Insulin-like Growth Factor-1 Receptor Protein Levels Research Articles

Insulin-like growth factor 1 knockdown attenuates high glucose-induced podocyte injury by promoting the JAK2/STAT signalling-mediated autophagy.

Podocyte injury plays a crucial role in the development of diabetic nephropathy (DN). A high serum level of insulin-like growth factor 1 (IGF-1) has been observed in patients with DN. This paper is to study the role and mechanism of IGF-1 in high glucose (HG)-induced podocyte injury. Mouse podocytes MPC-5 were treated with HG to establish a DN model in vitro. db/db diabetic mice and db/m nondiabetic mice were used to evaluate the IGF-1 role in vivo. Western blotting was used for measuring protein levels of IGF-1 receptor, Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway-related markers, podocyte markers podocin and nephrin, apoptosis- and autophagy-related markers in MPC-5 cells. Immunofluorescence staining was implemented for measuring the expression of nephrin and the autophagy marker LC3. Flow cytometry was used for detecting podocyte apoptosis. IGF-1 expression was increased in HG-stimulated MPC-5 cells and the kidney of db/db diabetic mice compared with corresponding controls. Knocking down IGF-1 downregulated IGF-1R and inhibited JAK2/STAT signalling pathway in HG-treated MPC-5 cells and db/db diabetic mice. IGF-1 silencing attenuated HG-induced podocyte injury, apoptosis and reduction in autophagy. Activating the JAK2/STAT signalling pathway or inhibiting autophagy reversed the effects of IGF-1 silencing on HG-treated MPC-5 cells. Knocking down IGF-1 alleviates HG-induced podocyte injury and apoptosis by inactivating the JAK2/STAT signalling pathway and enhancing autophagy.

Role of let-7 family in the invasion and metastasis of osteosarcoma.

Role of let-7 family in the invasion and metastasis of osteosarcoma.

The clinicopathological and prognostic significances of IGF-1R and Livin expression in patients with colorectal cancer

BackgroundColorectal cancer (CRC) is the third most common cancer worldwide. However, limited effective biomarkers are associated with the tumorigenesis and prognosis of CRC.MethodsThe present study identified potential signatures from The Cancer Genome Atlas (TCGA) database and further validated the identified biomarkers in CRC tissues by immunohistochemistry (IHC).ResultsThe expression of insulin-like growth factor 1 receptor (IGF-1R) and Livin gene was significantly upregulated in CRC samples compared to the adjacent normal samples in the TCGA dataset. IHC indicated that IGF-1R and Livin protein levels are increased in CRC and adenoma tissues compared to normal tissues. Notably, the IGF-1R protein levels differed significantly between adenoma and CRC. The elevated IGF-1R and Livin expression was associated with CRC clinicopathological features, including age, gender, histological subtype, individual cancer stages, nodal metastasis, and TP53-mutant in TCGA. Additionally, the IGF-1R promoter methylation level was closely related to CRC. Consistent with the TCGA study, IHC indicated that overexpressed IGF-1R and Livin proteins were independent risk factors for stage and metastasis. A marked correlation was established between IGF-1R and Livin expression in CRC, while the survival map showed no significant correlation with CRC. Kaplan–Meier survival curves showed that CRC patients with high IGF-1R or Livin expression had a prolonged overall disease-free survival than those with low expression in TCGA.ConclusionIGF-1R and Livin are associated with CRC tumorigenesis and might be valuable for novel biomarker identification and targeted therapeutic strategy development.

Increase of E3 ubiquitin ligase NEDD4 expression leads to degradation of its target proteins PTEN/IGF1R during the formation of goose fatty liver.

Goose fatty liver may have a unique protective mechanism as it does not show a pathological injury even in the case of severe steatosis. Although neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) participates in repair and regeneration of injured liver through its target proteins, its role in nonalcoholic fatty liver disease remains unknown. Using quantitative polymerase chain reaction (PCR) and immunoblot analyses, here, we found that the messenger RNA (mRNA) and protein expressions of NEDD4 were induced in goose fatty liver compared with normal liver. The mRNA expression of the gene of phosphate and tension homology deleted on chromosome ten (PTEN) and insulin-like growth factor 1 receptor (IGF1R) was also induced in goose fatty liver; however, their protein expression was or tended to be suppressed. Moreover, the co-immunoprecipitation analysis indicated that there was a physical association between NEDD4 and PTEN in goose liver, which was consistent with the ubiquitination of PTEN in goose fatty liver. Furthermore, NEDD4 overexpression in goose primary hepatocytes suppressed the PTEN and IGF1R protein levels without a significant effect on their mRNA expression. In conclusion, the increased expression of NEDD4 leads to the degradation of PTEN and IGF1R proteins through ubiquitination in goose fatty liver, suggesting that NEDD4 may protect goose fatty liver from severe steatosis-associated injury via its target proteins during the development of goose fatty liver.

High IGF1R protein expression correlates with disease-free survival of patients with stage III colon cancer

The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. Hundred and fifty-one CC patients were included (median age, 66.6years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p= 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p= 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p= 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.

Seasonal expressions of growth hormone receptor, insulin-like growth factor 1 and insulin-like growth factor 1 receptor in the scented glands of the muskrats (Ondatra zibethicus)

The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) system plays an important role in regulating the cellular growth and organ development. The present study investigated the seasonal expressions of growth hormone receptor (GHR), IGF-1 and insulin-like growth factor 1 receptor (IGF-1R) in the scented glands of the muskrats. Morphological changes in the scented glands of the muskrats were observed significantly between the breeding and non-breeding seasons. Immunohistochemically, the expressions of GH, GHR, IGF-1 and IGF-1R were found in glandular cells and epithelial cells of the scented glands in both seasons. The protein and mRNA expression levels of GHR, IGF-1 and IGF-1R in the scented glands during the breeding season were noticeably higher than those of the non-breeding season. In parallel, the levels of GH and IGF-1 in the sera and scented glands were remarkably higher during the breeding season. In addition, small RNA sequencing showed that the predicted targets of the significantly changed hsa-miR-5100 and mmu-miR-6937-5p might regulate the expressions of Ghr, Igf-1 or Igf-1r. These results suggested that the morphological changes in the scented glands of the muskrats during the different seasons might be related to the expression levels of GHR, IGF-1 and IGF-1R. Meanwhile, GHR/IGF-1 system might regulate the scented glandular functions via endocrine or autocrine/paracrine manners.

Jinmaitong, a Traditional Chinese Compound Prescription, Ameliorates the Streptozocin-Induced Diabetic Peripheral Neuropathy Rats by Increasing Sciatic Nerve IGF-1 and IGF-1R Expression.

Jinmaitong (JMT) is a Traditional Chinese Compound Prescription for the treatment of diabetic peripheral neuropathy (DPN). This study aims to investigate the effect of JMT on the insulin-like growth factor 1 (IGF-1) and the insulin like growth factor 1 receptor (IGF-1R) expression in sciatic nerves of diabetic rats. Firstly, the chemical profile of JMT was characterized by UPLC/Q-TOF-MS analysis. A total of 72 compounds were putatively identified. Secondly, streptozotocin (STZ)-induced diabetic rats were treated with neurotropin (NTP, 2.67 NU/kg/day) or JMT at low-dosage (0.4375 g/kg/day), medium-dosage (0.875 g/kg/day), and high-dosage (1.75 g/kg/day) for continuous 16 weeks. Blood glucose and body weight were detected every 4 weeks during the experiment. The mechanical pain and morphological change on sciatic nerves were detected by pain measurement instrument and microscopy. The IGF-1 level in serum and tissues were measured though ELISA and immunohistochemistry. The mRNA and protein expressions of IGF-1, IGF-1R, peripheral myelin protein zero (P0), and peripheral myelin protein 22 (PMP22) in the tissues were measured by qRT-PCR and western blot. As a result, JMT had no significant effect on body weight, but reduced the fasting blood glucose levels of diabetic rats. Besides, the pathological morphology, mechanical pain thresholds, serum level and tissue expression of IGF-1, mRNA, and protein levels of IGF-1R, P0, and PMP22 were significantly improved in JMT group at middle dosage. In conclusion, JMT could ameliorate the behavioristics and morphology changes in DPN rats by promoting IGF-1 and IGF-1R gene and protein expressions in sciatic nerves, as well as regulating the peripheral nerve remyelination genes P0 and PMP22 expressions, which provides scientific evidence for the clinical application of JMT in DPN patients.

Combined effects of 17β-estradiol and exercise training on cardiac apoptosis in ovariectomized rats.

BackgroundThe purpose of this study was to investigate the combined 17β-estradiol (E2) and exercise training on cardiac pro-survival and anti-apoptotic pathways in ovariectomized rats.MethodsFifty-six female Sprague–Dawley rats were divided into a sham-operated (Sham), a bilaterally ovariectomized (OVX), an OVX treated with E2 (OVX-E2; 10μg/kg/day), and an OVX with E2 and treadmill exercise training (OVX-E2-EX; 60 min/day, 5 days/week) for 10 weeks. Following 10 weeks of exercise training, rat hearts were isolated for the evaluation of Histopathological analysis, TUNEL assay, and Western blotting.ResultsThe protein levels of estrogen receptor α (ERα), estrogen receptor β (ERβ), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-phosphatidylinositol 3-kinase (p-PI3K) (estrogen receptors/IGF-1-related survival pathway) were significantly increased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. The protein levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL) and phosphorylated-Bad (p-Bad) (Bcl-2 family survival pathway) were significantly increased in the OVX-E2-EX group when compared with the OVX group. Only the p-Bad was significantly increased in the OVX-E2 group when compared with the OVX group. The protein levels of truncation of Bid (t-Bid), Bcl-2-associated death promotor (Bad), Bcl-2-associated X protein (Bax), Cytochrome c, caspases-9, and caspases-3 (mitochondria-dependent apoptotic pathway), as well as the protein levels of tumor necrosis factor-α (TNF-α), Fas ligand, Fas receptors, Fas-associated death domain (FADD), activated caspase-8 and activated caspase-3 (Fas receptor–dependent apoptotic pathway) were significantly decreased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. Furthermore, when compared with the OVX-E2 group, the protein levels of ERβ, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced in the OVX-E2-EX group as well as the protein levels of Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased in the OVX-EX-E2 group.ConclusionsCombined E2 and exercise training exhibited a positive effect of protection on ovariectomy-induced cardiac apoptosis by enhancing ERβ-related survival pathways, which might provide a more effective therapeutic effect on cardiac protection in bilaterally oophorectomized or menopausal women than E2 treatment only.

Decreased expression of microRNA-223 promotes cell proliferation in hepatocellular carcinoma cells via the insulin-like growth factor-1 signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most harmful types of cancer. Previous studies have demonstrated that microRNA (miR)-223 is downregulated in the serum and tumor tissue of patients with HCC. The present study aimed to investigate the regulatory role of miR-223 on insulin-like growth factor-1 receptor (IGF-1R) and downstream factors in HCC. The Hep3B cell line was transfected with miR-223 mimic and inhibitor. Following transfection, cell proliferation was analyzed using a cell counting kit 8 assay and cellular apoptosis was assessed using flow cytometry. The expression of key molecules in the IGF-1 signaling pathway, including IGF-1R, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The results demonstrated that the mRNA and protein levels of IGF-1R were decreased in cells transfected with miR-223. Transfection with miR-223 also decreased cell proliferation and promoted cell apoptosis. Expression of total Akt and ERK, and their active forms phosphorylated Akt and ERK, were also downregulated following transfection with miR-223. By contrast, transfection with miR-223 inhibitor did not induce any effects on Hep3B cell proliferation and apoptosis, and did not affect the expression of key molecules in the IGF-1 pathway. Therefore, the results of the present study indicate that miR-223 decreases the proliferation and promotes the apoptosis of HCC cells. Its molecular mechanism of action may at least partially occur via the direct regulation of IGF-1R and indirect reduction of the downstream molecules Akt and ERK.

Abstract P3-04-11: ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors

Abstract The mTORC1 inhibitor everolimus (afinitor) is approved for the treatment of patients with advanced/metastatic ER+/HER2- breast cancer in combination with the steroidal aromatase inhibitor exemestane following progression on a non-steroidal aromatase inhibitor. The BOLERO-2 and TAMRAD studies demonstrated that combined everolimus/anti-estrogen therapy provided longer PFS compared to anti-estrogen alone. However, it has not been clarified whether continued treatment with an anti-estrogen backbone is beneficial in the setting of mTORC1 inhibition. Upon activation by mTORC1, p70S6K phosphorylates the insulin-like growth factor-1 receptor (IGF-1R)/insulin receptor (InsR) effector IRS-1 to promote IRS-1 degradation, which in turn decreases activation of phosphatidylinositol 3-kinase (PI3K), AKT, and mTORC1. IGF1R, IRS1, and IRS2 are ER-inducible genes, and crosstalk between the ER and IGF-1R pathways has been described. We hypothesized that mTORC1 inhibition with everolimus will upregulate IGF-1R/InsR/IRS-1/2 signaling to activate PI3K/AKT and promote cancer cell survival, while combined inhibition of ER and mTORC1 will block PI3K/AKT activation by decreasing IGF-1R and IRS-1/2, providing rationale for combined targeting of ER and mTORC1. In 3 ER+ breast cancer cell lines, everolimus treatment increased phospho-AKT levels. ER inhibition with fulvestrant suppressed the induction of P-AKT by everolimus. IGF-1R/InsR inhibition with OSI-906, and RNAi-mediated knockdown of IGF-1R, InsR, or IRS-1/2, decreased everolimus-induced P-AKT. Everolimus sensitized IGF-1R/InsR to IGF-1 that was suppressed by fulvestrant but enhanced by 17b-estradiol. Although fulvestrant decreased IGF-1R and InsR protein levels, phospho-receptor tyrosine kinase profiling showed that fulvestrant increased P-IGF-1R and P-InsR. Acting downstream of IGF-1R/InsR, fulvestrant prevented everolimus-induced PI3K/AKT activation by blocking binding between the p85 regulatory subunit of PI3K and IRS-1, possibly by decreasing IRS-1/2 levels. In summary, everolimus-induced activation of PI3K/AKT requires IGF-1R/InsR/IRS-1/2 signaling facilitated by ER. Combined treatment with fulvestrant and everolimus synergistically inhibited growth in 4 ER+ cell lines. To determine whether ER promotes PI3K/AKT activation induced by mTORC1 inhibition in patients' tumors without exposing patients to everolimus, we analyzed live tumor tissues from post-menopausal patients with ER+/HER2- breast cancer treated +/- letrozole for 10-21 d before surgical tumor resection. Tumor cores (1 mm diameter) were used for ex vivo culture in DMEM +/- everolimus +/- OSI-906 for 1 h, and lysates were analyzed by immunoblot. Everolimus significantly increased P-AKT in tumors from untreated patients (n=10). OSI-906 did not affect P-AKT, but OSI-906 suppressed everolimus-induced P-AKT. In tumors from letrozole-treated patients (n=7), neither everolimus nor OSI-906 affected P-AKT. These data collectively suggest that ER activation is required for activation of PI3K/AKT induced by mTORC1 inhibition, and provide rationale for therapeutic combinations of anti-estrogens and mTORC1 inhibitors. Citation Format: Yang W, Chen VS, Schwartz GN, Marotti JD, Rosenkranz KM, Gui J, Miller TW. ER is required for mTORC1 inhibitor-induced feedback activation of PI3K/AKT in ER+ breast cancer cells and patients' tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-11.

Expression of YAP and Inhibitory Effect By IGF-1R Suppressor in Diffuse Large B-Cell Lymphoma

Introduction: Yes-associated protein (YAP), a direct downstream effector of the tumor suppressive Hippo signaling, functions as a transcriptional coactivator in the modulation of cell growth, proliferation and apoptosis in a number of cancers. Given that Hippo signaling does not have an extracellular ligand nor a specific membrane receptor, its activation depends on the crosstalk mechanisms. Receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF-1R) is implicated in various tumor entities by modulating the proliferation, survival and metastasis of cancer cells. Strategies to block IGF-1R pathway in solid malignancies are being tested in clinical trials. However, the potential significance of IGF-1R and YAP in DLBCL remains ill defined. We assume that IGF-1R modulates the proliferation and apoptosis of DLBCL cells partly by regulating YAP in DLBCL. We aimed to investigate the functional significance of IGF-1R and its regulatory effect on YAP in DLBCL.Methods: Lymph node biopsies from 30 de novo DLBCL patients and 20 reactive hyperplasia cases were collected with informed consents. Immunohistochemistry (IHC) was performed to assess the expression of YAP and IGF-1R in lymphoma tissues. Protein expression levels of YAP and IGF-1R in DLBCL cell lines were detected by western blotting. Peripheral blood mononuclear cells were isolated with informed consents from healthy volunteers. Effects of AG1024 on cell viabilities and apoptosis were assessed by cell counting kit-8 and Annexin-V/7-AAD staining. Propidium iodide staining was conducted to assess cell cycle of DLBCL cells.Results: Compared to the reactive hyperplasia group, DLBCL patients revealed significantly higher protein levels of both YAP and IGF-1R (Fig. 1A). Overexpression of YAP and IGF-1R protein in DLBCL cell lines (LY1 and LY8) were further confirmed by immunoblotting (Fig. 1B). AG1024, a selective inhibitor of IGF-1R, significantly restrained the viabilities of LY1 and LY8 cells in a dose dependent manner (Fig. 1C). Apoptotic rates in LY1 and LY8 cells treated with IGF-1R inhibitor were markedly increased (Fig. 1D). Moreover, addition of IGF-1Rsuppressor induced remarkable cell cycle arrest in G2/M phase in DLBCL cells (Fig. 1E). In LY1 and LY8 cells treated with AG1024, we observed that the reduced phosphorylation of IGF-1R was accompanied by significantly declined level of YAP protein level (Fig. 1F). Results of the immunofluorescense verified the inhibitory effect of AG1024 on expression of YAP protein in LY1 and LY8 cell (Fig. 1G).Conclusions: We identified the aberrant overexpression of YAP and IGF-1R in DLBCL patients and cell lines, which suggesting the dysregulation of Hippo and IGF-1 signaling in DLBCL. IGF-1R inhibitor exhibits proliferation-inhibitory and pro-apoptotic effect via inhibiting the activation YAP. We provided a novel mechanism involved in the function of IGF-1R in DLBCL. Further interrogation on the biological function of YAP in DLBCL will highlight the crosstalk between these two pathways and presents a promising therapeutic strategy in DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P41 Exploring the biological role of insulin and insulin-like growth factor-1 hybrid receptors in the endothelium

Endothelial cells co-express insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) homodimers. Furthermore, association of insulin αβ half receptors and IGF-1 αβ half receptors also occur resulting in insulin/IGF-1 hybrid receptors. An abundance of hybrid receptors has been associated with reduced insulin sensitivity in metabolic tissue. The aim of this project is to study the differential expression of vascular derived insulin receptors, IGF-1 receptors and insulin/IGF-1 hybrids during insulin resistance in a diet-induced model of obesity (DIO). C57BL/6 mice were fed on high-fat or low-fat diets ad libitum for 16-weeks. Obese mice showed significant metabolic abnormalities indicative of insulin resistance when compared with lean littermates. A significant increase in hybrid receptor levels were observed in aorta from obese mice (immunoprecipitation and Western blotting). Total IR and IGF-1R protein levels were reduced in obese mice (Western blotting) however, no change was observed in IR and IGF-1R mRNA levels (real time-PCR). Insulin-stimulated (4.5 nmol/kg injected) in vivo serine phosphorylation of Akt was significantly blunted in aortae of obese mice, whereas IGF-1-stimulated (90 nmol/kg injected) remained unaffected. We have shown that IR and IGF1R homodimers are preferentially titrated into hybrid receptors in a model of DIO and this is accompanied with a reduction in insulin sensitivity but not IGF-1 sensitivity in the aorta. Hybrid receptors may provide a novel target for the prevention of CVD progression in vulnerable patients.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

MiR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2 and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.

Insulin-like Growth Factor-1 Receptor (IGF-1R) As a Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

Introduction: The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is dysregulated in various tumor entities and hematological malignancies including chronic lymphocytic leukemia and mantle cell lymphoma. The implication of IGF1R in the development and progression of cancer has led to its current evaluation in clinical trials as a potential therapeutic target for solid tumors. However, its functional significance in diffuse large B-cell lymphoma (DLBCL) remains poorly characterized. We hypothesized that IGF1R plays a key role in the pathogenesis and progression of DLBCL. In this present study, we evaluated the expression and function of IGF1R in both B cell lines and DLBCL tissues, as well as assessed the proliferation and apoptosis of DLBCL cells when treated with IGF-1R inhibitor, AG1024.Methods: Expression of IGF1R in B-cell lymphoma cell lines (LY1, LY8, Mino, Jeko-1, and SP53) was evaluated by Western blotting. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Blood samples and araffin-embedded tissues from 30 initial-diagnosed DLBCL patients prior to therapeutic interventions as a study group, and from 15 patients with reactive hyperplasia lymphnode as a control group were collected with informed consents. Immunohistochemisty (IHC) was conducted to assess the expression of IGF-1R in lymphoma tissues. Correlations between IGF1R expression and the clinical characteristics of DLBCL patients were further analyzed. DLBCL cell lines (LY1 and LY8) were treated with an IGF1R specific small molecular inhibitor, AG1024, cell proliferation was analyzed by cell counting kit (CCK-8). Effects of inhibitor or stimulator on the apoptosis of LY1 and LY8 cells were assessed by Annexin-V/PI and Annexin-V/7AAD, respectively. Expression of apoptosis-related protein, including Caspase-3 and Mcl-1, was evaluated by western blotting. Protein levels of downstream targets of IGF-1 signaling were also detected.Results: Significantly upregulation of both phoaphprylated and total IGF1R protein levels were found in B-cell lymphoma cells (LY1, LY8, Mino, Jeko-1 and SP53) (Fig 1.A). IHC was conducted and revealed significantly enhancement of IGF1R expression in DLBCL patients (Fig 1.B). Among the included DLBCL patients and control group with inreactive hyperplastic lymphadenitis, the positive rate of IGF1R was 90% and 20%, respectively. We then investigated the function of IGF1R inhibitors on the proliferation and apoptosis of DLBCL cells. LY8 cells were treated with different doses of AG1024 at 24-96 hours. Cell proliferation was inhibited by 60% when treated with AG1024 at the concentration of 15µM for 72-hours (Fig 1.C). Culture of LY1 and LY8 cells in the presence of 10µM and 15µM AG1024 concentration for 24-hours resulted in 13% (p<0.05) and 33% (p<0.001) cell apoptosis, respectively (Fig 1.D). Inhibition of IGF1R by AG1024 also resulted in induction of cleaved-Caspase-3, as well as reduction of Mcl-1(Fig 1.E-F). In order to investigate the mechanisms involved in the dysregultaion of IGF1R in DLBCL, LY8 cells were treated with 5 to 15 µM AG1024, the results revealed that AG1024 caused a dose-dependent decrease in the levels of phosphorylated IGF1R, AKT and ERK (Fig 1.G). Treatment of LY8 cells with recombinant human IGF-1 led to enhanced phosporylation levels of IGF1R, AKT and ERK (Fig 1.H).Conclusions: Our investigation observed that expression levels of IGF-1R were up-regulated in both B-cell lymphoma cells and DLBCL tissues. DLBCL cells treated with IGF-1R inhibitor, AG1024, revealed reduced proliferation and increased apoptosis rate. In addition, induction of cleaved-Caspase-3 was also found in LY1 treated with AG1024. AG1024 caused a dose-dependent decrease in the phosphorylation levels of IGF1R, AKT and ERK. This study suggests that IGF1R could be a potential molecular target for the treatment of DLBCL. The IGF-1R inhibitor is a promising therapeutic approach for DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-term treadmill training ameliorates endothelium-dependent vasorelaxation mediated by insulin and insulin-like growth factor-1 in hypertension.

Dysfunction of insulin and insulin-like growth factor-1 (IGF-1) is associated with the pathophysiology of hypertension. The influence of long-term exercise on vascular dysfunction caused by hypertension remains unclear. We investigated whether long-term treadmill training improved insulin- and IGF-1-mediated vasorelaxation in hypertensive rats. Eight-week-old male spontaneously hypertensive rats (SHR) were randomly divided into sedentary and exercise (SHR-EX) groups. The SHR-EX group was trained on a treadmill for 60 min/day, 5 days/wk, for 8 wk. Wistar-Kyoto rats (WKY) were used as the normal control group. After training, aortic insulin- and IGF-1-mediated vasorelaxation was evaluated in organ baths. Additionally, the roles of phosphatidylinositol 3-kinase (PI3K), nitric oxide synthase (NOS), and aortic protein expression were examined in the three groups. Compared with sedentary SHR and WKY groups, insulin- and IGF-1-mediated vasorelaxation was significantly enhanced to a nearly normal level in the SHR-EX group. After endothelial denudation, blunted and comparable vasorelaxation was found among the three groups. Pretreatment with selective PI3K and NOS inhibitors attenuated insulin- and IGF-1-mediated vasorelaxation, and no significant difference was found among the three groups after the pretreatment. The aortic protein levels of the insulin receptor (IR), IGF-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1), and endothelial NOS (eNOS) were also significantly increased in the SHR-EX group compared with the other two groups. These results suggested that treadmill training elicited the amelioration of endothelium-dependent insulin/IGF-1-mediated vasorelaxation partly via the increased activation of PI3K and NOS, as well as the enhancement of protein levels of IR, IGF-1R, IRS-1, and eNOS, in hypertension.

MiR-143 and MiR-145 regulate IGF1R to suppress cell proliferation in colorectal cancer.

Insulin-like growth factor 1 receptor (IGF1R) is a transmembrane receptor that is activated by insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. It belongs to the large class of tyrosine kinase receptors and plays an important role in colorectal cancer etiology and progression. In this study, we used bioinformatic analyses to search for miRNAs that potentially target IGF1R. We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3′-untranslated region (3′-UTR) of the IGF1R gene. These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity. Consistent with the bioinformatic analyses, we identified an inverse correlation between miR-143/145 levels and IGF1R protein levels in colorectal cancer tissues. By overexpressing miR-143/145 in Caco2, HT29 and SW480 colorectal cancer cells, we experimentally validated that miR-143/145 directly recognizes the 3′-UTR of the IGF1R transcript and regulates IGF1R expression. Furthermore, the biological consequences of the targeting of IGF1R by miR-143/145 were examined by cell proliferation assays in vitro. We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells. Taken together, our findings provide evidence for a role of the miR-143/145 cluster as a tumor suppressor in colorectal cancer through the inhibition of IGF1R translation.

Alcohol alters insulin-like growth factor-1-induced transforming growth factor β1 synthesis in the medial basal hypothalamus of the prepubertal female rat.

Insulin-like growth factor-1 (IGF-1) and transforming growth factor β1 (TGFβ1) are produced in hypothalamic astrocytes and facilitate luteinizing hormone-releasing hormone (LHRH) secretion. IGF-1 stimulates release by acting directly on the LHRH nerve terminals and both peptides act indirectly through specific plastic changes on glial/tanycyte processes that further support LHRH secretion. Because the relationship between these growth factors in the hypothalamus is not known, we assessed the ability of IGF-1 to induce TGFβ1 synthesis and release and the actions of alcohol (ALC) on this mechanism prior to the onset of puberty. Hypothalamic astrocytes were exposed to medium only, medium plus IGF-1 (200ng/ml), or medium plus IGF-1 with 50mM ALC. After 18hours, media were collected and assayed for TGFβ1. For the in vivo experiment, prepubertal female rats were administered either ALC (3g/kg) or water via gastric gavage at 07:30hours and at 11:30hours. At 09:00hours, saline or IGF-1 was administered into the third ventricle. Rats were killed at 15:00hours and the medial basal hypothalamus (MBH) was collected for assessment of TGFβ1, IGF-1 receptor (IGF-1R), and Akt. IGF-1 induced TGFβ1 release (p<0.01) from hypothalamic astrocytes in culture, an action blocked by ALC. In vivo, IGF-1 administration caused an increase in TGFβ1 protein compared with controls (p<0.05), an action blocked by ALC as well as a phosphatidylinositol 3 kinase/Akt inhibitor. IGF-1 stimulation also increased both total (p<0.01) and phosphorylated (p)-IGF-1R (p<0.05) protein levels, and phosphorylated (p)-Akt levels (p<0.01), which were also blocked by ALC. This study shows that ALC blocks IGF-1 actions to stimulate synthesis and release of hypothalamic TGFβ1, total and p-IGF-1R, and p-Akt levels further demonstrating the inhibitory actions of ALC on puberty-related events associated with hypothalamic LHRH release.

Abstract 2863: TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer

Abstract INTRODUCTION: Insulin like growth factor 1 receptor (IGF-1R) plays a critical role in different tumors including prostate cancer (PCa). Recently, a fusion gene involving the androgen receptor-regulated gene TMPRSS2 and one of the ETS transcription factors family member, predominantly ERG, has been described in half of PCa patients. TMPRSS2-ERG results in the overproduction of a truncated ERG protein and cooperates in the establishment of a neoplastic phenotype in prostate cells. The aim of this study was to evaluate TMPRSS2-ERG impact on IGF-1R expression and to verify the value of IGF-1R as biomarker of PCa progression. MATERIAL AND METHODS: IGF-1R expression was evaluated in a panel of six prostate cell lines, including malignant VCaP, DU-145, PC-3, LNCaP, 22RV1 and non malignant RWPE-1 prostate cells by qRT-PCR, western blot and immunofluorescence. ERG siRNA was transiently transfected in VCaP cells and ERG down-regulation was assessed by western blot. Total RNA was extracted from a cohort of 270 paraffin-embedded PCa samples with more than 5 years of follow-up and IGF-1R expression was analysed by qRT-PCR. The prognostic value of IGF-1R was evaluated both in the univariate and multivariate analysis. The clinicopathological informations as well as the TMPRSS2-ERG fusion gene status were considered in the statistical analysis. RESULTS: The IGF-1R expression profile analysis was performed in PCa cells and pointed out that VCaP cell line, harboring the TMPRSS2-ERG fusion gene, carries the highest IGF-1R expression levels both considering mRNA, total protein and membrane expression levels. Interestingly, IGF-1R protein levels were found to be down-regulated upon ERG silencing in VCaP cells. PCa tumors did not show any remarkable difference in the expression of IGF-1R when compared with normal tissues (median=1.04) but IGF-1R was directly correlated with TMPRSS2-ERG status (p=0.027) in tumors. Log-rank tests showed an association between higher IGF-1R expression and better biochemical progression free survival (p=0.047).When dividing the population according to the TMPRSS2-ERG status we found higher fold-change in IGF-1R expression in tumors expressing TMPRSS2-ERG (median=1.25) compared to non-expressors (median=0.91) and a statistically stronger association between IGF-1R and prognosis in the TMPRSS2-ERG negative tumors (p=0.012). Moreover, multivariate analysis confirmed that higher expression of IGF-1R is an independent indicator of better prognosis in this subgroup (HR:0.48.IC95% [0.25-0.90].p=0.023). CONCLUSIONS: In this study we demonstrated that TMPRSS2-ERG affects IGF-1R expression both in vitro and in clinical samples. High expression of IGF-1R is significantly associated with a better clinical outcome of PCa patients. (Grants: FIRB RBAP11884M_005 to KS; FIS PI10/01206 and FPI11/00505 from ISCIII) Citation Format: Caterina Mancarella, Irene Casanova-Salas, Josè Antonio Lopez-Guerrero, Katia Scotlandi. TMPRSS2-ERG influences IGF-1R expression and affects its prognostic value in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2863. doi:10.1158/1538-7445.AM2014-2863

MiR-133a suppresses ovarian cancer cell proliferation by directly targeting insulin-like growth factor 1 receptor

The microRNA miR-133a is dysregulated in many types of cancer, but the underlying mechanism remains largely unknown. In this study, we showed that the expression level of miR-133a was reduced in ovarian cancer tissues compared with normal ovaries. Ectopic expression of miR-133a significantly inhibited ovarian cancer cell proliferation and colony formation, and induced G1-phase cell cycle arrest, whereas decreased miR-133a expression dramatically enhanced cell proliferation and colony formation. Importantly, miR-133a overexpression suppressed in vivo tumor growth in nude mice models. Through in silico search, we found that the 3'-untranslated region (UTR) of insulin-like growth factor 1 receptor (IGF1R) contains an evolutionarily conserved miR-133a binding site. miR-133a overexpression repressed IGF1R-3'UTR reporter activity, and reduced the mRNA and protein levels of endogenous IGF1R. Rescue experiments showed that ectopic expression of IGF1R significantly promoted the proliferation of ovarian cancer cells stably overexpressing miR-133a. Taken together, these findings indicate that miR-133a is an important regulator in ovarian cancer, and that its suppressive effects are mediated by targeting IGF1R.