. By evaluating the molecular regulation of these pathways, as well as surveying the gut-associated microbiota, effects not detectable in short-term feeding trials may be elucidated which explain subtle differences in performance, such as FCR, as observed in the present study.

Effect of Olea europaea leaves extract on streptozotocin induced diabetes in male albino rats.

The present study was aimed to evaluate the effect of olive (Olea europaea) leaves extract on streptozotocin (STZ)-induced diabetic male rats. The experimental rats were divided into six groups. Rats of the first group were served as normal controls. Rats of the second group were diabetic control. The third and fourth groups were diabetic rats, treated with olive leaves extract at low and high doses respectively. The fifth and sixth groups were non diabetic rats, subjected to olive leaves extract at the same doses given to the third and fourth groups respectively. The minimum of body weigh gain was noted in diabetic rats of the second group. the levels of serum glucose, insulin, total protein, albumin, triglycerides, cholesterol, low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), creatine kinase (CK), lactate dehydrogenase (LDH) and malondialdehyde (MDA) were significantly increased, while the levels of high density lipoprotein cholesterol (HDL-C), superoxide dismutase, (SOD) glutathione (GSH) and catalase (CAT) were statistically decreased in diabetic rats of the second group. The levels of liver insulin receptor substrate 1 (IRS1) and insulin receptor A (IRA) were significantly declined in diabetic rats of the second group. The diabetic pancreatic sections from diabetic rats of the second group showed several histopathological changes. Administration of low and high doses of olive leaves extract improved the observed physiological, molecular and histopathological alterations. Collectively, the obtained results confirmed that the protective effects of olive leaves extract are attributed to the antioxidant activities of olive leaves extract and its active constituents.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. To define differential gene expression and signaling pathways in leiomyoma cell populations. Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Research laboratory. Eight African American women. None. Gene expression patterns, cell proliferation, and differentiation. A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Insulin receptor trafficking steers insulin action.

Insulin receptor trafficking steers insulin action.

Nephrin Contributes to Insulin Secretion and Affects Mammalian Target of Rapamycin Signaling Independently of Insulin Receptor.

Nephrin belongs to a family of highly conserved proteins with a well characterized function as modulators of cell adhesion and guidance, and nephrin may have a role in metabolic pathways linked to podocyte and pancreatic β-cell survival. However, this role is incompletely characterized. In this study, we developed floxed nephrin mice for pancreatic β-cell-specific deletion of nephrin, which had no effect on islet size and glycemia. Nephrin deficiency, however, resulted in glucose intolerance in vivo and impaired glucose-stimulated insulin release ex vivo Glucose intolerance was also observed in eight patients with nephrin mutations compared with three patients with other genetic forms of nephrotic syndrome or nine healthy controls.In vitro experiments were conducted to investigate if nephrin affects autocrine signaling through insulin receptor A (IRA) and B (IRB), which are both expressed in human podocytes and pancreatic islets. Coimmunoprecipitation of nephrin and IRB but not IRA was observed and required IR phosphorylation. Nephrin per se was sufficient to induce phosphorylation of p70S6K in an phosphatidylinositol 3-kinase-dependent but IR/Src-independent manner, which was not augmented by exogenous insulin. These results suggest a role for nephrin as an independent modulator of podocyte and pancreatic β-cell nutrient sensing in the fasting state and the potential of nephrin as a drug target in diabetes.

MEDI-573, Alone or in Combination with Mammalian Target of Rapamycin Inhibitors, Targets the Insulin-like Growth Factor Pathway in Sarcomas

MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma.

Abstract 4493: Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas

Abstract MEDI-573 is a human antibody that neutralizes the insulin-like growth factors, IGF-1 and IGF-2. IGFs are over-expressed in multiple types of cancer; their over-expression is a potential mechanism for resistance to IGF-1 receptor (IGF-1R)-targeting therapy. Effects of IGFs on cell proliferation, differentiation, and survival are mediated through their binding to and activation of IGF-1R or insulin receptor A (IR-A). In this study, we studied the anti-tumor activity and mechanism of action of MEDI-573 in models of sarcoma. MEDI-573 potently inhibited in vitro proliferation of several sarcoma cell lines, with Ewing's sarcoma cell lines being the most sensitive. This inhibition also occurred after growth stimulation with added IGF-1- and IGF-2. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGF-1R, pIR, and pAKT, and significantly blocked IGF-1- and IGF-2-induced activation of the IGF-1R and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo, and inhibition correlated with neutralization of IGF-1 and IGF-2. Combination of MEDI-573 with either rapamycin or another mTOR inhibitor, AZD2014, significantly enhanced the anti-tumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by secreted IGF-1 and IGF-2, and inhibition of IGF-1 and IGF-2 by MEDI-573 results in potent anti-tumor activity in several sarcoma models. Our data provide evidence for evaluation of MEDI-573 and mTORi combinations in clinical studies of sarcoma patients. Note: This abstract was not presented at the meeting. Citation Format: Haihong Zhong, Christine Fazenbaker, Shannon Breen, Cui Chen, Jiaqi Huang, Morehouse Chris, Yihong Yao, Robert Hollingsworth. Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2014-4493

Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion

The objective of this study was to employ genetically engineered IGF-II analogs to establish which receptor(s) mediate the stemness promoting actions of IGF-II on mouse subventricular zone neural precursors. Neural precursors from the subventricular zone were propagated in vitro in culture medium supplemented with IGF-II analogs. Cell growth and identity were analyzed using sphere generation and further analyzed by flow cytometry. F19A, an analog of IGF-II that does not bind the IGF-2R, stimulated an increase in the proportion of neural stem cells (NSCs) while decreasing the proportion of the later stage progenitors at a lower concentration than IGF-II. V43M, which binds to the IGF-2R with high affinity but which has low binding affinity to the IGF-1R and to the A isoform of the insulin receptor (IR-A) failed to promote NSC growth. The positive effects of F19A on NSC growth were unaltered by the addition of a functional blocking antibody to the IGF-1R. Altogether, these data lead to the conclusion that IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R.

Mitogenic Insulin Receptor-A Is Overexpressed in Human Hepatocellular Carcinoma due to EGFR-Mediated Dysregulation of RNA Splicing Factors

Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.

Abstract 533: Differential expression of IGF-II in Vietnamese women in triple negative breast cancer is dependent on biallelic or monoallelic IGF-II with ApaI SNP.

Abstract Triple negative breast cancer (TNBC) is more likely to be advanced, poorly differentiated, larger, and present in women of younger age and of lower socioeconomic status. Moreover, TNBC patients have worse outcomes when treated with currently available adjuvant chemotherapy. We have demonstrated that Breast cancer (BC) tumors from AA patients express significantly higher levels of IGF-II compared to Caucasian women (CA), and show a higher activation of the IGF signaling pathways in tumors progression (Kalla et.al., 2010). Thus we designed a study in younger Vietnamese women where TNBC incidence is increasing. A total of 24 paired BC samples were analyzed by gDNA PCR for IGF-II Apa I polymorphism to correlate with IGF-II mRNA and protein levels. The levels of IGF-II correlated with the imprinting status, i.e., Biallelic (BA; n=14)&amp;gt;&amp;gt;, Monoallelic with SNP (MAS; n=5)&amp;gt;, and Monoallelic non-ApaI SNP (MA; n=5)&amp;gt;. Higher IGF-II levels were expressed in tumor as compared to normal BC tissue samples. The free proIGF-II protein and mRNA levels were higher in biallelic tumor tissues as compared to the proIGF-II levels detected in monoallelic tumor samples. The growth factor receptors; Insulin receptor A (IRA), Insulin receptor B (IRB), Vascular endothelial growth factor receptor (VEGFR), Estrogen receptor-1 (ESR1), Progesterone receptor (PGR) and Human epidermal growth factor receptor (ERBB2) levels were determined to show if IGF-II monoallelic or biallelic dosage regulated the receptor levels. We conclude that biallelic IGF-II expression in TNBC tumors results in higher IGF-II mRNA, protein and related receptors levels than in monoallelic samples. Citation Format: Vinodh Kumar Radhakrishnan, Qianwei Tan, Marino A. De León, Daisy D. De León. Differential expression of IGF-II in Vietnamese women in triple negative breast cancer is dependent on biallelic or monoallelic IGF-II with ApaI SNP. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2013-533