IGFBP3 Knockdown Research Articles

Abstract P1150: Integrative Transcriptomics And Cell Systems Analyses Reveal Protective Pathways Controlled By Igfbp-3 In Anthracycline-induced Cardiotoxicity

Rationale: Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents. Their use however is hindered by the risk of anthracycline-induced cardiotoxicity (AIC). Our understanding of protective pathways activated by cardiomyocytes following AIC remains incomplete. Insulin-like growth factor binding protein (Igfbp)-3, the most abundant Igfbp family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cell types. Whereas Igfbp-3 is significantly induced by Dox in the heart, its role in AIC remains ill-defined. Methods and Results: We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 overexpression leads to a significant reduction of γ-H2AX, a marker of DNA damage (56% reduction vs. control, n=3, P<0.05), and impedes topoisomerase (Top)-2β expression which forms a Top2β-Dox-DNA cleavage complex leading to DNA double-strand breaks (44% reduction vs. control, n=3, P<0.01). Next generation sequencing and gene network analyses of iPSC-CMs treated with Dox reveal that Igfbp-3 perturbation modulates genes implicated in microtubule function and cardiomyocyte contraction. Stimulated Emission Depletion (STED) microscopy and immunoblotting further indicate that Igfbp-3 knockdown exacerbates detyrosinated microtubule accumulation (STED, 21% increase vs. control, n=16-19, P<0.05; immunoblotting, 62% increase vs. control, n=6, P<0.01) – a hallmark of increased cardiomyocyte stiffness and heart failure – and favorably affects contractility following Dox treatment. Conclusion: Our delineation of the protective role of Igfbp-3 in AIC provides novel insight into pathways induced by cardiomyocytes to mitigate DNA damage and apoptosis, and improve contractile function.

GATA4 inhibits odontoblastic differentiation of dental pulp stem cells through targeting IGFBP3

ObjectiveThe odontogenic differentiation of human dental pulp stem cells (HDPSCs) is associated with reparative dentinogenesis. Transcription factor GATA binding protein 4 (GATA4) is proved to be essential for osteoblast differentiation and bone remodeling. This study clarified the function of GATA4 in HDPSCs odontoblast differentiation. MethodsThe change in GATA4 expression during reparative dentin formation was detected by immunohistochemistry staining. The expression of GATA4 during HDPSCs odontoblastic differentiation was detected by western blot and quantitative polymerase chain reaction. The effect of GATA4 on odontoblast differentiation was investigated following overexpression lentivirus transfection. RNA sequencing, dual luciferase assay and chromatin immunoprecipitation (CHIP) were conducted to verify downstream targets of GATA4. GATA4 overexpression lentivirus and small interference RNA targeting IGFBP3 were co-transfected to investigate the regulatory mechanism of GATA4. ResultsUpregulated GATA4 was observed during reparative dentin formation in vivo and the odontoblastic differentiation of HDPSCs in vitro. GATA4 overexpression suppressed the odontoblastic potential of HDPSCs, demonstrated by decreased alkaline phosphatase activity (p < 0.0001), mineralized nodules formation (p < 0.01), and odonto/osteogenic differentiation markers levels (p < 0.05). RNA sequencing revealed IGFBP3 was a potential target of GATA4. CHIP and dual luciferase assays identified GATA4 could activate IGFBP3 transcription. Additionally, IGFBP3 knockdown recovered the odontoblastic differentiation defect caused by GATA4 overexpression (p < 0.05). ConclusionsGATA4 inhibited odontoblastic differentiation of HDPSCs via activating the transcriptional activity of IGFBP3, identifying its promising role in regulating HDPSCs odontoblast differentiation and reparative dentinogenesis.

IGFBP3 epigenetic promotion induced by METTL3 boosts cardiac fibroblast activation and fibrosis

Cardiac fibrosis remains an unresolved problem in heart disease. Its etiology is directly caused by the activation and proliferation of cardiac fibroblasts (CFs). However, there is limited information regarding the biological role of cardiac fibroblasts in cardiac fibrosis. Herein, we screened out a gene, IGFBP3, whose expression significantly increased in TGF-β1-stimulated human primary CFs by mining RNA-Seq data for differential and WGCNA. We verified the IGFBP3's expression in transverse aortic constriction (TAC) surgery, isoproterenol (ISO)-induced cardiac fibrosis models, and TGFβ1-stimulated mouse primary CFs. We also found that the knockdown of IGFBP3 could inhibit the migration and proliferation ability of CFs. Furthermore, we found that aberrant N6-methyladenosine(m6A) mRNA modifications in the animal model and activated CFs may regulate the expression of IGFBP3 in developing cardiac fibrosis. Silencing METTL3 could downregulate the expression of IGFBP3 and inhibit the activation of CFs and the degree of cardiac fibrosis both in vitro and in vivo. Indeed, we also verified the expression of METTL3 and IGFBP3 in the atrial tissues of patients with atrial fibrillation (AF). Thus, METTL3 may regulate IGFBP3's expression and CFs activation via RNA epigenetic modifications, laying the foundation for a specific and novel therapeutic target in cardiac fibrosis.

Opposing Roles of IGFBP-3 and Heparanase in Regulating A549 Lung Cancer Cell Survival.

In this study, we examined the roles of heparanase and IGFBP-3 in regulating A549 and H1299 non-small-cell lung cancer (NSCLC) survival. We found that H1299 cells, known to be p53-null with no expression of IGFBP-3, had higher heparanase levels and activity and higher levels of heparan sulfate (HS) in the media compared to the media of A549 cells. Inhibiting heparanase activity or its expression using siRNA had no effect on the levels of IGFBP-3 in the media of A549 cells, reduced the levels of soluble HS fragments, and led to decreased interactions between IGFBP-3 and HS in the media. HS competed with HA for binding to IGFBP-3 or IGFBP-3 peptide (215-KKGFYKKKQCRPSKGRKR-232) but not the mutant peptide (K228AR230A). HS abolished the cytotoxic effects of IGFBP-3 but not upon blocking HA-CD44 signaling with the anti-CD44 antibody (5F12). Blocking HA-CD44 signaling decreased the levels of heparanase in the media of both A549 and H1299 cell lines and increased p53 activity and the levels of IGFBP-3 in A549 cell media. Knockdown of p53 led to increased heparanase levels and reduced IGFBP-3 levels in A549 cell media while knockdown of IGFBP-3 in A549 cells blocked p53 activity and increased heparanase levels in the media.

APOE ε4-dependent effects on the early amyloid pathology in induced neurons of patients with Alzheimer’s disease

BackgroundThe ε4 allele of apolipoprotein E (APOE ε4) is the strongest known genetic risk factor for late-onset Alzheimer’s disease (AD), associated with amyloid pathogenesis. However, it is not clear how APOE ε4 accelerates amyloid-beta (Aβ) deposition during the seeding stage of amyloid development in AD patient neurons.MethodsAD patient induced neurons (iNs) with an APOE ε4 inducible system were prepared from skin fibroblasts of AD patients. Transcriptome analysis was performed using RNA isolated from the AD patient iNs expressing APOE ε4 at amyloid-seeding and amyloid-aggregation stages. Knockdown of IGFBP3 was applied in the iNs to investigate the role of IGFBP3 in the APOE ε4-mediated amyloidosis.ResultsWe optimized amyloid seeding stage in the iNs of AD patients that transiently expressed APOE ε4. Remarkably, we demonstrated that Aβ pathology was aggravated by the induction of APOE ε4 gene expression at the amyloid early-seeding stage in the iNs of AD patients. Moreover, transcriptome analysis in the early-seeding stage revealed that IGFBP3 was functionally important in the molecular pathology of APOE ε4-associated AD.ConclusionsOur findings suggest that the presence of APOE ε4 at the early Aβ-seeding stage in patient iNs is critical for aggravation of sporadic AD pathology. These results provide insights into the importance of APOE ε4 expression for the progression and pathogenesis of sporadic AD.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.219 Antisense oligonucleotide-mediated knockdown of IGFBP3 to increase IGF-1 signalling in dystrophic muscle

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by the absence of dystrophin protein. Upon exhaustion of the regenerative capacity of muscle, there is an imbalance between muscle damage and repair. Insulin-like growth factor 1 (IGF-1) plays an essential role in increasing muscle mass and strength, reducing degeneration, inhibiting chronic inflammation and increasing the proliferation potential of satellite cells. Although liver is the main source of IGF-1, it is also expressed in different tissues including skeletal muscle. Both in circulation and the extracellular matrix, IGF-1 is bound to one of its binding proteins (IGFBPs). The majority of IGF-1 transport is associated with IGFBP3. Other IGFBPs are also found in the circulation and complexes of these IGFBPs with IGF-1 can exit the circulation easily, making them more available to target tissues. By contrast, IGF-1+IGFBP3 complexes, unless cleaved by specific proteases, cannot cross vascular endothelium. IGFBP3 is thought to act by inhibiting the availability of IGF-1 to its receptor, which initiates most of the actions of IGF-1. More recently, studies revealed that IGFBP-3 can be inhibitory to myoblast proliferation even in the absence of IGF signalling. Therefore, decreasing IGFBP3 levels would increase IGF-1 action and prevent IGF-1 independent antiproliferative effect of IGFBP3, making it a potential therapy for DMD by improving muscle repair and growth. This can be achieved by downregulation of IGFBP3 using antisense oligonucleotides (AONs) that skip out of frame exon(s), thus preventing the expression of functional protein. In this study, two different AONs were designed to target exon 2 of Igfbp3 and transfected using lipofectamine in mouse C2C12 cell cultures. Cells were harvested after 24 hours and total RNA was isolated. Exon skipping was confirmed by RT-PCR on RNA level. In addition, time course and western blot experiments are ongoing. In vivo studies are being planned.

IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis

PurposeWe have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis.MethodsEffects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34+ tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum.ResultsKnockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling.ConclusionsSerum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.

Suppression of tumor growth via IGFBP3 depletion as a potential treatment in glioma.

Despite intensive medical treatment, patients with glioblastoma (grade IV glioma [GBM]) have a low 5-year survival rate of 5.5%. In this study, the authors tried to improve currently used therapies by identification of a therapeutic target, IGFBP3, for glioma treatment. IGFBP3 RNA expression in 135 patients newly diagnosed with glioma was correlated with clinicopathological factors. Immunohistochemical analysis was performed to determine IGFBP3 protein expression in glioma specimens. The effect of IGFBP3 depletion on cell proliferation was examined using IGFBP3 knockdown glioma cells. Intracranial infusion of IGFBP3 siRNAs was performed to evaluate the effect of IGFBP3 depletion in mouse intracranial xenograft models. We demonstrated higher IGFBP3 expression in GBM than in tumor margin and grade II glioma. IGFBP3 expression was not only positively correlated with tumor grades but also associated with tumor histology and IDH1/2 mutation status. Additionally, higher IGFBP3 expression predicted shorter overall survival in glioma and GBM proneural subgroup patients. In vitro cell culture studies suggested IGFBP3 knockdown suppressed cell proliferation and induced cell cycle G2/M arrest as well as apoptosis in glioma cells. Also, accumulation of DNA double-strand breaks and γH2AX was observed in IGFBP3 knockdown cells. IGFBP3 knockdown delayed in vivo tumor growth in mouse subcutaneous xenograft models. Furthermore, convection-enhanced delivery of IGFBP3 siRNA to mouse brain suppressed intracranial tumor growth and prolonged survival of tumor-bearing mice. Our findings suggest IGFBP3 predicts poor outcome of glioma patients and is a potential therapeutic target for which depletion of its expression suppresses tumor growth through inducing apoptosis and accumulation of DNA damage in glioma cells.

IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells.

IGF-binding protein (IGFBP)-3 is a multifunctional protein that can exert IGF-independent effects on apoptosis. Anisomycin (ANS) is a potent inducer of IGFBP-3 production in bovine mammary epithelial cells (MECs), and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. IGFBP-3 is present in the nucleus and the conditioned media in response to ANS. The goal of this study was to determine whether ribotoxic stress induced by ANS or a second ribotoxin, deoxynivalenol (DON), specifically regulates transport of IGFBP-3 to the nucleus and to determine the pathway by which it traffics. In ribotoxin-treated cells, both endogenous IGFBP-3 and transfected IGFBP-3 translocated to the nucleus. Inhibition of the nuclear transport protein importin-β with importazole reduced ribotoxin-induced nuclear IGFBP-3. Immunoprecipitation studies showed that ANS induced the association of IGFBP-3 and importin-β, indicating that ribotoxins specifically induce nuclear translocation via an importin-β‒dependent mechanism. To determine whether secretion of IGFBP-3 is required for nuclear localization, cells were treated with Pitstop 2 or brefeldin A to inhibit clathrin-mediated endocytosis or overall protein secretion, respectively. Neither inhibitor affected nuclear localization of IGFBP-3. Although the IGFBP-3 present in both the nucleus and conditioned media was glycosylated, secreted IGFBP-3 exhibited a higher molecular weight. Deglycosylation experiments with endoglycosidase Hf and PNGase indicated that secreted IGFBP-3 completed transit through the Golgi apparatus, whereas intracellular IGFBP-3 exited from the endoplasmic reticulum before transit through the Golgi. In summary, ANS and DON specifically induced nuclear localization of nonsecreted IGFBP-3 via an importin-β‒mediated event, which may play a role in their ability to induce apoptosis in MECs.

Abstract 1095: Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) regulates mitochondrial dynamics, EMT and angiogenesis in progression of prostate cancer

Abstract Prostate cancer (PCa) is a major epithelial cancer among men and with the second highest incidence rate, worldwide. The high ratio of IGF-1/IGFBP-3 correlates with increased risk of many cancers including prostate cancer. In present study, we have evaluated the role of IGFBP-3 and effect of fisetin, a phytochemical, active constituent of strawberry, apple etc. in the progression of prostate cancer. The exogenous expression of IGFBP-3 moderately decreased the cell growth and it further decreased with addition of fisetin in DU145 and LNCaP cells. The restoration of IGFBP-3 in DU145 cells inhibited the clonogenic potential which is further decreased with fisetin treatment. Increased ROS content was observed in IGFBP-3 overexpression condition, however fisetin treatment reversed the ROS content in DU145, shows antioxidant behaviour. Morphological examination of mitochondria revealed that IGFBP-3 overexpression destabilizes the mitochondrial dynamics by reduction in active DRP1 level which has been reversed by fisetin treatment. We evaluated the effect on mitochondrial mass which was decreased in IGFBP-3 overexpressing DU145 cells which further decreased with addition of fisetin at 12 and 24 h. IGFBP-3 overexpression decreased the migratory potential of DU145 cells under normoxic conditions and under hypoxic condition it increased the migration of DU145 cells. Furthermore, IGFBP-3 overexpression decreased the VEGF expression as compared to vector control which can inhibit the expansion of tumor cells. Under the hypoxic conditions (1% oxygen), cells showed increased levels of IGFBP-3 when compared to normoxic conditions (21% Oxygen) in time dependent manner. The down-regulation of IGFBP-3 in PC3 cells, increased the expression of E-cadherin, a biomarker of epithelial to mesenchymal transition. The wound scratch assay showed the pro-migratory role of IGFBP-3 in PC3 cells and treatment with fisetin inhibited the migration of these cells under the hypoxic condition. The knockdown of IGFBP3 resulted in the decreased invasive potential of PC3 in comparison to cells in hypoxic state. Together with these observation, IGFBP-3 have shown biphasic character depending on normoxic and hypoxic condition in controlling the prostate cancer progression. Citation Format: Arpit Dheeraj, Dhanir Tailor, Gagan Deep, Rana P. Singh. Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) regulates mitochondrial dynamics, EMT and angiogenesis in progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1095.

IGFBP3 deposited in the human umbilical cord mesenchymal stem cell-secreted extracellular matrix promotes bone formation.

The extracellular matrix (ECM) contains rich biological cues for cell recruitment, proliferationm, and even differentiation. The osteoinductive potential of scaffolds could be enhanced through human bone marrow mesenchymal stem cell (hBMSC) directly depositing ECM on surface of scaffolds. However, the role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSC)‐secreted ECM in bone formation remain unknown. We tested the osteoinductive properties of a hUCMSC‐secreted ECM construct (hUCMSC‐ECM) in a large femur defect of a severe combined immunodeficiency (SCID) mouse model. The hUCMSC‐ECM improved the colonization of endogenous MSCs and bone regeneration, similar to the hUCMSC‐seeded scaffold and superior to the scaffold substrate. Besides, the hUCMSC‐ECM enhanced the promigratory molecular expressions of the homing cells, including CCR2 and TβRI. Furthermore, the hUCMSC‐ECM increased the number of migrated MSCs by nearly 3.3 ± 0.1‐fold, relative to the scaffold substrate. As the most abundant cytokine deposited in the hUCMSC‐ECM, insulin‐like growth factor binding protein 3 (IGFBP3) promoted hBMSC migration in the TβRI/II‐ and CCR2‐dependent mechanisms. The hUCMSC‐ECM integrating shRNA‐mediated silencing of Igfbp3 that down‐regulated IGFBP3 expression by approximately 60%, reduced the number of migrated hBMSCs by 47%. In vivo, the hUCMSC‐ECM recruited 10‐fold more endogenous MSCs to initiate bone formation compared to the scaffold substrate. The knock‐down of Igfbp3 in the hUCMSC‐ECM inhibited nearly 60% of MSC homing and bone regeneration capacity. This research demonstrates that IGFBP3 is an important MSC homing molecule and the therapeutic potential of hUCMSC‐ECM in bone regeneration is enhanced by improving MSC homing in an IGFBP3‐dependent mechanism.

Abstract 2122: Pharmacologic interconversion of EMT to MET for prostate cancer

Abstract Introduction and objection: Dysregulation of transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) axis has been linked to reactive stroma dynamics in the tumor microenvironment during prostate cancer progression. IGFBP3 induction is initiated by stroma remodeling and represents a potential therapeutic target for advanced prostate cancer. A lead quinazoline-based Doxazosin® derivative, DZ-50, generated in our laboratory (US Patent # 8377948), inhibits prostate tumor growth via inducing anoikis and disrupting focal adhesions. Molecular profiling revealed that the process of epithelial-mesenchymal-transition (EMT) is targeted by DZ-50. In this study, we investigated the effect of DZ-50 on EMT landscape, EMT to mesenchymal-epithelial-transition (MET) conversion, and invasive properties of prostate cancer cells. Methods: Human prostate cancer cells LNCaP, LNCaP overexpressing TGF-β type II receptor (TβRII), and cancer associated fibroblasts (CAFs) derived from human prostate cancer specimens, were used. The antitumor effect of DZ-50 against prostate cancer epithelial cells and CAFs was evaluated using cell viability assays. Effect of the drug on EMT key regulators (including IGFBP3) was determined using RT-PCR and Western blot analysis. Drug-induced phenotypic conversions of EMT were evaluated by confocal microscopy. Impact of TGF-β from the stroma microenvironment or exogenous cytokine, on prostate tumor cell migration and invasion, was assessed in co-cultures with CAFs. The functional contribution of IGFBP3 to EMT-MET interconversion in response to DZ-50 was assessed using siRNA approaches. Results: DZ-50 induced cell death in prostate cancer epithelial cells and CAFs, in a concentration-dependent manner. DZ-50 downregulated IGFBP3 mRNA and protein expression and promoted EMT-MET conversion in both LNCaP and LNCaPTβRII cells. IGFBP3 knockdown in LNCaPTβRII cells led to E-cadherin upregulation and MET induction, implicating IGFBP3 as a potential target of DZ-50 to reverse EMT to MET. Moreover exposure to TGF-β reversed DZ-50-induced MET by upregulating IGFBP3 in LNCaPTβRII cells. Co-cultures of LNCaPTβRII with CAFs promoted prostate cancer cell invasion via TGF-β and IGFBP3, an effect that was inhibited by the drug. Conclusions: Treatment of prostate cancer cells with the novel agent DZ-50 inhibits cell migration and invasion and causes reversal of EMT to MET by regulating IGFBP3. This study integrates IGFBP3 as new signaling effector driving the antitumor action of DZ-50 via targeting the EMT-MET phenotypic landscape in the prostate tumor microenvironment. Ongoing work in pre-clinical models will establish the therapeutic value of this novel compound in advanced metastatic prostate cancer. Citation Format: Zheng Cao, Shahriar Koochekpour, Stephen E. Strup, Natasha Kyprianou. Pharmacologic interconversion of EMT to MET for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2017-2122

Pancreatic cancer cell-derived IGFBP-3 contributes to muscle wasting.

BackgroundProgressive loss of skeletal muscle, termed muscle wasting, is a hallmark of cancer cachexia and contributes to weakness, reduced quality of life, as well as poor response to therapy. Previous studies have indicated that systemic host inflammatory response regarding tumor development results in muscle wasting. However, how tumor directly regulates muscle wasting via tumor-derived secreted proteins is still largely unknown.MethodsIn this study, we performed bioinformatics analysis in two datasets of pancreatic ductal adenocarcinoma, which causes cancer cachexia and muscle wasting with the highest prevalence, and uncovered that IGFBP3, which encodes IGF-binding protein-3 (IGFBP-3), is dramatically up-regulated in pancreatic tumor samples. We also verified the wasting effect of IGFBP-3 on C2C12 muscle cells with biochemical and genetic assays.ResultsIGFBP-3 potently leads to impaired myogenesis and enhanced muscle protein degradation, the major features of muscle wasting, via IGF signaling inhibition. Moreover, conditioned medium from Capan-1 pancreatic cancer cells, which contains abundant IGFBP-3, significantly induces muscle cell wasting. This wasting effect is potently alleviated by IGFBP3 knockdown in Capan-1 cells or IGFBP-3 antibody neutralization. Strikingly, compared to muscle cells, IGF signaling and proliferation rate of Capan-1 cells were rarely affected by IGFBP-3 treatment.ConclusionsOur results demonstrated that pancreatic cancer cells induce muscle wasting via IGFBP-3 production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0317-z) contains supplementary material, which is available to authorized users.

Endogenous IGFBP-3 Mediates Intrinsic Apoptosis Through Modulation of Nur77 Phosphorylation and Nuclear Export

In nontransformed bovine mammary epithelial cells, the intrinsic apoptosis inducer anisomycin (ANS) induces IGFBP-3 expression and nuclear localization and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. Others have shown in prostate cancer cells that exogenous IGFBP-3 induces apoptosis by facilitating nuclear export of the orphan nuclear receptor Nur77 and its binding partner, retinoid X receptor-α (RXRα). The goal of the present work was to determine whether endogenous IGFBP-3 plays a role in ANS-induced apoptosis by facilitating nuclear transport of Nur77 and/or RXRα in nontransformed cells. Knockdown of Nur77 with siRNA decreased ANS-induced cleavage of caspase-3 and -7 and their downstream target, PARP, indicating a role for Nur77 in ANS-induced apoptosis. In cells transfected with IGFBP-3, IGFBP-3 associated with RXRα but not Nur77 under basal conditions, however, IGFBP-3 co-precipitated with phosphorylated forms of both proteins in ANS-treated cells. Indirect immunofluorescence and cell fractionation techniques showed that ANS induced phosphorylation and transport of Nur77 from the nucleus to the cytoplasm and these effects were attenuated by knockdown of IGFBP-3. These data suggest that endogenous IGFBP-3 plays a role in intrinsic apoptosis by facilitating phosphorylation and nuclear export of Nur77 to the cytoplasm where it exerts its apoptotic effect. Whether this mechanism involves a physical association between endogenous IGFBP-3 and Nur77 or RXRα remains to be determined.

Genome-wide association and functional studies identify a role for IGFBP3 in hip osteoarthritis

ObjectivesTo identify genetic associations with hip osteoarthritis (HOA), we performed a meta-analysis of genome-wide association studies (GWAS) of HOA.MethodsThe GWAS meta-analysis included approximately 2.5 million imputed HapMap single nucleotide polymorphisms...

Gene Expression Profiling of Early Hepatic Stellate Cell Activation Reveals a Role for Igfbp3 in Cell Migration

BackgroundScarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells. Aim and methodsTo identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. ResultsGene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp) 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. ConclusionIgfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.

OR04-5 Prohibitin-2 is a binding protein for IGFBP-6 on rhabdomyosarcoma cell membranes and modulates IGFBP-6-dependent migration

to examine whether phosphorylation of IGFBP-3 by DNA-PK is necessary for its apoptotic effect. ATM, which phosphorylates similar sites to DNA-PK, was also investigated. Methods: Two triple-negative breast cancer cell lines (Hs578T and MDAMB468) were treated for 1–48 hours with doxorubicin (0–10 mM), etoposide (0–50 mM) or ionising radiation (0–10 Gy), alone or after pretreatment (30–60 min) with inhibitors of ATM (KU55933; 0–30 mM) or DNA-PK (NU7441; 0–10 mM). DSB were monitored by gH2AX formation, DNA repair processes by kinase autophosphorylation and p53 activation, and apoptosis by caspase-3 activity. Endogenous IGFBP-3, ATM and DNA-PK were downregulated using siRNA. Knockdown (≥80%) was confirmed by qRT-PCR and immunoblot. Results: Although IGFBP3 is reported to be p53-inducible, IGFBP-3 protein was downregulated by doxorubicin treatment in MDAMB468 cells and upregulated in Hs578T cells, even though p53 was activated in both cell lines. This may be due to the different p53 mutations in each cell line. DSB were induced within 1 hour after addition of DNA-damaging agents. DNA damage-stimulated gH2AX and p53 phosphorylation was inhibited by KU55933 but increased by NU7441. DNA damage-induced apoptosis was significantly enhanced in both cell lines by kinase inhibition. In IGFBP-3-silenced cells, doxorubicin-induced apoptosis was significantly attenuated (20%), but neither KU55933 nor NU7441 altered the decreased apoptotic response, suggesting that IGFBP-3 does not need to be phosphorylated by ATM or DNA-PK for its apoptotic effect. In DNA-PK-silenced cells, IGFBP-3 knockdown no longer attenuated doxorubicin-induced apoptosis, suggesting that IGFBP-3 cannot carry out its apoptotic functions in the absence of DNA-PK. Conclusion: We conclude that DNA damaging therapy in breast cancer cells is enhanced by DNA repair inhibitors and is most effective in the presence of both DNA-PK and IGFBP-3. This suggests that cancers positive for IGFBP-3 may be most amenable to this therapy. Further work will employ mass spectrometric methods to determine the specific sites on IGFBP-3 that may become phosphorylated during these treatments. This work was supported by the Australian Research Council (DP0984232) (RCB); and a University of Sydney Postgraduate Award (KAM).

IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos

IGFBP3 is a multi-functional protein that has IGF-dependent and IGF-independent actions in cultured cells. Here we show that the IGF binding domain (IBD), nuclear localization signal (NLS) and transactivation domain (TA) are conserved and functional in zebrafish Igfbp3. The in vivo roles of these domains were investigated by expression of Igfbp3 and its mutants in zebrafish embryos. Igfbp3, and its NLS and TA mutants had equally strong dorsalizing effects. Human IGFBP3 had similar dorsalizing effects in zebrafish embryos. The activities of IBD and IBD+NLS mutants were lower, but they still caused dorsalization. Thus, the IGF-independent action of Igfbp3 is not related to NLS or TA in this in vivo model. We next tested the hypothesis that Igfbp3 exerts its IGF-independent action by affecting Bmp signaling. Co-expression of Igfbp3 with Bmp2b abolished Bmp2b-induced gene expression and inhibited its ventralizing activity. Biochemical assays and in vitro experiments revealed that IGFBP3 bound BMP2 and inhibited BMP2-induced Smad signaling in cultured human cells. In vivo expression of Igfbp3 increased chordin expression in zebrafish embryos by alleviating the negative regulation of Bmp2. The elevated level of Chordin acted together with Igfbp3 to inhibit the actions of Bmp2. Knockdown of Igfbp3 enhanced the ventralized phenotype caused by chordin knockdown. These results suggest that Igfbp3 exerts its IGF-independent actions by antagonizing Bmp signaling and that this mechanism is conserved.

Transcriptional profiling of ErbB signalling in mammary luminal epithelial cells - interplay of ErbB and IGF1 signalling through IGFBP3 regulation

BackgroundMembers of the ErbB family of growth factor receptors are intricately linked with epithelial cell biology, development and tumourigenesis; however, the mechanisms involved in their downstream signalling are poorly understood. Indeed, it is unclear how signal specificity is achieved and the relative contribution each receptor has to specific gene expression.MethodsGene expression profiling of a human mammary luminal epithelial cell model of ErbB2-overexpression was carried out using cDNA microarrays with a common RNA reference approach to examine long-term overlapping and differential responses to EGF and heregulin beta1 treatment in the context of ErbB2 overexpression. Altered gene expression was validated using quantitative real time PCR and/or immunoblotting. One gene of interest was targeted for further characterisation, where the effects of siRNA-mediated silencing on IGF1-dependent signalling and cellular phenotype were examined and compared to the effects of loss of ErbB2 expression.Results775 genes were differentially expressed and clustered in terms of their growth factor responsiveness. As well as identifying uncharacterized genes as novel targets of ErbB2-dependent signalling, ErbB2 overexpression augmented the induction of multiple genes involved in proliferation (e.g. MYC, MAP2K1, MAP2K3), autocrine growth factor signalling (VEGF, PDGF) and adhesion/cytoskeletal regulation (ZYX, THBS1, VCL, CNN3, ITGA2, ITGA3, NEDD9, TAGLN), linking them to the hyper-poliferative and altered adhesive phenotype of the ErbB2-overexpressing cells. We also report ErbB2-dependent down-regulation of multiple interferon-stimulated genes that may permit ErbB2-overexpressing cells to resist the anti-proliferative action of interferons. Finally, IGFBP3 was unique in its pattern of regulation and we further investigated a possible role for IGFBP3 down-regulation in ErbB2-dependent transformation through suppressed IGF1 signalling. We show that IGF1-dependent signalling and proliferation were enhanced in ErbB2-overexpressing cells, whilst loss of ErbB2 expression by siRNA silencing reduced IGF1 signalling. Furthermore, IGFBP3 knockdown resulted in basal ERK and Akt activation in luminal epithelial cells and increased invasiveness and anchorage-independent colony formation in SKBR3 cells.ConclusionsThese data show IGFBP3 as a negative regulator of transformation and that its down-regulation enhances IGF1-dependent signalling. They also show that ErbB2 can up-regulate IGF1-dependent signalling, possibly via the regulated expression of IGFBP3.

Insulin-like growth factor-binding protein-3 promotes transforming growth factor-β1-mediated epithelial-to-mesenchymal transition and motility in transformed human esophageal cells

Insulin-like growth factor-binding protein (IGFBP)-3 is overexpressed frequently in esophageal squamous cell carcinoma. Yet, the role of IGFBP3 in esophageal tumor biology remains to be elucidated. We find that IGFBP3 facilitates transforming growth factor (TGF)-beta1-mediated epithelial-to-mesenchymal transition (EMT) in transformed human esophageal epithelial cells, EPC2-hTERT-EGFR-p53(R175H). In organotypic 3D culture, a form of human tissue engineering, laser-capture microdissection revealed concurrent upregulation of TGF-beta target genes, IGFBP3 and EMT-related genes in the cells invading into the stromal compartment. IGFBP3 enhanced TGF-beta1-mediated EMT as well as transcription factors essential in EMT by allowing persistent SMAD2 and SMAD3 phosphorylation. TGF-beta1-mediated EMT and cell invasion were enhanced by ectopically expressed IGFBP3 and suppressed by RNA interference directed against IGFBP3. The IGFBP3 knockdown effect was rescued by IGFBP3(I56G/L80G/L81G), a mutant IGFBP3 lacking an insulin-like growth factor (IGF)-binding capacity. Thus, IGFBP3 can regulate TGF-beta1-mediated EMT and cell invasion in an IGF or insulin-like growth factor 1 receptor-independent manner. IGFBP3(I56G/L80G/L81G) also promoted EMT in vivo in a Ras-transformed human esophageal cell line T-TeRas upon xenograft transplantation in nude mice. In aggregate, IGFBP3 may have a novel IGF-binding independent biological function in regulation of TGF-beta1-mediated EMT and cell invasion.