MicroRNA let-7i induced autophagy to protect T cell from apoptosis by targeting IGF1R

MicroRNA let-7i is up-regulated in T cells from patients with Ankylosing Spondylitis (AS). In this study, we investigated the role of let-7i in T cells survival. Our results demonstrated down-regulation of insulin-like growth factor-1 receptor (IGF1R) in T cells from patients with AS. Luciferase reporter assay suggested IGF1R as direct target of let-7i. Overexpression of let-7i in Jurkat cells significantly suppressed IGF1R expression, which mimicked the action of IGF1R siRNA. IGF1R inhibition led to a strinking decrease in phosphorylation of mTOR and Akt, down-regulation of Bcl-2, up-regulation of Bax and cleavage of caspase 3 and PARP. Meanwhile, IGF1R inhibition induced autophagy. Autophagy induced by let-7i overexpression contributed to protect cells from apoptosis. Our data indicated that let-7i might control T cells fates in AS by targeting IGF1R.     

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.     

An Integrin Binding-defective Mutant of Insulin-like Growth Factor-1 (R36E/R37E IGF1) Acts as a Dominant-negative Antagonist of the IGF1 Receptor (IGF1R) and Suppresses Tumorigenesis but Still Binds to IGF1R

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvβ3 and α6β4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.     

Impaired IGF1R signaling in cells expressing longevity-associated human IGF1R alleles

Dampening of insulin/insulin-like growth factor-1 (IGF1) signaling results in the extension of lifespan in invertebrate as well as murine models. The impact of this evolutionarily conserved pathway on the modulation of human lifespan remains unclear. We previously identified two IGF1R mutations (Ala-37-Thr and Arg-407-His) that are enriched in Ashkenazi Jewish centenarians as compared to younger controls and are associated with the reduced activity of the IGF1 receptor as measured in immortalized lymphocytes. To determine whether these human longevity-associated IGF1R mutations affect IGF1 signaling, we engineered mouse embryonic fibroblasts (MEFs) expressing the different human IGF1R variants in a mouse Igf1r null background. The results indicate that MEFs expressing the human longevity-associated IGF1R mutations attenuated IGF1 signaling, as demonstrated by significant reduction in phosphorylation of both IGF1R and AKT after IGF1 treatment, in comparison with MEFs expressing the wild-type IGF1R. The impaired IGF1 signaling caused by the IGF1R mutations resulted in the reduced induction of the major IGF1-activated genes in MEFs, including EGR1, mCSF, IL3Rα, and TDAG51. Furthermore, the IGF1R mutations caused a delay in cell cycle progression after IGF1 treatment, indicating a dysfunctional physiological response to a cell proliferation signal. These results demonstrate that the human longevity-associated IGF1R variants are reduced-function mutations, implying that dampening of IGF1 signaling may be a longevity mechanism in humans.     

Development of molecular agents for IGF receptor targeting.

The type 1 insulin-like growth factor receptor (IGF1R) is a promising anticancer treatment target, being frequently overexpressed by tumours, and mediating proliferation, motility and apoptosis protection. Design of specific kinase inhibitors is problematic because of homology between the IGF1R and insulin receptor. This obstacle can be circumvented using sequence-specific molecular agents including antisense, triplex and ribozymes. Recent studies indicate that profound sequence-specific IGF1R gene silencing can be induced by small interfering RNAs that mediate RNA interference in mammalian cells. IGF1R downregulation blocks tumour growth and metastasis, and enhances sensitivity to cytotoxic drugs and irradiation. In murine melanoma cells, radiosensitisation is associated with impaired activation of Atm, which is required for initiation of cell cycle checkpoints and DNA repair pathways after double-strand DNA breaks. Furthermore, tumour cells killed in vivo following IGF1R downregulation can provoke an immune response, protecting against tumour rechallenge. After years of studying the role of the IGF system in tumour biology, novel agents for IGF1R targeting will soon be available for clinical testing. This review summarises the development of molecular agents, and considers factors that will influence clinical activity, including the requirement of established tumours for IGF signalling, and the efficacy and toxicity of IGF1R inhibitors.     

Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis

RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.     

Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs

Antisense DNA target sites can be selected by the accessibility of the mRNA target. It remains unknown whether a mRNA site that is accessible to an antisense DNA is also a good candidate target site for a siRNA. Here, we reported a parallel analysis of 12 pairs of antisense DNAs and siRNA duplexes for their potency to inhibit reporter luciferase activity in mammalian cells, both of the antisense DNA and siRNA agents in a pair being directed to same site in the mRNA. Five siRNAs and two antisense DNAs turned out to be effective, but the sites targeted by those effective siRNAs and antisense DNAs did not overlap. Our results indicated that effective antisense DNAs and siRNAs have different preferences for target sites in the mRNA.     

Repression of malignant tumor progression upon pharmacologic IGF1R blockade in a mouse model of insulinoma

NVP-AEW541, a specific ATP-competitive inhibitor of the insulin-like growth factor-1 receptor (IGF1R) tyrosine kinase, has been reported to interfere with tumor growth in various tumor transplantation models. We have assessed the efficacy of NVP-AEW541 in repressing tumor growth and tumor progression in the Rip1Tag2 transgenic mouse model of pancreatic β-cell carcinogenesis. In addition, we have tested NVP-AEW541 in Rip1Tag2;RipIGF1R double-transgenic mice which show accelerated tumor growth and increased tumor malignancy compared with Rip1Tag2 single-transgenic mice. Previously, we have shown that high levels of IGF-2, a high-affinity ligand for IGF1R, are required for Rip1Tag2 tumor cell survival and tumor growth. Unexpectedly, treatment of Rip1Tag2 mice with NVP-AEW541 in prevention and intervention trials neither did affect tumor growth nor tumor cell proliferation and apoptosis. Yet, it significantly repressed progression to tumor malignancy, that is, the rate of the transition from differentiated adenoma to invasive carcinoma. Treatment of Rip1Tag2;RipIGF1R double-transgenic mice resulted in moderately reduced tumor volumes and increased rates of tumor cell apoptosis. Sustained expression of IGF-2 and of the IGF-2-binding form of insulin receptor (IR-A) in tumor cells suggests a compensatory role of IR-A upon IGF1R blockade. The results indicate that inhibition of IGF1R alone is not sufficient to efficiently block insulinoma growth and imply an overlapping role of IGF1R and insulin receptor in executing mitogenic and survival stimuli elicited by IGF-2. The reduction of tumor invasion upon IGF1R blockade on the other hand indicates a critical function of IGF1R signaling for the acquisition of a malignant phenotype.     

Cryo-EM Structure Reveals Polymorphic Ligand-bound States of IGF1R

Type 1 insulin-like growth factor receptor (IGF1R) plays an important role in regulating cellular metabolism and cell growth and has been identified as an anticancer drug target. Although previous studies have revealed some structures of IGF1R with different ligands, the continuous dynamic conformation change remains unclear. Here, we report 10 distinct structures (7.9–3.6 Å) of IGF1R bound to IGF1 or insulin to reveal the polymorphic conformations of ligand-bound IGF1R. These results showed that the α-CT², disulfide bond (C670-C670′), and FnIII-2 domains had the most flexible orientations for the conformational change that occurs when ligands bind to the receptor. In addition, we found one special conformation (tentatively named the diverter-switch state) in both complexes, which may be one of the apo-IGF1R forms under ligand-treatment conditions. Hence, these results illustrated the mechanism of how different ligands could bind to human IGF1R and provided a rational template for drug design.     

MiR-122 Inhibits Cell Proliferation and Tumorigenesis of Breast Cancer by Targeting IGF1R

miRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, microRNA-122 (miR-122) has been proved to play an important role in hepatocellular carcinoma, but its functions in the context of breast cancer (BC) remain unknown. In this study, we report that miR-122 is commonly downregulated in BC specimens and BC cell lines with important functional consequences. Overexpression of miR-122 not only dramatically suppressed cell proliferation, colony formation by inducing G1-phase cell-cycle arrest in vitro, but also reduced tumorigenicity in vivo. We then screened and identified a novel miR-122 target, insulin-like growth factor 1 receptor (IGF1R), and it was further confirmed by luciferase assay. Overexpression of miR-122 would specifically and markedly reduce its expression. Similar to the restoring miR-122 expression, IGF1R downregulation suppressed cell growth and cell-cycle progression, whereas IGF1R overexpression rescued the suppressive effect of miR-122. To identify the mechanisms, we investigated the Akt/mTOR/p70S6K pathway and found that the expression of Akt, mTOR and p70S6K were suppressed, whereas re-expression of IGF1R which did not contain the 3′UTR totally reversed the inhibition of Akt/mTOR/p70S6K signal pathway profile. We also identified a novel, putative miR-122 target gene, PI3CG, a member of PI3K family, which further suggests miR-122 may be a key regulator of the PI3K/Akt pathway. In clinical specimens, IGF1R was widely overexpressed and its mRNA levels were inversely correlated with miR-122 expression. Taken together, our results demonstrate that miR-122 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting IGF1R and regulating PI3K/Akt/mTOR/p70S6K pathway. Given these, miR-122 may serve as a novel therapeutic or diagnostic/prognostic-target for treating BC.     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

Background

Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling.

Methodology/Principal Findings

A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists.

Conclusions/Significance

The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.     

Insulin-like Growth Factor (IGF) I Down-Regulates Type 1 IGF Receptor (IGF 1R) and Reduces the IGF I Response in A549 Non-Small-Cell Lung Cancer and Saos-2/B-10 Osteoblastic Osteosarcoma Cells

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.     

siRNA作用效果的靶点依赖性

小分子双链RNA(siRNA)可以高效、特异地沉默目的基因表达 ,为基因功能研究及基因治疗提供了新工具。近年来研究表明针对基因mRNA不同位点随机设计的siRNA在作用效果上存在差异 ,siRNA作用效果有序列依赖性 ,而且与其在基因mRNA上的结合部位的高级结构有关 ,与反义核酸发挥作用的靶点依赖性类似。这一性质对设计高效siRNA为基因功能和基因治疗研究提供指导作用。     

应用RNAi技术沉默大转录本基因LRP1B

低密度脂蛋白受体相关蛋白1B(Lipoprotein receptor-related protein 1 B, LRP1B)是脂蛋白受体家族庞大受体亚群的成员之一, LRP1B基因编码一个长达16.5 kb的大转录本。由于LRP1B的转录产物过大限制了其功能研究。为研究LRP1B基因与肿瘤转移的相关性, 文章应用RNAi技术特异地封闭了LRP1B基因的表达。在设计siRNA过程中, 根据高效siRNA序列特征和靶序列处的mRNA二级结构特点, 对LRP1B基因多达1 100个的siRNA候选靶序列进行双重评价, 最终在基因转录本的不同位置选取了6个siRNA靶位点。针对这些靶位点, 文章构建了6个表达shRNA的pSilencer4.1重组体, 稳定转染HEK 293细胞并采用半定量RT-PCR检测各表达载体的沉默效果。结果表明, 所构建的6个shRNA-pSilencer4.1重组体中有5个对LRP1B基因形成了有效沉默(＞50%), 并得到了多个完全封闭LRP1B基因表达的HEK 293细胞单克隆。