Knockdown Of Insulin-like Growth Factor 1 Receptor Research Articles

Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria invivo and podocyte cell death invitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes.

Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 291 kinases are regulating DENV infection. Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of EPHB4, ERBB2, FGFR2, or IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

Insulin-like growth factor 1 regulates excitatory synaptic transmission in pyramidal neurons from adult prefrontal cortex

Insulin-like growth factor 1 (IGF1) influences synaptic function in addition to its role in brain development and aging. Although the expression levels of IGF1 and IGF1 receptor (IGF1R) peak during development and decline with age, the adult brain has abundant IGF1 or IGF1R expression. Studies reveal that IGF1 regulates the synaptic transmission in neurons from young animals. However, the action of IGF1 on neurons in the adult brain is still unclear. Here, we used prefrontal cortical (PFC) slices from adult mice (∼8 weeks old) to characterize the role of IGF1 on excitatory synaptic transmission in pyramidal neurons and the underlying molecular mechanisms. We first validated IGF1R expression in pyramidal neurons using translating ribosomal affinity purification assay. Then, using whole-cell patch-clamp recording, we found that IGF1 attenuated the amplitude of evoked excitatory postsynaptic current (EPSC) without affecting the frequency and amplitude of miniature EPSC. Furthermore, this decrease in excitatory neurotransmission was blocked by pharmacological inhibition of IGF1R or conditional knockdown of IGF1R in PFC pyramidal neurons. In addition, we determined that IGF1-induced decrease of EPSC amplitude was due to postsynaptic effect (internalization of a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors [AMPAR]) rather than presynaptic glutamate release. Finally, we found that inhibition of metabotropic glutamate receptor subtype-1 (mGluR1) abolished IGF1-induced attenuation of evoked EPSC amplitude and decrease of AMPAR expression at synaptic membrane, suggesting mGluR1-mediated endocytosis of AMPAR was involved. Taken together, these data provide the first evidence that IGF1 regulates excitatory synaptic transmission in adult PFC via the interaction between IGF1R-dependent signaling pathway and mGluR1-mediated AMPAR endocytosis.

Aggravation of pulmonary fibrosis after knocking down the aryl hydrocarbon receptor in the insulin-like growth factor 1 receptor pathway.

Idiopathic pulmonary fibrosis is a devastating disease with multiple contributing factors. Insulin-like growth factor 1 receptor (IGF1R), with a reciprocal function to aryl hydrocarbon receptor (AhR), is involved in airway inflammation. The exact relationship between IGF1R and AhR in lung fibrogenesis is unclear. This study aimed to investigate the cascade pathway involving IGF1R and AhR in idiopathic pulmonary fibrosis. The AhR and IGF1R expressions were determined in the lungs of idiopathic pulmonary fibrosis patients and in a rodent fibrosis model. Pulmonary fibrosis was evaluated in bleomycin (BLM)-induced lung injury in wild type and AhR knockout (Ahr-/- ) mice. The effects of IGF1R inhibition and AhR activation in vitro on TGF-β1-induced epithelial-mesenchymal transition (EMT) in Beas2B cells and in vivo on BLM-exposed mice were also examined. There were increased IGF1R levels but AhR expression decreased in the lung of idiopathic pulmonary fibrosis patients and BLM-induced mice. Knockout of AhR aggravated lung fibrosis, while the use of IGF1R inhibitor and AhR agonist significantly attenuated such effects and inhibited TGF-β1-induced epithelial-mesenchymal transition in Beas2B cells. Both TGF-β1 and BLM markedly suppressed AhR expression through endoplasmic reticulum stress and consequently, IGF1R activation. The IGF1R inhibitor and specific knockdown of IGF1R reversed the activation of the TGF-β1 signal pathway. In the development of idiopathic pulmonary fibrosis, AhR and IGF1R play opposite roles via the TGF-β/Smad/STAT signalling cascade. The AhR/IGF1R axis is a potential target for the treatment of lung injury and fibrosis.

MiR-448 suppresses cell proliferation and glycolysis of hepatocellular carcinoma through inhibiting IGF-1R expression.

Microribonucleic acids (miRNAs) have been shown to play important roles in hepatocellular carcinoma (HCC) progression. MiR-448 has frequently been shown to be a tumor suppressor, and is abnormally expressed in HCC tumor tissues. However, little is known about the role of miR-448 in HCC development. In this article, the regulatory role of miR-448 on insulin-like growth factor 1 receptor (IGF-1R) in modulating hepatoma cell viability and glycolysis was investigated. The expression of miR-448 profiles in clinical tumor tissues and cell lines was examined using quantitative real-time polymerase chain reaction (qRT-PCR). HepG2 and Huh7 cells were transfected with miR-448 mimics, inhibitors, and scramble sequences. Cell viability and apoptosis were determined by a Cell Counting Kit-8 assay and a flow cytometry analysis. IGF-1R, a potential target of miR-448, was selected following a bioinformatic analysis, and the regulatory effects of miR-448 on IGF-1R expression was confirmed by luciferase reporter assay, qRT-PCR, and western blot. Glucose uptake, lactate production, and adenosine triphosphate (ATP) generation were detected by corresponding kits. Decreased miR-448 expression was observed in both HCC patients' tumor tissues and hepatoma cells in vitro. The overexpression of miR-448 in HepG2 and Huh7 cells decreased cell viability and increased apoptosis. Additionally, the overexpression of miR-448 or the knockdown of IGF-1R lowered the level of glucose uptake, lactate production, and ATP generation, while the knockdown of miR-448 increased glycolysis. Further, aberrantly expressed miR-448 downregulated IGF-1R levels, while the inhibition of miR-448 resulted in the upregulation of IGF-1R in both HepG2 and Huh7 cells. In addition, miR-448 interacted with the wild-type 3'untranslated regions (3'UTRs) of IGF-1R, but had no effect on the mutant 3'UTRs. The expression of IGF-1R was increased in HCC patients' tumor tissues and serum, and was inversely correlated with miR-448 expression. The increased expression of miR-448 appears to downregulate the expression of IGF-1R by interacting with the 3'UTR in HCC progression. These findings highlight its role as a potential target for HCC therapy.

Circular RNA ERBB2 Contributes to Proliferation and Migration of Airway Smooth Muscle Cells via miR-98-5p/IGF1R Signaling in Asthma.

BackgroundAsthma belongs to chronic inflammatory respiratory diseases characterized by airway inflammation and remodeling. Circular RNAs (circRNAs) are promising therapeutic targets for various diseases, including asthma. In this work, we aim to investigate the role of circular RNA Erb-B2 receptor tyrosine kinase 2 (circERBB2) during progression of asthma.MethodsHuman airway smooth muscle cells (ASMCs) were treated with platelet-derived growth factor BB (PDGF-BB) to mimic cell remodeling. The expression of circERBB2, microRNA-98-5p (miR-98-5p), and insulin-like growth factor 1 receptor (IGF1R) was measured by qRT-PCR. Cell proliferation, migration and apoptosis were determined by cell counting-8 (CCK-8), transwell, and flow cytometry. Protein levels of PCNA, MMP-9, IGF1R were evaluated using Western blotting. The levels of tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6 were detected by enzyme‐linked immunosorbent assay (ELISA). Luciferase reporter gene experiment was adopted to evaluate the targeting relationship between miR-98-5p with circERBB2 and IGF1R. Interaction between RNAs was determined by RNA pulldown and RIP assay.ResultsThe depletion of circERBB2 attenuated the proliferation, migration, and levels of inflammatory factors induced by PDGF-BB and cell apoptosis. CircERBB2 was identified to directly interact with miR-98-5p, and overexpression of miR-98-5p abolished the function of circERBB2 on PDGF-BB-stimulated ASMCs. IGF1R was identified as a target of miR-98-5p, and knockdown of IGF1R relieved the PDGF-BB-induced ASMCs proliferation and migration.ConclusionOur work disclosed that knockdown of circERBB2 suppressed PDGF-BB-caused proliferation, migration and inflammatory response of ASMCs, through regulating miR-98-5p/IGF1R signaling, presented circERBB2 as a promising therapeutic target for asthma.

Abstract 3088: Identification of insulin-like growth factor 1 receptor as a novel and specific target of heparin hexasaccharide that selectively inhibits cancer stem cells

Abstract Introduction: Earlier, we discovered that heparin hexasaccharide (HS06), but not shorter or longer polymeric glycosaminoglycan (GAGs), selectively inhibit cancer stem cells (CSCs). Insulin-like growth factor-1 (IGF1) receptor (IGF1R) plays a critical role in regulating CSC phenotype. However, unlike many growth factor receptors, GAG and IGF1R interaction and consequences have not been characterized. Methods: Colon cancer HT29 and HCT-116 cells were grown on ornithine as spheroids. Stable IGF1R knockdown cells (KD) were generated using shRNA transfection and selection. Immunofluorescence (IF) studies were performed with fluorescent-tagged HS06, GAG polymer, and pIGF1R antibody. Images were obtained using a Cytation 5 and analyzed using Gen5. Computational and molecular dynamics studies were employed to determine the binding site and nature of the interaction between HS06 and IGF1R. The impact of this interaction on activation and CSC phenotype was determined using standard techniques such as western blotting and Q-PCR. Results: Fluorescent-tagged HS06 and polymeric GAG, both bound to the colon cancer cell membrane (CM) in a dose-dependent fashion. Intriguingly, HS06 but not polymeric GAG's binding to the cell surface was abolished by IGF1R knockdown, suggesting IGF1R as a specific target HS06. Computational studies identified L1 and CR domain in the N-terminus of IGF1R as a binding site for HS06 but not multimeric GAGs. Intriguingly, there is an overlap between HS06 and IGF1 binding sites on IGF1R. Pretreatment with three different IGF1R N-terminus targeting antibodies reversed HS06 binding to CM, suggesting the N-terminus domain as a likely site of HS06 and IGF1R interactions. Additionally, IGF1 pretreatment also reversed the binding of HS06 in a dose-dependent fashion. Functionally, HS06 (but not polymeric GAG) inhibited IGF1R phosphorylation and CSC phenotype (2° spheroids and/or CSC markers). Both these effects were reversed by pretreatment with IGF1 suggesting competitive inhibition of IGF1 induced activation of IGF1R by HS06. Molecular dynamics studies identified key N-terminus residues in the L1 and CR domain of the N-terminus portion of the IGF1R distinct from IGF1 binding residues. Key basic amino acid residues that were predicted to bind specifically to HS06 in MD simulation have been observed to be mutated in cancer, and IF studies in HT29 cells expressing HS06 binding-site mutant proteins are ongoing. Conclusion: We have uncovered a novel, specific and functionally relevant interaction between HS06 and the N-terminal domain of IGF1R. The HS06-IGF1R interaction induces competitive inhibition of IGF1 induced activation of IGF1R and CSC phenotype. The findings significantly impact an understanding of GAG mediated regulation of the CSC phenotype and present a highly novel mechanism of targeting IGF1R to develop novel anti-cancer agents Citation Format: Rio S. Boothello, Jyothi C. Sistla, Nehru Viji Sankaranarayanan. Identification of insulin-like growth factor 1 receptor as a novel and specific target of heparin hexasaccharide that selectively inhibits cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3088.

SOX4 Mediates BRAF Inhibitor Resistance in Melanoma through Regulation of IGF-1R Signaling: In Vitro Study

Abstract Objective: SOX4, a transcription factor, has been found to contribute to tumorigenesis in several cancers. This study was performed to determine whether SOX4 mediates BRAF inhibitor resistance in melanoma. Methods: Melanoma cell lines with acquired resistance to BRAF inhibitor (SK-MEL-5R, SK-MEL-28R, and A375R) were generated by adding escalating concentrations of PLX4032 into parental SK-MEL-5, SK-MEL-28, and A375 cells for >6 months. The expression of SOX4 and insulin-like growth factor 1 receptor (IGF-1R) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The downstream signaling of IGF-1R was detected by Western blotting. SOX4 and IGF-1R overexpression or knockdown was conducted by lentivirus transfection. Cell viability and apoptosis were demonstrated by MTT and flow cytometry, respectively. The binding ability of SOX4 to IGF-1R promoter was determined by chromatin immunoprecipitation quantitative PCR assay. Results: SOX4 was upregulated in BRAF inhibitor-resistant melanoma cells as compared with parental cells (SK-MEL-5 group, 1.02 vs. 6.33; SK-MEL-28 group, 1.03 vs. 3.22; A375 group, 1.00 vs. 1.86; t =°7.069, 29.26, and 5.291, respectively; all P < 0.01), and PLX4032 treatment could not alter the expression of SOX4 in resistant cells. SOX4 overexpression attenuated the response of parental cells to PLX4032 (for cell viability, SK-MEL-5 group: 77.76% vs. 104.28%, F = 91.50; SK-MEL-28 group: 60.59% vs. 93.13%, F = 171.8; A375 group: 62.50% vs. 80.87%, F = 47.15. For apoptosis rates, SK-MEL-5 group: 34.90% vs. 14.31%, F = 4.781; SK-MEL-28 group, 40.8% vs. 29.4%, F = 13.32, P = 0.063; A375 group: 40.20% vs. 17.09%, F = 11.39; all P < 0.05, otherwise indicated). While SOX4 knockdown enhanced the response of resistant cells to PLX4032 (for cell viability, SK-MEL-5R group: 93.75% vs. 69.53%, F = 94.45, SK-MEL-28R group: 95.60% vs. 66.79%, F = 30.41, A375R group: 95.51% vs. 59.98%, F = 111.6; for apoptosis rates, SK-MEL-5R group: 16.2% vs. 44.4%, F = 25.67, SK-MEL-28R group: 26.59% vs. 44.20%, F = 158.0, A375R group: 5.98% vs. 31.51%, F = 14.35, and all P < 0.01). Chromatin immunoprecipitation quantitative PCR assay demonstrated that SOX4 binded to the promoter of IGF-1R (1.04 vs. 1.94 [−1044 to −920 bp] and 0.110 vs. 0.139 [GAPDH], F = 534.5, P < 0.01). In addition, SOX4 overexpression increased IGF-1R and its downstream phosphorylated ERK, phosphorylated AKT, and phosphorylated STAT3 expression, while SOX4 knockdown exerted the opposite effects. Moreover, IGF-1R knockdown overcame SOX4 overexpression-induced PLX4032 resistance (cell viability: 35.85% vs. 52.79% vs. 37.84% [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; apoptosis rates: 25.30% vs. 9.56% vs. 22.26 [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; F = 13.01 and 41.87, respectively; all P < 0.01), while IGF-1R overexpression abrogated SOX4 knockdown-induced response enhancement to PLX4032 for comparison of negative control group, sh-SOX4 group and sh-SOX4 + IGF-1R overexpressing group (cell viability: 96.62% vs. 86.86% vs. 97.26% (A375R), 98.15% vs. 81.63% vs. 98.49% [SK-MEL-5R]; apoptosis rates: 13.81% vs. 32.00% vs. 12.16 [A375R], 29.70% vs. 41.40% vs. 26.10% [SK-MEL-5R]; F = 13.56, 12.86, 38.81, and 39.85, respectively; all P < 0.01). Conclusion: SOX4 mediates BRAF inhibitor resistance in melanoma through regulation of IGF-1R signaling. SOX4 might serve as a potential target for the treatment of BRAF inhibitor-resistant melanoma.

Long non‑coding RNA BANCR mediates esophageal squamous cell carcinoma progression by regulating theIGF1R/Raf/MEK/ERK pathway via miR‑338‑3p.

Esophageal squamous cell carcinoma (ESCC) is a type of digestive tract malignant tumor that severely threatens human health. The long non-coding RNA BRAF activated non-coding RNA (BANCR) and insulin-like growth factor 1 receptor (IGF1R) are associated with various types of cancer; however, it remains unclear whether BANCR can regulate IGF1R expression in ESCC. In the present study, the expression levels of BANCR, IGF1R mRNA and microRNA-338-3p (miRNA/miR-338-3p) in ESCC tissues or cells were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The levels of IGF1R, E-cadherin, N-cadherin, Vimentin, p-Raf-1, p-MEK1/2 and p-ERK1/2 were measured by western blot analysis. The proliferation, migration and invasion of ESCC cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) or Transwell assays. The relationship between miR-338-3p and BANCR or IGF1R was predicted using starBase2.0 and confirmed by dual-luciferase reporter assay. The role of BANCR in ESCC in vivo was confirmed through a tumor xenograft assay. It was found that BANCR and IGF1R were upregulated, while miR-338-3p was down-regulated in ESCC tissues and cells. Both BANCR and IGF1R knockdown suppressed the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells. IGF1R enhancement reversed BANCR knockdown-mediated effects on the proliferation, migration, invasion and EMT of ESCC cells. BANCR regulated the Raf/MEK/ERK pathway by regulating IGF1R expression. Notably, BANCR regulated IGF1R expression by sponging miR-338-3p. Moreover, BANCR silencing inhibited tumor growth in vivo. On the whole, the findings of the present study demonstrate that BANCR inhibition blocks ESCC progression by inactivating the IGF1R/Raf/MEK/ERK pathway by sponging miR-338-3p.

Tetradecyl 2,3-dihydroxybenzoate alleviates oligodendrocyte damage following chronic cerebral hypoperfusion through IGF-1 receptor

Currently, there is no effective therapy for chronic cerebral hypoperfusion-induced subcortical ischemic vascular dementia (SIVD), which displays cognitive deficits and progressive white matter damage. Tetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead compound derived from gentisides with neuritogenic activity. In this report, we intended to investigate the effect of ABG-001 on the SIVD experimental model through right unilateral common carotid arteries occlusion (rUCCAO) in mice. We found that ABG-001 remarkably alleviated white matter damage and cognitive deficits after cerebral hypoperfusion induced by rUCCAO. The protection of ABG-001 on the white matter was related to an amelioration of the oligodendrocyte apoptosis and demyelination rather than promoting remyelination. Molecular docking study showed that ABG-001 possesses a high affinity for insulin-like growth factor-1 receptor (IGF-1R), but not for tropomyosin receptor kinase A (TrkA). The protection of ABG-001 against oligodendrocyte damage was abrogated by IGF-1R antagonist or knockdown of IGF-1R through shRNA, but not TrkA antagonist. Moreover, ABG-001 did not induce hematological, renal or hepatic toxicity after chronic treatment. The present study indicates that ABG-001 protects oligodendrocytes through IGF-1R to relieve demyelination following chronic cerebral hypoperfusion, which could be represented as an encouraging treatment for SIVD.

Association of IGF1R polymorphisms (rs1546713) with susceptibility to age-related cataract in a Han Chinese population.

To explore the susceptible association between the insulin-like growth factor-1 receptor (IGF1R) single nucleotide polymorphism (SNP) and age-related cataract (ARC), and investigate the underlying mechanisms in human lens epithelium (HLE) cells. Totally 1190 unrelated participants, comprising 690 ARC patients and 500 healthy individuals in Han Chinese population were recruited and genotyped for target SNP. The χ 2-test was used to detect genotypic distribution between the patient and control groups and the logistic regression was performed to adjust the age and gender. Meanwhile, different biological experimental methods, such as cell counting kit 8 (CCK-8) assay, flow cytometry, quantitative real time polymerase chain reaction (Q-PCR) and Western blot, were used to detect cell viability, cell cycle progression and apoptosis in HLE cells or IGF1R knockdown HLE cells. The rs1546713 in IGF1R gene was identified (P=0.046, OR: 1.606, 95%CI: 1.245-2.071), which shown a significant relevance with ARC risk under the dominant model. The results demonstrated that IGF1R knockdown inhibited cell proliferation by inducing cell cycle arrested at S phase and promoting apoptosis. Mechanistically, the cell cycle blocked at S phase was linked with the alterations of cyclin A, cyclin B, cyclin E and P21. The pro-apoptosis function of IGF1R may related with stimulating the activation of Caspase-3 and altering the expression levels of apoptotic proteins, including Bcl-2, Bax and Caspase-3. This study first report that IGF1R polymorphisms may affect susceptibility to ARCs in Han Chinese population and provide new clues to understand the pathogenic mechanism of ARCs. Notably, IGF1R is likely a potential target for ARC prevention and treatment.

MiR-4458 directly targets IGF1R to inhibit cell proliferation and promote apoptosis in hemangioma.

Hemangiomas (HAs) are benign neoplasms of the vasculature. MicroRNA-4458 (miR-4458) has been reported to function as a tumor suppressor in multiple malignancies, but its biological function in HAs remains unknown. In the present study, the potential role of miR-4458 in HA-derived endothelial cells (HDECs) was investigated. Firstly, reverse-transcription-quantitative PCR analysis was used to confirm the expression of miR-4458 in HDECs following transfection with miR-4458 mimics or inhibitor. Subsequently, MTT and EdU assays were performed and subsequently determined that miR-4458 overexpression significantly inhibited proliferation, and knockdown promoted cell proliferation in HDECs. Flow cytometry analysis revealed that miR-4458 overexpression induced cell cycle arrest, whereas knockdown reversed G0/G1 phase arrest and apoptosis. Furthermore, insulin-like growth factor 1 receptor (IGF1R) was identified as a target of miR-4458. IGF1R knockdown enhanced the effects of miR-4458 on cell proliferation, cell cycle G0/G1 phase arrest and apoptosis in HDECs. Taken together, the results revealed that miR-4458 targeting of IGF1R may serve as a novel therapeutic strategy for treating patients with HAs.

Targeting Insulin-Like Growth Factor 1 Receptor Delays M-Phase Progression and Synergizes with Aurora B Inhibition to Suppress Cell Proliferation.

The insulin-like growth factor 1 receptor (IGF1R) is a receptor-type tyrosine kinase that transduces signals related to cell proliferation, differentiation, and survival. IGF1R expression is often misregulated in tumor cells, but the relevance of this for cancer progression remains unclear. Here, we examined the impact of IGF1R inhibition on cell division. We found that siRNA-mediated knockdown of IGF1R from HeLa S3 cells leads to M-phase delays. Although IGF1R depletion causes partial exclusion of FoxM1 from the nucleus, quantitative real-time PCR revealed that the transcription of M-phase regulators is not affected by decreased levels of IGF1R. Moreover, a similar delay in M phase was observed following 2 h of incubation with the IGF1R inhibitors OSI-906 and NVP-ADW742. These results suggest that the M-phase delay observed in IGF1R-compromised cells is not caused by altered expression of mitotic regulators. Live-cell imaging revealed that both prolonged prometaphase and prolonged metaphase underlie the delay and this can be abrogated by the inhibition of Mps1 with AZ3146, suggesting activation of the Spindle Assembly Checkpoint when IGF1R is inhibited. Furthermore, incubation with the Aurora B inhibitor ZM447439 potentiated the IGF1R inhibitor-induced suppression of cell proliferation, opening up new possibilities for more effective cancer chemotherapy.

Activation of insulin-like growth factor-1 receptor confers acquired resistance to osimertinib in non-small cell lung cancer with EGFR T790M mutation.

BackgroundOsimertinib (AZD9291) is a third‐generation EGFR‐tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR‐mutant non‐small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable.MethodsWe established osimertinib‐resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR‐mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole‐exome sequencing and multiple phospho‐receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR‐mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis.ResultsWhole‐exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho‐RTK array revealed that insulin‐like growth factor‐1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR‐mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment.ConclusionsIGF1R activation could occur following treatment with osimertinib in EGFR‐mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation.Key points Significant findings of the study: Using osimertinib‐resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR‐mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR‐mutant NSCLC patient with acquired osimertinib resistance. What this study adds: IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

MicroRNA-939-5p directly targets IGF-1R to inhibit the aggressive phenotypes of osteosarcoma through deactivating the PI3K/Akt pathway.

The dysregulation of microRNA-939-5p (miR-939) is involved in the development of multiple types of human cancer. However, the expression and roles of miR-939 in osteosarcoma (OS) have yet to be clarified. The expression level of miR-939 in OS was measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR). A Cell Counting Kit-8 assay, flow cytometry analysis, Transwell migration and invasion assays, and a tumor xenograft assay were employed to explore the effects of miR-939 in OS cells. Bioinformatics analysis, RT-qPCR, western blot analysis and luciferase reporter assays were performed to explore its underlying mechanism. Expression of miR-939 was decreased in both OS tissues and cell lines. The decreased miR-939 expression was notably correlated with clinical stage and distant metastasis in patients with OS, where low miR-939 levels were correlated with lower overall survival. miR-939 overexpression decreased OS cell proliferation, migration and invasion in vitro; induced cell apoptosis, and impaired tumor growth in vivo. Mechanistically, insulin-like growth factor 1 receptor (IGF-1R) was characterized as direct target gene of miR-939 in OS. The tumor-suppressing effects of miR-939 in OS cells were imitated by IGF-1R knockdown. In addition, exogenous IGF-1R expression abolished the tumor suppressive roles of miR-939 in OS cells. miR-939 was implicated in the deactivation of the PI3K/Akt pathway in OS in vitro and in vivo through regulating IGF-1R expression. The present study demonstrated that miR-939 exerted tumor-suppressing roles in the malignancy of OS cells by directly targeting IGF-1R and inactivating the PI3K/AKT pathway. Therefore, this miRNA may be a promising target for anticancer therapy in patients with OS.

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.

<p>IGF1R facilitates epithelial-mesenchymal transition and cancer stem cell properties in neuroblastoma via the STAT3/AKT axis</p>

BackgroundNeuroblastoma (NB) displays the most heterogeneity in clinical manifestation. The insulin-like growth factor 1 receptor (IGF1R) has long been recognized for its role in tumourigenesis and growth. The IGF/IGF1R pathway is important in maintaining cell survival. It is reported that IGF1R participates in the occurrence of NB, but the mechanism is still unclear.MethodsHuman NB cell lines IMR-32 and SH-SY5Y were recruited in this study. IGF1R was knocked down by transfection with short hairpin RNA. Signal transducer and activator of transcription 3 (STAT3) expression was inhibited by Cryptotanshinone treatment. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The cancer stem cell properties were characterized by tumour sphere formation assay and colony formation assay. The mRNA and protein expression levels of related proteins were detected by RT-PCR and Western blot, respectively.ResultsThe knockdown of IGF1R inhibits NB cell tumourigenesis and the epithelial-mesenchymal transition (EMT) of NB cells. Additionally, IGF1R was found to stimulate cancer stem cell-like properties in NPC cells. The knockdown of IGF1R significantly reduced the phosphorylation of AKT, and STAT3, indicating that the activation of the AKT and STAT3 pathways was inhibited by IGF1R knockdown. Furthermore, IGF1R was demonstrated to stimulate cancer stem cell-like properties in NB cells via the regulation of the STAT3/AKT axis.ConclusionIGF1R promotes cancer stem cell properties to facilitate EMT in neuroblastoma via the STAT3/AKT axis.

MiRNA‑30a‑3p inhibits metastasis and enhances radiosensitivity in esophageal carcinoma by targeting insulin‑like growth factor 1 receptor.

It has been demonstrated that microRNAs (miRNAs) serve important roles in various biological processes, such as tumorigenesis. In the present study, the role of miR-30a-3p in the pathogenesis of esophageal carcinoma (EC) was investigated. Reverse transcription-quantitative polymerase chain reaction was performed to determine the levels of miR-30a-3p expression in EC tissues and cell lines. Then, the effects of miR-30a-3p on the migration, invasion and radiosensitivity of EC cells were investigated using scratch-wound, Transwell and radiosensitivity assays, respectively. A dual-luciferase reporter assay was performed to determine potential interactions between miR-30a-3p and the 3′-untranslated region (3′-UTR) of insulin-like growth factor 1 receptor (IGF-1R). The results demonstrated that the levels of miR-30a-3p expression in EC tissues and cell lines were significantly decreased compared with those in paired healthy tissues and a human esophageal epithelial cell line. Upregulation of miR-30a-3p expression significantly suppressed migration, invasion and epithelial-mesenchymal transition (EMT), and enhanced radiosensitivity in EC cells. Analysis of luciferase activity demonstrated that miR-30a-3p interacted with the 3′-UTR of IGF-1R, and knockdown of IGF-1R induced similar effects on the migration, invasion, EMT and radiosensitivity of EC cells. The results indicated that miR-30a-3p suppressed metastasis and enhanced the radiosensitivity of EC cells via downregulation IGF-1R, suggesting that miR-30a-3p may be a potential therapeutic target in the treatment of EC.

Heterozygous knockdown insulin‐like growth factor‐1 receptor (IGF‐1R) regulates mitochondrial functions and prevents colitis and colorectal cancer

Although insulin‐like growth factor‐1 receptor (IGF‐1R) has been accepted as a major determinant of tumorigenesis, the mechanisms of its roles in the pathogenesis of cancers have remained elusive. Herein, we demonstrate that IGF‐1R plays pivotal roles in regulation of mitochondrial functions and resistance to oxidative stress in colitis and colorectal tumorigenesis. Heterozygous knockdown IGF‐1R attenuated azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colitis and colitis associated cancer (CAC) in Igf1r+/− mice. Knockdown of IGF‐1R confers the resistance to oxidative stress‐induced damage on colorectal epithelium through protection the mitochondrial dynamics and structures. Knockdown of IGF‐1R develops the function of mitochondrial fusion under oxidative stress. Mechanically, an increase of mitochondrial coupling index RCI and oxidative phosphorylation index ADP/O was seen in the epithelial cells of Igf1r+/− mice. Seahorse XFe‐24 analyzer analysis of mitochondrial bioenergetics showed an increase of oxygen consumption rate (OCR) and a decrease of extracellular acidification rate (ECAR) in Igf1r+/− cells. Further analysis suggests the protection mechanisms of colorectal epithelial cells from oxidative stress through activating mitochondrial respiratory chain and the LKB1/AMPK pathways. These results support the roles of IGF‐1R at the nexus between oxidative damage and mitochondrial function and connect colitis and colorectal tumorigenesis.Support or Funding InformationThis work was supported by Natural Science Foundation of China (81673449, 91629303).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Knockdown of IGF-1R Triggers Viral RNA Sensor MDA5- and RIG-I-Mediated Mitochondrial Apoptosis in Colonic Cancer Cells

The important role of insulin-like growth factor-1 receptor (IGF-1R) in tumorigenesis has been well established. The classical model involves IGF-1R binding to IGF-1/2, the following activation of PI3K-Akt-signaling cascades, driving cell proliferation and apoptosis inhibition. Here we report a new signal transduction pathway of IGF-1R in the intestinal epithelium. Using heterozygous knockout mice (Igf1r+/−), we analyzed the expressions of viral RNA sensors MDA5 and RIG-I in the intestinal epithelium. Igf1r+/− mice exhibited higher MDA5 and RIG-I than wild-type (WT) mice, indicating that knockdown of IGF-1R could trigger MDA5 and RIG-I. IGF-1R knockdown-triggered MDA5 and RIG-I were further investigated in human colonic cancer cells. Increased MDA5 and RIG-I were clearly seen in the cytoplasm in cancer cells as well as normal human colonic cells with silenced IGF-1R. Notably, the upregulations of MDA5 and RIG-I was not affected by blockage of the PI3K-Akt pathway with LY294002. These results suggested a new signal transduction pathway of IGF-1R. Importantly, IGF-1R knockdown-triggered MDA5 and RIG-I resulted in colorectal cancer apoptosis through activation of the mitochondrial pathway. These in vitro observations were evidenced in the azoxymethane (AOM)-dextran sulfate sodium (DSS) colorectal cancer model of mice. In conclusion, knockdown of IGF-1R triggers viral RNA sensor MDA5- and RIG-I-mediated mitochondrial apoptosis in cancer cells.