Overexpression Of Insulin-like Growth Factor Research Articles

IGF-1 enhances BMSC viability, migration, and anti-apoptosis in myocardial infarction via secreted frizzled-related protein 2 pathway

BackgroundBone marrow mesenchymal stem cell (BMSC) transplantation represents a promising therapeutic strategy for ischemic heart disease. However, its effects are hampered by the poor viability of transplanted cells and the hostile microenvironment of the ischemic region. Insulin-like growth factor-1 (IGF-1) is an important paracrine growth factor of BMSC and plays an important role in the properties of BMSC. Here, we investigated whether overexpressing IGF-1 could enhance the BMSC viability, migration, anti-apoptosis, and protective effects of cardiomyocytes, and explore the underlying mechanisms’ focus on the role of the AKT/secreted frizzled-related protein 2 (SFRP2)/β-catenin pathway.MethodsWe constructed BMSCs overexpressing insulin-like growth factor-1 (BMSCs-IGF-1) or empty vector (BMSCs-NC) using lentivirus, and evaluated cell survival, proliferation, and migration under normoxic and hypoxic conditions. Co-culture of rat cardiomyoblasts with BMSCs was performed to explore the paracrine effect of BMSCs-IGF-1 for rescuing cardiomyoblasts under hypoxia. Transplantation of BMSCs in acute myocardial infarction rats was used to explore the effect of BMSCs-IGF-1 therapy.ResultsBMSCs-IGF-1 exhibited a higher cell proliferation rate, migration capacity, and stemness, and were more resistant to apoptosis under hypoxia. Overexpression of IGF-1 upregulated the expression of total and nuclear β-catenin via the AKT-secreted frizzled-related protein 2 (SFRP2) pathway, which enhanced cell survival. Inhibition of AKT or SFRP2 knockdown by siRNA significantly antagonized the effect of IGF-1 and decreased the expression of β-catenin. The expression of β-catenin target genes, including cyclin D1 and c-Myc, were accordingly decreased. Moreover, BMSCs-IGF-1 could rescue cardiomyoblasts from hypoxia-induced apoptosis and preserve cell viability under hypoxia. Transplantation of BMSCs-IGF-1 into myocardial infarction rats greatly reduced infarct volume than BMSCs-NC, with significantly greater expression of SFRP2 and β-catenin.ConclusionsThese results suggest that in BMSCs overexpressing IGF-1, SFRP2 is an important mediator for the enhancement of stem cell viability via activating, rather than antagonizing, the Wnt/β-catenin pathway.

Effect of insulin-like growth factor 1 on hepatocyte senescence in carbon tetrachloride-induced liver fibrosis rats

Objective To investigate the development of hepatocyte senescence during liver fibrogenesis and to explore the effect and possible mechanism of insulin-like growth factor 1 (IGF-1) on hepatocyte senescence and liver fibrosis. Methods A total of 42 male Sprague Dawley (SD) rats were selected. Eighteen rats were induced by carbon tetrachloride (CCl4) to establish the rat model of liver fibrosis. On the day 0, six and 28 after the establishment of the model, six rats were executed respectively to analyze the liver fibrosis and hepatocyte senescence in CCl4-induced liver fibrosis rat models. Twenty-four rats were divided into control group, CCl4 group, CCl4+ lentivirus vector (LV-CTR) group and CCl4+ LV-IGF-1 group, with six rats in each group.The rats were sacrificed on the 28th day after the establishment of the model. The liver tissues were obtained and the inferior vena cava blood was collected to analyze the effect of IGF-1 overexpression on liver fibrosis and hepatocyte senescence. Analysis variance (ANOVA), least significant difference (LSD) and Dunnett T3 test were performed for statistical analysis. Results Steatohepatitis on the 6th day and early stage of hepatic fibrosis on the 28th day, which indicated the model was successfully established. The results of the effects of IGF-1 overexpression on hepatic fibrosis and hepatocyte senescence showed that on the 28th day, compared with those of control group, both the score of Ishak liver inflammation and necrosis and the score of Ishak liver fibrosis were increased in the CCl4 group, CCl4+ LV-CTR group and CCl4+ LV-IGF-1 group (0, 14.55±1.94, 15.43±2.19 and 10.29±1.47, respectively; 0, 3.51±0.51, 3.21±0.79 and 1.32±0.40, respectively). The area of liver tissues by Masson staining (0.45±0.40, 5.62±1.08, 6.03±0.65 and 2.88±1.54), SA-β-Gal staining (1.75±0.80, 4.28±1.19, 4.92±1.14, 3.11±0.79), p53 (2.02±0.81, 4.36±1.02, 4.72±0.72 and 3.58±0.70) and progerin (0.72±0.40, 4.52±1.01, 4.01±1.25 and 2.66±0.80) all were increased. The levels of serum IGF-1 all were decreased ((632.00±6.04), (503.00±40.42), (508.00±21.94) and (572.40±5.94) ng/L). However the levels of ALT all were increased ((11.20±5.97), (214.00±73.90), (245.00±76.06) and (30.00±5.00) U/L). The relative expression levels of p53 (0.58±0.06, 1.78±0.18, 1.72±0.10 and 1.23±0.22) and progerin (0.12±0.02, 0.78±0.15, 1.32±0.20 and 0.81±0.16) in the primary hepatocytes were increased. The differences were all statistically significant (F=91.674, 90.778, 32.982, 9.726, 10.640, 17.029, 103.910, 30.059, 64.707 and 97.457, all P<0.05). Compared with those of CCl4+ LV-CTR group, the score of Ishak liver inflammation and necrosis and the score of Ishak liver fibrosis were decreased in the rats′ liver tissues of CCl4+ LV-IGF-1 group, the areas of Masson staining, SA-β-Gal staining, p53 and progerin in the liver tissues were decreased, the level of serum IGF-1 was increased, the level of ALT was decreased, and the relative expression levels of p53 and progerin in primary hepatocytes both were decreased. The differences were all statistically significant (all P<0.05, respectively). Conclusions Hepatocyte senescence increases in the process of liver fibrosis induced by CCl4. Overexpression of IGF-1 may alleviate liver injury, improve hepatocyte senescence and liver fibrogenesis by regulating the nuclear p53/progerin pathway. Key words: Liver cirrhosis; Insulin-like growth factor 1; Hepatocyte senescence; p53; Rat

Insulin-Like Growth Factor 1 Regulates Acute Inflammatory Lung Injury Mediated by Influenza Virus Infection.

The acute inflammatory lung injury is an important cause of death due to influenza A virus (IAV) infection. Insulin-like growth factor 1 (IGF1) played an important role in the regulation of inflammation in the immune system. To investigate the role of IGF1 in IAV-mediated acute inflammatory lung injury, the expression of IGF1 and inflammatory cytokines was tested after IAV A/Puerto Rico/8/1934 (H1N1; abbreviated as PR8) infection in A549 cells. Then, a BALB/c mouse model of PR8 infection was established. On days 3, 5, 7, and 9 post-infection, the mice lung tissue was collected to detect the expression changes in IGF1 mRNA and protein. The mice were divided into four groups: (1) PBS (abbreviation of phosphate buffered saline); (2) PR8 + PBS; (3) PR8 + IGF1; and (4) PR8 + PPP (abbreviation of picropodophyllin, the IGF1 receptor inhibitor). The body weight and survival rate of the mice were monitored daily, and the clinical symptoms of the mice were recorded. On day 5 post-infection, the mice were sacrificed to obtain the serum and lung tissues. The expression of inflammatory cytokines in the serum was detected by enzyme linked immunosorbent assay; lung injury was observed by hematoxylin-eosin staining; the viral proliferation in the lung was detected by real-time quantitative PCR; and the protein expression of the main molecules in the phosphatidylinositol-3-kinases/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways was detected by Western blot. It was found that IGF1 expression is upregulated in A549 cells and BALB/c mice infected with PR8, whereas IGF1 regulated the expression of inflammatory cytokines induced by PR8 infection. Overexpression of IGF1 aggravated the IAV-mediated inflammatory response, whereas the inhibition of IGF1 receptor reduced such inflammatory response. The phosphorylation of IGF1 receptor triggered the PI3K/AKT and MAPK signaling pathways to induce an inflammatory response after IAV infection. Therefore, IGF1 plays an important immune function in IAV-mediated acute inflammatory lung injury. IGF1 may provide a therapeutic target for humans in response to an influenza outbreak, and inhibition of IGF1 or IGF1 receptor may represent a novel approach to influenza treatment.

Insulin-Like Growth Factor 2 (IGF2) Signaling in Colorectal Cancer-From Basic Research to Potential Clinical Applications.

Colorectal cancer (CRC) is one of the most common cancers in men and women worldwide as well as is the leading cause of death in the western world. Almost a third of the patients has or will develop liver metastases. While genetic as well as epigenetic mechanisms are important in CRC pathogenesis, the basis of the most cases of cancer is unknown. High spatial and inter-patient variability of the molecular alterations qualifies this cancer in the group of highly heterogeneous tumors, which makes it harder to elucidate the mechanisms underlying CRC progression. Determination of highly sensitive and specific early diagnosis markers and understanding the cellular and molecular mechanism(s) of cancer progression are still a challenge of the current era in oncology of solid tumors. One of the accepted risk factors for CRC development is overexpression of insulin-like growth factor 2 (IGF2), a 7.5-kDa peptide produced by liver and many other tissues. IGF2 is the first gene discovered to be parentally imprinted. Loss of imprinting (LOI) or aberrant imprinting of IGF2 could lead to IGF2 overexpression, increased cell proliferation, and CRC development. IGF2 as a mitogen is associated with increased risk of developing colorectal neoplasia. Higher serum IGF2 concentration as well as its tissue overexpression in CRC compared to control are associated with metastasis. IGF2 protein was one of the three candidates for a selective marker of CRC progression and staging. Recent research indicates dysregulation of different micro- and long non-coding RNAs (miRNAs and lncRNAs, respectively) embedded within the IGF2 gene in CRC carcinogenesis, with some of them indicated as potential diagnostic and prognostic CRC biomarkers. This review systematises the knowledge on the role of genetic and epigenetic instabilities of IGF2 gene, free (active form of IGF2) and IGF-binding protein (IGFBP) bound (inactive form), paracrine/autocrine secretion of IGF2, as well as mechanisms of inducing dysplasia in vitro and tumorigenicity in vivo. We have tried to answer which molecular changes of the IGF2 gene and its regulatory mechanisms have the most significance in initiation, progression (including liver metastasis), prognosis, and potential anti-IGF2 therapy in CRC patients.

Insulin Receptor Isoform A Modulates Metabolic Reprogramming of Breast Cancer Cells in Response to IGF2 and Insulin Stimulation.

Previously published work has demonstrated that overexpression of the insulin receptor isoform A (IR-A) might play a role in cancer progression and metastasis. The IR has a predominant metabolic role in physiology, but the potential role of IR-A in cancer metabolic reprogramming is unknown. We aimed to characterize the metabolic impact of IR-A and its ligand insulin like growth factor 2 (IGF2) in human breast cancer (BC) cells. To establish autocrine IGF2 action, we generated human BC cells MCF7 overexpressing the human IGF2, while we focused on the metabolic effect of IR-A by stably infecting IGF1R-ablated MCF7 (MCF7IGF1R-ve) cells with a human IR-A cDNA. We then evaluated the expression of key metabolism related molecules and measured real-time extracellular acidification rates and oxygen consumption rates using the Seahorse technology. MCF7/IGF2 cells showed increased proliferation and invasion associated with aerobic glycolysis and mitochondrial biogenesis and activity. In MCF7IGF1R-ve/IR-A cells insulin and IGF2 stimulated similar metabolic changes and were equipotent in eliciting proliferative responses, while IGF2 more potently induced invasion. The combined treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) and the mitochondrial inhibitor metformin blocked cell invasion and colony formation with additive effects. Overall, these results indicate that IGF2 and IR-A overexpression may contribute to BC metabolic reprogramming.

MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells

Numerous studies have described the critical role played by microRNAs (miRNAs) in cancer progression and the potential of these small non-coding RNAs for diagnostic or therapeutic applications. However, the mechanisms responsible for the altered expression of miRNAs in malignant cells remain poorly understood. Herein, via epigenetic unmasking, we identified a group of miRNAs located in the imprinted delta like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3 (MEG3) locus that were repressed in hepatic tumor cells. Notably, miR-493-5p epigenetic silencing was correlated with hypermethylation of the MEG3 differentially regulated region (DMR) in liver cancer cell lines and tumor tissues from patients. Experimental rescue of miR-493-5p promoted an anti-cancer response by hindering hepatocellular carcinoma (HCC) cell growth in vitro and tumor progression in vivo. We found that miR-493-5p mediated part of its tumor-suppressor activity by abrogating overexpression of insulin-like growth factor 2 (IGF2) and the IGF2-derived intronic oncomir miR-483-3p in HCC cells characterized by IGF2 loss of imprinting (LOI). In summary, this study describes an unknown miRNA-dependent regulatory mechanism between two distinct imprinted loci and a possible therapeutic window for liver cancer patients exhibiting IGF2-miR-483 LOI and amplification.

Cardiomyocyte targeted overexpression of IGF1 during detraining restores compromised cardiac condition via mTORC2 mediated switching of PKCδ to PKCα

Altered cardiac adaptation of physiologically hypertrophied heart during detraining remained obscure for long time. We had previously reported the switching of protein kinase C (PKC) isoforms (-α to -δ) associated with functional deterioration of heart at detraining in mice undergone swim exercise. Here we report that, myocardium targeted overexpression of insulin-like growth factor 1 (IGF1) and knockdown of insulin-like growth factor 1 receptor (IGF1R) during detraining and exercise respectively altered the activation of PKCs and eventual cardiac condition. Moreover, downregulation of mammalian target of rapamycin complex 2 (mTORC2) was recorded in both IGF1R knockdown or detraining groups. Additionally, knocking down of mTORC2 during exercise exhibited impaired cardiac condition. Interestingly, significantly increased interactions of mTORC2 with both PKCα and δ was recorded exclusively in exercise group. This interaction resulted into hydrophobic motif phosphorylation of both PKCs (Serine657-PKCα; Serine662-PKCδ). Serine phosphorylation on one hand activated PKCα mediated cell survival and on the other hand alleviated the apoptotic activity of PKCδ during exercise. Mutation of Serine662 of PKCδ in exercised mice showed higher Tyrosine311 phosphorylation with increased apoptotic load similar to that in detrained animals. These observations confirmed that differential and conditional activation of PKCs depend upon IGF1 induced mTORC2 activation. Furthermore, blocking of PKCα resulted in activated p53 which in turn repressed IGF1 expression during swim, mimicking the condition of detrained heart. In conclusion, this is the first report to unravel the intricate molecular mechanism of switching a physiologically hypertrophied heart to a pathologically hypertrophied heart during exercise withdrawal.

Long non-coding RNA HAND2-AS1 downregulation predicts poor survival of patients with end-stage dilated cardiomyopathy.

ObjectiveInsulin-like growth factor-1 (IGF-1) signaling is inhibited in end-stage dilated cardiomyopathy (DCM), and recombinant IGF-1 improves cardiac function in DCM patients. Long non-coding (lnc)RNA HAND2-AS1 was previously shown to be involved in cancer development, but its role in DCM is unknown. The present study investigated the involvement of IGF-1 and HAND2-AS1 in DCM.MethodsPlasma HAND2-AS1 was detected by quantitative real-time PCR. IGF-1 plasma levels were measured by a human IGF-1 enzyme-linked immunosorbent assay. All experiments were performed in triplicate. Pearson’s correlation coefficient analyzed correlations between levels of IGF-1 and HAND2-AS1. Patients were divided into high and low IGF-1 or lncRNA HAND2-AS1 groups, and survival curves were plotted and compared by the Kaplan–Meier method and log-rank test, respectively.ResultsPlasma levels of IGF-1 and lncRNA HAND2-AS1 were significantly lower in end-stage DCM patients than in healthy controls, and were only positively correlated in end-stage DCM patients. A follow-up study revealed that low levels of IGF-1 or lncRNA HAND2-AS1 were significantly associated with poor survival. In the human cardiomyocyte cell line AC16, lncRNA HAND2-AS1 overexpression failed to alter IGF-1 levels and IGF-1 overexpression did not affect lncRNA HAND2-AS1 expression.ConclusionsLncRNA HAND2-AS1 may participate in end-stage dilated cardiomyopathy.

IGF-1 overexpression improves mesenchymal stem cell survival and promotes neurological recovery after spinal cord injury

BackgroundSurvival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI.MethodsUsing a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes.ResultsBMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment.ConclusionsOverexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.

MicroRNA 615-3p Inhibits the Tumor Growth and Metastasis of NSCLC via Inhibiting IGF2.

MicroRNAs are essential regulators of cancer-associated genes at the posttranscriptional level, and their expression is altered in cancer tissues. Herein we sought to identify the regulation of miR-615-3p in NSCLC progression and its mechanism. miR-615-3p expression was significantly downregulated in NSCLC tissue compared to control normal tissue. Exogenous overexpression of miR-615-3p inhibited the growth and metastasis of NSCLC cells. In addition, the in vivo mouse xenograft model showed that overexpression of miR-615-3p inhibited NSCLC growth and lung metastasis, whereas decreased expression of miR-615-3p caused an opposite outcome. Furthermore, we revealed that insulin-like growth factor 2 (IGF2) expression was negatively correlated with the miR-615-3p level in NSCLC specimens, and IGF2 knockdown mimicked the effect of miR-615-3p inhibition on NSCLC cell proliferation, migration, and invasion. In addition, overexpression of IGF2 rescued the inhibition of miR-615-3p in NSCLC cells. Together, our results indicated that miR-615-3p played important roles in the regulation of NSCLC growth and metastasis by targeting IGF2.

MiR-491-5p functions as a tumor suppressor by targeting IGF2 in colorectal cancer.

BackgroundDysregulation of miRNAs is critically implicated in tumorigenesis, and aberrant expression of miR-491-5p has been reported to play a key role in initiation and progression of various cancers. However, the biological function and underlying mechanism of miR-491-5p in colorectal cancer (CRC) remain elusive.MethodsQuantitative real-time PCR (qRT-PCR) was employed to evaluate the levels of miR-491-5p and IGF2 mRNA expression in CRC tissues, cell lines and plasma. Cell counting kit-8 and colony formation assays were used to detect the effects of miR-491-5p on CRC cell growth. Luciferase reporter assays were applied to confirm the miR-491-5p target gene. In vivo experiments were conducted in nude mice.ResultsmiR-491-5p was found to be obviously downregulated in CRC tissues and cell lines, and decreased miR-491-5p expression level was shown to be associated with differentiation, TNM stage and poor overall survival (OS). miR-491-5p overexpression suppressed CRC cell proliferation both in vitro and in vivo. Mechanically, insulin-like growth factor 2 (IGF2) was identified to be a direct target of miR-491-5p in CRC cells, and overexpression of IGF2 rescued the miR-491-5p-induced suppression of proliferation in CRC cells. Finally, we demonstrated that plasma miR-491-5p expression was decreased in CRC when compared to healthy controls and might be an effective diagnostic biomarker for CRC.ConclusionThese data showed that miR-491-5p functioned as a tumor suppressor by targeting IGF2 in CRC, and miR-491-5p could serve as a potential diagnostic and prognostic biomarker for CRC.

Safety, tolerability, and preliminary efficacy of an IGF-1 mimetic in patients with spinal and bulbar muscular atrophy: a randomised, placebo-controlled trial

Spinal and bulbar muscular atrophy is an X-linked neuromuscular disease caused by CAG repeat expansion in the androgen receptor gene. Patients with this disease have low concentrations of insulin-like growth factor-1 (IGF-1), and studies of overexpression and administration of IGF-1 showed benefit in a transgenic model; thus the IGF-1 pathway presents as a potential treatment target. We assessed safety, tolerability, and preliminary efficacy of BVS857, an IGF-1 mimetic, in patients with spinal and bulbar muscular atrophy. In this randomised, double-blind, placebo-controlled trial, we recruited patients from neuromuscular centres in Denmark (Copenhagen), Germany (Ulm), Italy (Padova), and three sites within the USA (Bethesda, MD; Irvine, CA; and Columbus, OH). Eligible patients were 18 years or older with a confirmed genetic diagnosis of spinal and bulbar muscular atrophy, were ambulatory, had symptomatic weakness, and had serum IGF-1 concentrations of 170 ng/mL or lower. Patients were randomly assigned (2:1) to study drug or placebo by a number scheme. Patients, investigators, and study personnel were masked to treatment assignment. After a safety and tolerability assessment with eight patients, BVS857 was administered once a week (0·06 mg/kg intravenously) for 12 weeks. Primary outcome measures were safety, tolerability, and the effects of BVS857 on thigh muscle volume (TMV) measured by MRI. The ratio of TMV at day 85 to baseline was analysed with ANCOVA per protocol. Secondary outcomes of muscle strength and function were measured with the Adult Myopathy Assessment Tool, lean body mass through dual energy x-ray absorptiometry, and BVS857 pharmacokinetics. This trial was registered with ClinicalTrials.gov, NCT02024932. 31 patients were assessed for eligibility, 27 of whom were randomly assigned to either BVS857 treatment (n=18) or placebo (n=9), and 24 were included in the preliminary efficacy analysis (BVS857 group, n=15; placebo group, n=9). BVS857 was generally safe with no serious adverse events. No significant differences were found in adverse events between the BVS857 and placebo groups. Immunogenicity was detected in 13 (72%) of 18 patients in the BVS857 group, including crossreacting antibodies with neutralising capacity to endogenous IGF-1 in five patients. TMV decreased from baseline to day 85 in the placebo group (-3·4% [-110 cm3]) but not in the BVS857 group (0% [2 cm3]). A significant difference in change in TMV was observed in the BVS857 group versus the placebo group (geometric-mean ratio 1·04 [90% CI 1·01-1·07]; p=0·02). There were no differences between groups in measures of muscle strength and function. TMV remained stable in patients with spinal and bulbar muscular atrophy after being given BVS857 for 12 weeks. The intervention was associated with high incidence of immunogenicity and did not improve muscle strength or function. Additional studies might be needed to assess the efficacy of activating the IGF-1 pathway in this disease. Novartis Pharmaceuticals and the US National Institutes of Health.

Abstract PR14: Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors

Abstract Mutations in microRNA processing genes arise in ~20% of Wilms tumors, the most common pediatric kidney cancer. These mutations block microRNA production, yet the key microRNA target genes that drive tumorigenesis in this setting are unknown. To address this question, we performed whole-transcriptome and small RNA sequencing in Wilms tumors. In tumors with microRNA processing mutations, the most significantly overexpressed gene was Pleomorphic adenoma gene 1 (PLAG1). PLAG1 has known oncogenic activity in other cancers but has never been studied in Wilms tumor. This transcription factor is normally expressed only in the developing kidney and liver, where it transactivates insulin-like growth factor 2 (IGF2). Because IGF2 overexpression plays a major role in Wilms tumor pathogenesis, we hypothesized that microRNA pathway mutations contribute to Wilms tumor formation by de-repressing PLAG1, thus overexpressing IGF2. To determine the effect of ectopic PLAG1 expression in vivo, we generated mice that overexpress PLAG1 throughout the developing kidney (Wt1-Cre; LSL-PLAG1). These mice develop grossly enlarged kidneys and die by 4 weeks of age. Prenatally, nephron production is impaired, resulting in fewer glomeruli and dilated tubules. Importantly, PLAG1-overexpressing kidneys progressively acquire several neoplastic characteristics. The kidney parenchyma becomes replaced by cysts that develop polyps and dysplastic thickening. As these dysplastic cells continue to proliferate and fill the cyst lumen, they form nodules that appear precancerous and exhibit markers of active proliferation (Ki-67) and Igf2 signaling (phospho-Erk1/2, phospho-S6). Thus, ectopic PLAG1 expression in the developing kidney recapitulates the two key features of Wilms tumor: blocked differentiation and neoplastic proliferation. We next confirmed that PLAG1 is a microRNA target gene that promotes proliferation of Wilms tumor cells in vitro. Luciferase reporter assays demonstrated that highly conserved sequences in the PLAG1 3’UTR can be targeted by miR-16 and miR-34a. These two miRNAs are strongly downregulated in Wilms tumors with microRNA processing mutations. In addition, mimics and inhibitors of miR-16 and miR-34a modulate endogenous PLAG1 and IGF2 expression in the Wilms tumor cell line WiT49. Finally, PLAG1 overexpression in WiT49 produced increased IGF2 expression, increased activating phosphorylation of its receptor, and increased proliferation. Lastly, we interrogated PLAG1 levels in publicly available data from the TARGET consortium. In 75 relapsed favorable-histology Wilms tumors, microRNA pathway mutations were seen in 14 cases. However, other genomic aberrations were also associated with high PLAG1 expression. For instance, chromosome 8p12, containing the PLAG1 locus, is amplified in 15 cases. Additionally, nine cases show loss of chromosome 1p, which includes the miR-34a locus. These copy number changes rarely overlap with each other or with microRNA pathway mutations. Together, however, these three changes account for 35 of the 75 tumors, and all 35 display high PLAG1 expression. Regardless of mechanism, high levels of PLAG1 correlated with earlier relapse, implying that high PLAG1 expression drives more aggressive tumors. In sum, Wilms tumors can produce high levels of the oncogenic transcription factor PLAG1 through 1p loss, 8p12 gain, or microRNA processing blockade. PLAG1 activates IGF2 transcription and enhances proliferation of Wilms tumor cells in vitro. Overexpression of PLAG1 in developing mouse kidneys produces impaired differentiation and neoplastic growth. These data establish PLAG1 as a novel mediator of Wilms tumor formation and a potential therapeutic target. Citation Format: Kenneth S. Chen, Emily K. Stroup, Albert Budhipramono, Joshua T. Mendell, James F. Amatruda. Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR14.

Overexpression of IGF2 and IGF2 receptor in malignant solitary fibrous tumor with hypoglycemia: a case report

BackgroundSolitary fibrous tumor (SFT) is a prototypical mesenchymal neoplasm that induces non-islet cell tumor hypoglycemia (NICTH) due to overproduction of insulin-like growth factor 2 (IGF2). We here report the case of a malignant SFT associated with a hypoglycemia attack.Case presentationAn 81-year-old man with a large subphrenic mass presented with hypoglycemia and loss of consciousness. His serum insulin and IGF1 levels were relatively low, suggesting an excessively high serum IGF2 levels. Preoperative Western blotting of serum confirmed the overproduction of high-molecular-weight IGF2. After total tumor resection, the patient recovered from hypoglycemia without the need for further treatment. Histological examination revealed proliferation of spindle cells and frequent nuclear mitoses with STAT6 and CD34 immunoreactivity, which led to the diagnosis of malignant SFT. IGF2 was strongly upregulated in the tumor upon immunohistochemistry, consistent with the report of NICTH. In addition, the tumor expressed IGF2 receptor (IGF2R) but not IGF1R.ConclusionsThe present results indicate that the tumor co-expressed IGF2 and IGF2R. IGF2R has not previously been recognized as a tyrosine kinase receptor participating in cell signal transduction. Thus, further case series are required to determine whether IGF2R overexpression reflects the action of an unknown autocrine/paracrine system involving IGF2 for cell proliferation or for the scavenging and degradation of IGF2.

Effect and mechanism of mulberry leaf polysaccharide on type 1 diabetic nephropathy in rats

Objective: To observe the effects of mulberry leaf polysaccharide (MLP) on insulin-like growth factor 1 (IGF-1) and insulin-like growth factor blinding protein 3 (IGFBP-3) as well as the expression of IGF-1 and IGFBP-3 mRNA in the kidney of type 1 Diabetic Nephropathy (DN) rats, and to investigate its therapeutic effects and underlying mechanisms. Methods: Type 1 DN rat model was established by intraperitoneally injecting streptozocin (STZ). SD rats were randomly divided into the control, model, insulin and MLP groups, with eight rats in each group. Rats in MLP group were given orally with MLP 200 mg/kg daily for 8 weeks and insulin group rats were given subcutaneously injection of short acting insulin 1 U daily for 8 weeks. The changes in body weight, blood and urine parameters were recorded. Extracellular matrix (ECM) was calculated. The contents of IGF-1 and IGFBP-3 in blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of IGF-1 and IGFBP-3 in the kidney were evaluated by fluorescence quantitative PCR. Results: Compared with rats in control group, blood glucose, triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein, 24 h urine protein, serum creatinine and urea nitrogen in the model group rats were significantly increased (all P<0.01), and these parameters of MLP group were significantly lower than the model group (all P<0.01). The contents of IGF-1 and IGFBP-3 in the blood serum of the model group were significantly higher than those in the control group (both P<0.001), while in the MLP group they were lower than the model group[IGF-1: (0.777±0.018) ng/ml vs (0.864±0.022) ng/ml, P<0.001; IGFBP-3: (0.759±0.016) ng/ml vs (0.846±0.021) ng/ml, P<0.001]. The mRNA expressions of IGF-1 and IGFBP-3 in the kidney of the model group were significantly higher than those in the control group (both P<0.001), while in the MLP group they were lower than in the model group (IGF-1: 1.450±0.032 vs 1.810±0.090, P<0.001; IGFBP-3: 1.684±0.018 vs 1.968±0.044, P<0.001). Compared with the model group rats, there were fewer pathological changes of kidney in MLP group rats. Conclusion: MLP has a certain therapeutic effect on DN, which may be achieved by decreasing the contents of IGF-1 and IGFBP-3 in the blood serum and down-regulating the over-expression of IGF-1 and IGFBP-3 mRNA in the kidney.

Yak IGF2 Promotes Fibroblast Proliferation Via Suppression of IGF1R and PI3KCG Expression.

Insulin-like growth factor 2 (IGF2) recapitulates many of the activities of insulin and promotes differentiation of myoblasts and osteoblasts, which likely contribute to genetic variations of growth potential. However, little is known about the functions and signaling properties of IGF2 variants in yaks. The over-expression vector and knockdown sequence of yak IGF2 were transfected into yak fibroblasts, and the effects were detected by a series of assays. IGF2 expression in yak muscle tissues was significantly lower than that of other tissues. In yak fibroblasts, the up-regulated expression of IGF2 inhibits expression of IGF1 and insulin-like growth factor 2 receptor (IGF2R) and significantly up-regulates expression of IGF1R. Inhibition of IGF2 expression caused the up-regulates expression of IGF1, IGF1R and IGF2R. Both over-expression and knockdown of IGF2 resulted in up-regulation of threonine protein kinase 1 (Akt1) expression and down-regulation of phosphatidylinositol 3-kinase, catalytic subunit gamma (PIK3CG). Cell cycle and cell proliferation assays revealed that over-expression of IGF2 enhanced the DNA synthesis phase and promoted yak fibroblasts proliferation. Conversely, knockdown of IGF2 decreased DNA synthesis and inhibited proliferation. These results suggested that IGF2 was negatively correlated with IGF1R and PIK3CG and demonstrated an association with the IGFs-PI3K-Akt (IGFs-phosphatidylinositol 3-kinase- threonine protein kinase) pathway in cell proliferation and provided evidence supporting the functional role of IGF2 for use in improving the production performance of yaks.

Disruption of IGF‑1R signaling by a novel quinazoline derivative, HMJ‑30, inhibits invasiveness and reverses epithelial-mesenchymal transition in osteosarcoma U‑2 OS cells.

Osteosarcoma is the most common primary malignancy of the bone and is characterized by local invasion and distant metastasis. Over the past 20 years, long-term outcomes have reached a plateau even with aggressive therapy. Overexpression of insulin-like growth factor 1 receptor (IGF-1R) is associated with tumor proliferation, invasion and migration in osteosarcoma. In the present study, our group developed a novel quinazoline derivative, 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino)quinazoline (HMJ-30), in order to disrupt IGF-1R signaling and tumor invasiveness in osteosarcoma U-2 OS cells. Molecular modeling, immune-precipitation, western blotting and phosphorylated protein kinase sandwich ELISA assays were used to confirm this hypothesis. The results demonstrated that HMJ-30 selectively targeted the ATP-binding site of IGF-1R and inhibited its downstream phosphoinositide 3-kinase/protein kinase B, Ras/mitogen-activated protein kinase, and IκK/nuclear factor-κB signaling pathways in U-2 OS cells. HMJ-30 inhibited U-2 OS cell invasion and migration and downregulated protein levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9. An increase in protein levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was also observed. Furthermore, HMJ-30 caused U-2 OS cells to aggregate and form tight clusters, and these cells were flattened, less elongated and displayed cobblestone-like shapes. There was an increase in epithelial markers and a decrease in mesenchymal markers, indicating that the cells underwent the reverse epithelial-mesenchymal transition (EMT) process. Overall, these results demonstrated the potential molecular mechanisms underlying the effects of HMJ-30 on invasiveness and EMT in U-2 OS cells, suggesting that this compound deserves further investigation as a potential anti-osteosarcoma drug.

Central Infusion of Insulin-Like Growth Factor-1 Increases Hippocampal Neurogenesis and Improves Neurobehavioral Function after Traumatic Brain Injury.

Traumatic brain injury (TBI) produces neuronal dysfunction and cellular loss that can culminate in lasting impairments in cognitive and motor abilities. Therapeutic agents that promote repair and replenish neurons post-TBI hold promise in improving recovery of function. Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor capable of mediating neuroprotective and neuroplasticity mechanisms. Targeted overexpression of IGF-1 enhances the generation of hippocampal newborn neurons in brain-injured mice; however, the translational neurogenic potential of exogenously administered IGF-1 post-TBI remains unknown. In a mouse model of controlled cortical impact, continuous intracerebroventricular infusion of recombinant human IGF-1 (hIGF) for 7 days, beginning 15 min post-injury, resulted in a dose-dependent increase in the number of immature neurons in the hippocampus. Infusion of 10 μg/day of IGF-1 produced detectable levels of hIGF-1 in the cortex and hippocampus and a concomitant increase in protein kinase B activation in the hippocampus. Both motor function and cognition were improved over 7 days post-injury in IGF-1-treated cohorts. Vehicle-treated brain-injured mice showed reduced hippocampal immature neuron density relative to sham controls at 7 days post-injury. In contrast, the density of hippocampal immature neurons in brain-injured mice receiving acute onset IGF-1 infusion was significantly higher than in injured mice receiving vehicle and equivalent to that in sham-injured control mice. Importantly, the neurogenic effect of IGF-1 was maintained with as much as a 6-h delay in the initiation of infusion. These data suggest that central infusion of IGF-1 enhances the generation of immature neurons in the hippocampus, with a therapeutic window of at least 6 h post-injury, and promotes neurobehavioral recovery post-TBI.

A sensitive biomarker panel to distinguish pancreaticobiliary malignancies from benign disease.

238 Background: Biliary strictures present a diagnostic challenge to differentiate benign disease from hepatopancreaticobiliary (HPB) malignancies. Cytology from Endoscopic retrograde cholangiopancreatography (ERCP) or Endoscopic ultrasound with fine needle aspiration (EUS-FNA) have both been plagued by poor sensitivity and high false negative rates. In a review of over 80 adjunct molecular biology tests that have been attempted on ERCP and EUS-FNA samples, four immunohistochemistry biomarkers with sensitivities ranging 74-80% were identified; von Hippel Lindau loss of expression (VHL), over-expression of insulin-like growth factor 2 mRNA-binding Protein 3 (IMP3), and EF-hand Calcium 2+ binding S100 subfamily members A4 (S100A4) and P (S100P). We sought to determine if these results could be validated in a tumor explant model and furthermore if combining these tests into a biomarker panel could boost overall diagnostic sensitivity to 100%. Methods: Tumor tissue and normal surrounding pancreas from 27 pancreaticoduodenectomy specimens were selected by an experienced pathologist, subjected to immunohistochemistry staining with VHL, IMP3, S100A4, S100P, and the intensity and percent of cells staining was graded. Using ROC curve analysis, threshold criteria were chosen for each biomarker to differentiate between tumor and normal pancreas. Biomarkers were then evaluated as a panel for their ability to discriminate malignant from benign specimens. Results: Individual sensitivity of VHL, IMP3, S100A4, and S100P were found to be 75.0%, 79.2%, 45.8%, and 0%. When VHL, IMP3, and S100A4 were grouped into a panel, they were able to distinguish cancer from normal tissue with a sensitivity of 100% and a specificity of 96%. S100P was dispensable in our assay and only VHL, IMP3, and S100A4 staining were required to achieve 100% sensitivity. Conclusions: A panel of three biomarkers, VHL, IMP3, and S100A4, were able to distinguish pancreatic cancer from surrounding normal tissue with 100% sensitivity in our tumor explant model. Prospective studies on patient biopsy specimens are required to further validate the clinical use of this biomarker panel.

Inhibition of microRNA-19b promotes ovarian granulosa cell proliferation by targeting IGF-1 in polycystic ovary syndrome.

The purpose of the present study was to investigate the functional role of microRNA (miR)-19b in polycystic ovary syndrome (PCOS) and try to elucidate its underlying mechanisms. Expression of miR-19b and insulin-like growth factor 1 (IGF-1) was examined in ovarian cortexes [(from 18 women with PCOS and 10 who did not have PCOS (non-PCOS)] and KGN cells. Cell proliferation assays (cell viability and colony formation assay) were performed following overexpression or inhibition of miR-19b and IGF-1 or following insulin treatment in KGN cells. Expression levels of the cell cycle-associated protein cyclin D1 and cyclin-dependent kinase (CDK) 1 were analyzed following overexpression or inhibition of miR-19b and IGF-1. Potential miR-19b targets were identified by bioinformatics. Luciferase assay, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine whether IGF-1 was a target of miR-19b. miR-19b expression was significantly decreased in the PCOS ovarian cortex and KGN cells and its identified target, IGF-1, was upregulated. miR-19b overexpression inhibited cell proliferation at G2/M phrase. Overexpression of IGF-1 promoted cell viability and colony formation ability in KGN cells. The expression of cyclin D1 and CDK1 was statistically increased by inhibition of miR-19b and overexpression of IGF-1. High concentrations of insulin decreased levels of miR-19b, stimulated KGN cell proliferation, and elevated IGF-1 levels. Inhibition of miR-19b promoted ovarian granulosa cell proliferation by targeting IGF-1 in PCOS. Insulin decreased the expression levels of miR-19b and stimulated cell proliferation. The present study suggested that overexpression of miR-19b may be a potential therapeutic approach for PCOS.