IGF-1R Signaling Pathway Research Articles

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Abstract PO5-09-02: Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density

Abstract Introduction: Breast cancer incidence is rising in premenopausal, hence, there is a critical need to understand factors underlying premenopausal breast cancer development in order to guide targeted prevention. Mammographic breast density is a strong risk factor for, as well as an intermediate phenotype for premenopausal breast cancer. Yet, the molecular mechanisms underlying the associations of dense breasts with breast cancer are not well understood. Our objectives in this study are to perform proteomic analysis in breast tissues to (i) identify proteins that are associated with breast cancer development in premenopausal women; (ii) determine which of these proteins are also associated with dense breasts. Methods: We performed proteomic analysis on tumor and adjacent normal tissues from 50 premenopausal women with breast cancer who had breast tissue samples archived at the St. Louis Breast Tumor Registry. Samples were analyzed on Orbitrap Eclipse Tribrid coupled with Vanquish Neo LC system (Thermo Fisher Scientific). Database search was performed using MaxQuant and MS1 LFQ intensities were used for further data analysis. We performed multiple imputation using Bayesian Principal Component Analysis and hierarchical clustering analysis using ‘ConsensusClusterPlus’ package in R. Pathway enrichment analysis was performed using Reactome Pathway database. 10 times repeated 5-fold cross validation using Logistic Regression (LR) was performed for model development. Top 10 proteins were selected by variable importance in logistic regression, and recursive feature elimination was performed for feature selection. We corrected for multiple testing by setting the false discovery rate, FDR, p-value < 0.05. Results: After imputing for missing values, we identified 3,571 proteins that were expressed in tumor and normal breast tissues. We selected 1,640 proteins for further analysis using median absolute deviation analysis. In multivariable-adjusted analysis, 451 proteins were up-regulated, and 180 proteins were down-regulated in tumor tissues compared to adjacent normal tissues (FDR < 0.05). 53 of the 451 proteins that were upregulated in tumor tissues were also upregulated in dense breasts. Some of the top proteins that were upregulated in both tumor tissues and dense breasts were AIMP2, ALDH18A1, BZW1, CKAP5, COPG1, ERGIC1, GSPT1, ILF2, IPO7, LRBA, and PSMD12. Mammographic breast density protein clusters were highly correlated with hormone receptor positivity clusters and were enriched in ESR-mediated signaling, IGF1R signaling, NOTCH4, PTEN regulation, and NFkB pathway. Discussion: Using untargeted proteomics, we identified proteomic signatures of breast cancer development in premenopausal women. We further identified protein biomarkers and pathways that are shared between breast tumors and dense breast tissues in premenopausal women. Our novel findings highlight mammographic breast density as a strong intermediate phenotype that can be targeted in breast cancer prevention in premenopausal women. Larger studies are needed to validate our findings. Citation Format: Shaili Tapiavala, Minsoo Son, Graham Colditz, Ah Young Goo, Adetunji Toriola. Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-02.

In vivo CRISPR screen identifies LTN1 as a novel tumor suppressor ubiquitinating insulin-like growth factor 2 mRNA-binding protein 1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a frequent and aggressive kind of cancer. Although E3 ligases play important roles in HCC development, several E3 ligases remain unknown. Through in vivo CRISPR knockout (KO) screens targeting related E3 ligase genes in HCC nude mice models, we discovered LTN1 as a novel tumor suppressor in HCC. Co-IP paired with 2D-LC-MS/MS and subsequent western blotting in HCC cells were used to identify the interactome of LTN1. Compared to matched normal tissues, the expression of LTN1 was decreased in human HCC tissues (ANT) (157/209). Clinically, patients with HCC who expressed low levels of LTN1 had a poor prognosis. Forced expression of LTN1 decreased cell growth in vitro and in vivo, whereas knockdown of LTN1 increased cell growth. Mechanistically, elevated LTN1 expression inhibited HCC cell growth by ubiquitinating and destabilizing the IGF2BP1 protein, which inhibited the c-Myc and IGF-1R signaling pathways. There was a negative correlation between the LTN1 protein expression and the IGF2BP1 protein expression in HCC tissues (R2=0.2799, P=0.0165). LTN1 may be a crucial tumor suppressor for determining the prognosis and a possible therapeutic target since it inhibits the proliferation of HCC cells by ubiquitinating IGF2BP1.

CircDiaph3 influences PASMC apoptosis by regulating PI3K/AKT/mTOR pathway through IGF1R

The pathogenesis of pulmonary hypertension has not been elucidated. We investigated the role of a circular ribonucleic acid, circDiaph3, in the proliferation and migration of pulmonary artery smooth muscle cells during pulmonary hypertension. CircDiaph3 overexpression in blood samples of patients with pulmonary hypertension was analyzed by real-time quantitative polymerase chain reaction. Subsequently, a rat model of pulmonary arterial hypertension was established under hypoxic conditions. Pulmonary artery smooth muscle cells were harvested from the rat model for subsequent experiments with small interfering ribonucleic acid-mediated knockdown of circDiaph3. In cell model, we found that PI3K, AKT, mTOR and insulin-like growth factor 1 signaling pathway (IGF1R) and smooth muscle cell marker genes (α-SMA, Vcam1) were significantly downregulated. The overexpression of Igf1r in pulmonary artery smooth muscle cells rescued the downregulated smooth muscle cell genes, IGF1R signaling pathway proteins, increased smooth muscle cell proliferation, and reduced apoptosis. CircDiaph3 regulates the PI3K/AKT/mTOR signaling pathway via IGF1R to inhibit apoptosis and promote proliferation of smooth muscle cells. Additionally, adenovirus-mediated in vivo inhibition of circDiaph3 was carried out in rats with pulmonary arterial hypertension, followed by harvesting of their pulmonary artery smooth muscle cells for subsequent experiments. Excessive proliferation of smooth muscle cells in the pulmonary artery has narrowed the pulmonary artery lumen, thereby causing pulmonary hypertension, and our results suggest that circDiaph3 has important value in the treatment of pulmonary hypertension.

Abstract 2564: Targeting IGF-1/STAT3 signaling crosstalk to inhibit prostate cancer growth and metastasis

Abstract Introduction: Prostate cancer is the most prevalent cancer in American men with unknown etiology. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor, which has been implicated in the growth and metastasis of prostate cancer. Activation of STAT3 through phosphorylation modulates the expression of miR-21, an oncomir, by directly binding to their promoter region in response to various cytokines and growth factors. One such growth factor is insulin-like growth factor (IGF)-1, whose elevated levels are linked to the growth of prostate cancer. IGF-1 exerts its mitogenic effect by binding to its receptor, IGF-1R, whose expression on prostate cancer cells is also increased during neoplastic transformation. There is limited information concerning the crosstalk between IGF-1/IGF-1R and STAT3 signaling pathways which could explain their oncogenic roles in prostate cancer. In this study, we examined whether STAT3-mediated increase of miR-21 is critical for the proliferative effects of IGF-1. We further show that resveratrol, a polyphenolic antioxidant, attenuates prostate cancer growth and invasiveness through regulation of IGF-1/STAT3 pathway. Methods: In vitro studies were performed on DU145 cells, which are androgen receptor negative human prostate cancer cells. Cell viability was assessed by MTS assays, while invasion and migration of cells were tested by Boyden chamber assay and wound healing assay, respectively. Modulation of protein expression was determined by Western blotting. MiR-21 levels were determined by TaqMan assay. SiRNA transfections were performed to suppress the expression of specific proteins in order to elucidate the molecular mechanisms underlying the actions of IGF-1, STAT3 and resveratrol. Results: Acute IGF-1 treatment of DU145 cells increased phosphorylation of Akt which was correlated with the activation of STAT3. This effect of IGF-1 was through activation of IGF-1R, as siRNA-mediated knockdown of IGF-1R attenuated STAT3 activation and Akt phosphorylation. IGF-1 treatment also increased miR-21 levels and decreased the expression of its downstream protein, programmed cell death 4 (PDCD4). Effects of IGF-1 were completely attenuated by resveratrol. Furthermore, knockdown of IGF-1R and STAT3 mimicked the actions of resveratrol on DU145 cells suggesting that a signaling crosstalk exists between IGF-1 and STAT3 pathways which can be targeted to inhibit prostate cancer. Conclusion: This study demonstrates, for the first time, that IGF-1 treatment significantly activates STAT3 pathway and increases miR-21 expression in prostate cancer cells. Targeted inhibition of IGF-1-induced activation of STAT3 by resveratrol holds the promise for the development of novel therapeutic agents against prostate cancer. Citation Format: Sandeep Sheth, Raheem Al-Aameri, Vickram Ramkumar. Targeting IGF-1/STAT3 signaling crosstalk to inhibit prostate cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2564.

Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma

Abstract Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye. Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2557.

DAB2 promotes pulmonary fibrosis and may act as an intermediate between IGF‑1R and PI3K/AKT signaling pathways.

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous lung disease associated with high mortality. Disabled-2 (DAB2), an adapter protein, regulates cell-fibrinogen adhesion and fibrinogen uptake. DAB2 is differentially expressed in mouse fibrotic lungs induced by bleomycin according to a genome microarray analysis based on Gene Expression Omnibus database. However, the role of DAB2 in IPF has not been revealed. A bleomycin-induced mouse model of pulmonary fibrosis was constructed in the present study. It found that the expression of DAB2 was upregulated in bleomycin-induced fibrotic lung tissue with collagen fiber deposition and pulmonary interstitium thickening. Colocalization of DAB2 with α-smooth muscle actin (SMA) was observed in lung tissue sections. In vitro, human lung fibroblast MRC-5 cells were treated with TGF-β1 and the expression of DAB2 was increased. Knockdown of DAB2 suppressed cell proliferation and the expression of α-SMA, collagen I, collagen IV and fibronectin in TGF-β1-treated MRC-5 cells. The phosphorylation levels of PI3K and AKT were suppressed in DAB2-knockdown cells. IGF-1/IGF-1R has been reported to promote pulmonary fibrosis and activate the PI3K/Akt signaling. In the present study, the activation of IGF-1/IGF-1R signaling pathways in bleomycin-induced fibrotic lung tissues were positively associated with DAB2 expression. The phosphorylation level of IGF-1R was increased in MRC-5 cells with TGF-β1 treatment, and DAB2 expression was decreased by silencing of IGF-1R. This suggested that DAB2 might be a downstream target of the IGF-1R pathway and thus induced PI3K/AKT signaling activation and fibrogenesis. The current study demonstrated the importance of DAB2 in pulmonary fibrosis and suggested the potential of IGF-1R/DAB2/PI3K in the pathogenesis of IPF.

Effect of antibiotic monensin on cell proliferation and IGF1R signaling pathway in human colorectal cancer cells

Background/Aims Colorectal cancer is the third leading cause of death in patients with cancers in America. Monensin has represented anti-cancer effect on various human cancer cells. We seek to investigate the effect of monensin on proliferation of human colorectal cancer cells and explore whether IGF1R signaling pathway is involved in anti-cancer mechanism of monensin. Methods Cell proliferation and migration were assessed by crystal violet staining and cell wounding assay respectively. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell cycle progression was detected with the use of flow cytometry. Cancer-associated pathways were assessed with the use of pathway-specific reporters. Gene expression was detected by touchdown-quantitative real-time PCR. Inhibition of IGF1R was tested by immunofluorescence staining. Inhibition of IGF1R signaling was accomplished by adenovirus-mediated expression of IGF1. Results We found that monensin not only effectively inhibited cell proliferation, cell migration as well as cell cycle progression, but also induced apoptosis and G1 arrest in human colorectal cancer cells. Monensin was shown to target multiple cancer-related signaling pathways such as Elk1, AP1, as well as Myc/max, and suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. Conclusion Monensin could suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. It has the potential to be repurposed as an anti-colorectal cancer agent, but further studies are still required to investigate the detailed mechanisms of monensin underlying its anti-cancer motion. Key Messages Monensin inhibits the cell proliferation and the migration, induces apoptosis and inhibits cell cycle progression in human colorectal cancer cells. Monensin may exert anti-cancer activity by targeting multiple signaling pathways, including the IGF1R signaling pathway. Monensin has the potential to be repurposed as an anti-colorectal cancer agent.

Single-cell-led drug repurposing for Alzheimer’s disease

Alzheimer’s disease is the most common form of dementia. Notwithstanding the huge investments in drug development, only one disease-modifying treatment has been recently approved. Here we present a single-cell-led systems biology pipeline for the identification of drug repurposing candidates. Using single-cell RNA sequencing data of brain tissues from patients with Alzheimer’s disease, genome-wide association study results, and multiple gene annotation resources, we built a multi-cellular Alzheimer’s disease molecular network that we leveraged for gaining cell-specific insights into Alzheimer’s disease pathophysiology and for the identification of drug repurposing candidates. Our computational approach pointed out 54 candidate drugs, mainly targeting MAPK and IGF1R signaling pathways, which could be further evaluated for their potential as Alzheimer’s disease therapy.

14-3-3β is essential for milk composition stimulated by Leu/IGF-1 via IGF1R signaling pathway in BMECs

The cell proliferation of bovine mammary epithelial cells (BMECs) and consequent milk synthesis are regulated by multiple factors. The purpose of this study was to examine the effect of 14-3-3β on cellular proliferation and milk fat/β-casein synthesis in BMECs and reveal its underlying mechanisms. In this study, we employed gene function analysis to explore the regulatory effect and molecular mechanisms of 14-3-3β on milk synthesis and proliferation in BMECs. We found that leucine and IGF-1 enhance cell proliferation and milk synthesis in a 14-3-3β-dependent manner and only exhibiting such effect in the presence of 14-3-3β. We further determined that 14-3-3β interacts with the IGF1R self-phosphorylation site and it additionally mediated leucine and IGF-1 to stimulate the synthesis of milk through the IGF1R-AKT-mTORC1 signaling pathway. In summary, our data indicated that 14-3-3β mediates the expression of milk fat and protein stimulated by leucine and IGF-1, leading to lactogenesis through IGF1R signaling pathway in BMECs.

LINC02381-ceRNA exerts its oncogenic effect through regulation of IGF1R signaling pathway in glioma

LncRNAs play essential roles in the cellular and molecular biology of glioma. Some LncRNAs exert their role through sponging miRNAs and regulating multiple signaling pathways. LINC02381 is involved in several cancer types as either oncogene or tumor suppressor. Here, we intended to find the molecular mechanisms of the LINC02381 effect during the glioma progression in related cell lines. RNA-seq data analysis indicated the oncogenic characteristics of LINC02381, and RT-qPCR results confirmed its upregulation compared to normaltissues. Besides its expression was relatively stronger in invasive glioma cell lines. Furthermore, in silico analysis revealed LINC02381 is concentrated in the cytoplasm and predicted its sponging effect against miR-128 and miR-150, which was verified through dual luciferase assay. When LINC02381 was overexpressed in 1321N1, U87, and A172 cell lines, IGF1R and TrkC receptors as well as their downstream pathways (PI3K and RAS/MAPK), were upregulated, detected by RT-qPCR, and verified by western analysis. Consistently, LINC02381 overexpression was followed by an increased proliferation rate of transfected glioma cell lines, detected by flow cytometry and MTT assay, and RT-qPCR. It also resulted in elevated EMT and stemness markers expression level, increased migration rate, and reduced apoptosis rate, detected by RT-qPCR, western analysis, scratch test, and Annexin/PI flow cytometry analysis, respectively. The overall results indicated that LINC02381 exerts its oncogenic effect in glioma cells through sponging miR-128 and miR-150 to upregulate the IGF1R signaling pathway. Our results introduce LINC02381 and miR-128, and miR-150 as potential prognosis and therapy targets for the treatment of glioma.

Molecular regulation of phenolic compounds on IGF-1 signaling cascade in breast cancer.

Breast cancer is a highly aggressive and heterogeneous disease with complex features that remains a major health problem and undermines the span and quality of life of women worldwide. Primary literature has shown the role of phenolic compounds in controlling the onset of breast cancer. The mechanism of action of phenolic compounds can be explained by their interaction with signal transduction pathways that regulate cell proliferation and induction of apoptosis. One of the targets of phenolic compounds is the insulin like growth factor 1 (IGF-1) signaling cascade, which plays a significant role in the growth and development of mammary tissues by leading proliferative and anti-apoptotic events. Increasing research evidence points to the function of the IGF-1 cascade system in the commencement, progression, and metastasis of breast tissue malignancy. In this review, we mainly discuss the function of the IGF-1 system, and the role of phenolic compounds in regulating the IGF-1 signaling cascade and curbing breast malignancies.

Ginsenoside Rg1 enhances the healing of injured tendon in achilles tendinitis through the activation of IGF1R signaling mediated by oestrogen receptor

BackgroundDuring the pathogenesis of tendinopathy, the chronic inflammation caused by the injury and apoptosis leads to the generation of scars. Ginsenoside Rg1 (Rg1) is extracted from ginseng and has anti-inflammatory effects. Rg1 is a unique phytoestrogen that can activate the estrogen response element. This research aimed to explore whether Rg1 can function in the process of tendon repair through the estrogen receptor. MethodsIn this research, the effects of Rg1 were evaluated in tenocytes and in a rat model of Achilles tendinitis (AT). Protein levels were shown by western blotting. qRT-PCR was employed for evaluating mRNA levels. Cell proliferation was evaluated through EdU assay and cell migration was evaluated by transwell assay and scratch test assay. ResultsRg1 up-regulated the expression of matrix-related factors and function of tendon in AT rat model. Rg1 reduced early inflammatory response and apoptosis in the tendon tissue of AT rat model. Rg1 promoted tenocyte migration and proliferation. The effects of Rg1 on tenocytes were inhibited by ICI182780. Rg1 activates the insulin-like growth factor-I receptor (IGF1R) and MAPK signaling pathway. ConclusionRg1 promotes injured tendon healing in AT rat model through IGF1R and MAPK signaling pathway activation.

SAHA anti‐neoplastic effects in Glioblastoma via IGF‐1R signaling pathway

Glioblastoma Multiforme (GBM) is the most common primary malignant tumor and is aggressive, highly invasive, and angiogenic in nature. Median survival time of patients has not improved with current treatments and demands new therapies. SAHA; an HDAC inhibitor has been recently identified as a promising candidate to treat various neoplasms but has not been well studied in GBM. The Insulin-like growth factor IGFR, a glial growth factor, is highly overexpressed in malignant tissues and functions as an anti-apoptotic agent and promotes cell proliferation and migration. The present study investigated the anti-neoplastic effects of SAHA (HDACi) in U-87MG cell line (Human Primary Gliblastoma cell line) and elucidated probable mechanisms of action via IGF1R pathway in GBM. Results demonstrated that SAHA decreased the U-87MG cell viability in a dose dependent manner. SAHA imparted its effect via IGF1R signaling pathway. Cells treated with SAHA showed increased expression of Caspase-3; a hallmark of induction of apoptosis. SAHA also exhibited anti-migratory effect on U-87MG cells. This cytotoxic effect of SAHA was significantly lower in neuronal PC-12 cells compared to U-87MG cells. Overall, SAHA proved to be an anti-neoplastic agent in U-87MG cells and can be considered as a potential adjuvant therapy for GBM with further in-vivo based analysis.

Influence of high glucose in the expression of miRNAs and IGF1R signaling pathway in human myometrial explants

PurposeSeveral roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium.MethodsHuman myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR.ResultsMyometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged.ConclusionsThe human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

Extracellular Prolidase (PEPD) Induces Anabolic Processes through EGFR, β1-integrin, and IGF-1R Signaling Pathways in an Experimental Model of Wounded Fibroblasts.

The role of prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR) was studied in an experimental model of wound healing in cultured fibroblasts. The cells were treated with PEPD (1–100 nM) and analysis of cell viability, proliferation, migration, collagen biosynthesis, PEPD activity, and the expressions of EGFR, insulin-like growth factor 1 (IGF-1), and β1-integrin receptor including downstream signaling proteins were performed. It has been found that PEPD stimulated proliferation and migration of fibroblasts via activation of the EGFR-downstream PI3K/Akt/mTOR signaling pathway. Simultaneously, PEPD stimulated the expression of β1-integrin and IGF-1 receptors and proteins downstream to these receptors such as FAK, Grb2, and ERK1/2. Collagen biosynthesis was increased in control and “wounded” fibroblasts under PEPD treatment. The data suggest that PEPD-induced EGFR signaling may serve as a new attempt to therapy wound healing.

Knockdown of miR-130a-3p alleviates spinal cord injury induced neuropathic pain by activating IGF-1/IGF-1R pathway

BackgroundRecent studies have elucidated the instrumental role of microRNAs (miRNAs) in neuropathic pain (NP) progression. As one member of miRNAs, miR-130a-3p has been proved as a mediator in inflammation and neuronal maturation. The present study attempted to elucidate what effect miR-130a-3p exerts on NP. Materials and methodsThe miR-130a-3p expression in the spinal cord tissues of rat with spinal cord compression injury (SCI) and LPS-induced BV2 microglia was determined with RT-PCR, which was further applied to analyze the clinical relevance between miR-130a-3p and neuropathic pain. Besides, the expression of IGF-1, IL-1β, IL-6, and TNF-α in the spinal cord tissues of rats was measured using RT-PCR and ELISA after intrathecal injection of miR-130a-3p inhibitors and tail vein injection of IGF-1 low-expression lentivirus (Lv-shIGF-1). Further, neuronal apoptosis (labeled by Caspase3) and microglial activation (labeled by Iba1) were examined by immunohistochemistry (IHC), and the levels of IGF-1, IGF-1R, NF-κB were determined by western blot. Additionally, bioinformatic was employed to analyze the potential target genes of miR-130a-3p. Furthermore, the dual luciferase activity assay and RNA immunoprecipitation assay were conducted to further substantiate whether miR-130a-3p targets IGF-1. ResultsIn comparison with the sham group, the miR-130a-3p expression was remarkably up-regulated in the spinal cord lesions of SCI rats. The ELISA results showed that inhibiting the miR-130a-3p significantly reduced NP symptoms of SCI rats, mitigated neuronal apoptosis, microglial activation, repressed NF-κB phosphorylation and the IL-1β, IL-6 and TNF-α expressions in SCI rats. Contrarily, downregulation of miR-130a-3p increased IGF-1 and IGF-1R expression. What's more, we observed the same effects in BV2 microglia. In addition, the bioinformatics analysis showed that miR-130-3p targeted at the 3′-untranslated region of IGF-1 and inhibiting its expression. However, abolishing IGF-1 not only promoted the inflammatory responses in the SCI lesions, but also aggravated NP of SCI rats, while those effects were attenuated by the downregulation of miR-130a-3p. ConclusionThe inhibition of miR-130a-3p expression up-regulates the IGF-1/IGF-1R signaling pathway, thus reducing neuropathic pain caused by spinal cord injury.

The correlation between IGF-1R and HER-2 expression with mitosis count and histopathological grade in invasive breast carcinoma of no special type

BackgroundThe IGF-1R signaling pathways have cross-talk with HER-2 signaling pathways and was thought to be one of the resistance mechanism intrastuzumab therapy. This research was proposed to analyze the correlation between IGF-1R and HER-2 expression with mitosis count andhistopathological grade in invasive breast carcinoma of no special type.MethodsThis research was a cross-sectional design. A total of fifty-five invasive breast carcinoma of no special type cases diagnosed in theAnatomical Pathology Centre Diagnostic of Medical Faculty of Andalas University in a period of 2014-2015 were collected and stainedimmunohistochemically with IGF-1R and HER-2 antibodies. IGF-1R and HER-2 expression were examined and their correlation with mitosiscount and histopathological grade were statistically analyzed with T-test, Oneway Anova and Chi-Square test.ResultsIGF-1R cytoplasm and membranous expression were found positive in 18.2% and 34.5% cases respectively, meanwhile, HER-2 expressionswere found positive in 23.6% cases of invasive breast carcinoma of no special type. There were significant correlations between IGF-1Rcytoplasm expression with mitosis count (p=0.049). There were no significant correlations between IGF-1R membranous expression withmitosis count (p=0.641) with histopathological grade (p=1.00). There were no significant correlations between HER-2 expression withmitosis count (p=0,495) and histopathological grade (p=1.000).ConclusionIGF-1R expressions have a significant correlation with mitosis count rather than HER-2 expressions. Inhibition it’s signaling pathways mayhave therapeutic value in breast carcinoma. Combination therapy of anti-HER-2 and anti-IGF-1R are expected to overcome resistance withtrastuzumab in HER-2 positive breast carcinoma

Abstract 1894: Role of IGF1R in mediating resistance to MAPK pathway inhibitors in BRAF-mutant melanoma

Abstract Melanoma is the most aggressive form of skin cancer originating in the pigment producing melanocytes of the skin. The five year survival rate for patients diagnosed with metastatic melanoma is only 23%. Around 60% of the melanomas encompass the active BRAFV600E oncoprotein, which is critical for survival and proliferation of the melanoma cells as a consequence of hyperactivation of theMAPK pathway. This opened up avenues for development of BRAF inhibitors and subsequently the development of MEK inhibitors. Co-targeting of BRAF and MEK with Dabrafenib and Trametinib respectively, results in a response rate of about 70% in patients harboring the BRAF mutation. The use of these inhibitors is limited as patients relapse on treatment typically within 6 months due to the development of acquired resistance. Recent studies have identified various resistance mechanisms including involvement of various receptor tyrosine kinases. In order to decipher novel mechanisms of resistance, we generated double resistant cell lines to BRAF and MEK inhibitor in WM115, WM983 and A375 human melanoma cell lines harboring BRAFV600 mutations using Dabrafenib and Trametinib over a course of 9-11 months. The drug resistant cell lines exhibited increased viability and maintenance of downstream p-ERK protein as compared to their drug sensitive counterparts. Receptor tyrosine kinase array revealed an increase in phosphorylated HER3, AXL, EGFR and IGF1R in the drug resistant cells versus drug sensitive cells. Quantitative PCR data revealed an upregulation of these molecules along with an increase in the expression of some of their ligands at the mRNA level. RNA-seq analysis identified IGF1R and IGF2BP2 to be upregulatedin the resistant cell lines compared to parental cell lines.IGF2BP2 is a tumor promoter and drives cancer proliferation in a variety of malignancies and is correlated with poor survival.Our next experiments are focused on testing the efficacy of IGF1R inhibitors in the drug resistant cell lines and their downstream effector signaling molecules in vitro and in vivo. Further, we aim to decipher the mechanism by which IGF2BP2 regulates the IGF1R signaling pathway in MAPK inhibitors resistant melanoma setting. Delineating the role of IGF1R in mediating resistance to BRAF and MEK inhibitors will help expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients. Citation Format: Hima Patel, Rosalin Mishra, Natasa Broit, Nour Yacoub, Long Yuan, Samar Alanazi, Nicholas Hayward, Peter Johansson, Joan T. Garrett. Role of IGF1R in mediating resistance to MAPK pathway inhibitors in BRAF-mutant melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1894.

Risk and mechanism of glucose metabolism disorder in the offspring conceived by female fertility maintenance technology

Although female fertility maintenance technology (FFMT) provides an effective option for preserving fertility in patients with cancer suffering from fertility loss due to cancer treatment, previous studies have shown that the technique has certain potential risks and requires an assessment of the health status of the offspring since FFMT may lead to glucose metabolism disorder in offspring mice. The present animal study examined the glucose metabolism of adult mice offspring born from ovarian tissue cryopreservation and orthotopic allotransplantation. The mice were divided into three groups: normal, fresh ovary transplantation, and cryopreserved ovary transplantation. We recorded fasting blood glucose, glucose tolerance, and fasting serum insulin level for six months. Liver DNA, RNA, and proteins were extracted to detect the interaction between DNA methylation and Grb10 expression and insulin signaling pathway factors such as P-IGF1R, P-IRS2, P-AKT, and Grb10. Female recipient mice that received FFMT could successfully give birth after mating. The average litter size and total litter size of the cryopreserved and fresh groups showed marked differences compared with the normal group. Compared with the normal group, the fasting blood glucose and fasting serum insulin levels were higher in the cryopreserved and fresh groups. The mRNA and protein expressions of Grb10 were higher in the fresh and cryopreserved groups. Compared with the normal group, the DNA methylation status of four of the 11 sites of the Grb10 promoter was lower in the cryopreserved group. Grb10 overexpression inhibited the downstream phosphorylation protein factor expression (p-IGF-1R, p-IRS2, and p-Akt) of the IGF-1R signaling pathway. Female fertility maintenance technology (FFMT), including ovarian tissue cryopreservation (OTC), and orthotopic allotransplantation techniques might lead to glucose metabolism disorders in offspring mice.