IGF-I Receptor Levels Research Articles

Genome-Wide Analyses Identify Filamin-A As a Novel Downstream Target for Insulin and IGF1 Action.

Insulin analogs were developed to improve diabetes therapy. However, certain modifications introduced into the insulin molecule were shown to enhance their affinity to the insulin-like growth factor-1 receptor (IGF1R). Most tumors, including endometrial cancers, express high levels of IGF1R. The present study was aimed at identifying the entire set of genes that are differentially activated by insulin glargine or detemir, in comparison to insulin and IGF1, in Type 1 and Type 2 endometrial cancer cell lines (ECC-1 and USPC-1, respectively). Global gene expression analyses demonstrated a ligand-dependent upregulated expression of filamin-A (FLNA), a gene that encodes an actin filament cross-linking protein, in both endometrial cancer cell types. Silencing experiments linked to migration assays confirmed the role of FLNA in cell growth and motility. Our data suggest that the activation of distinct sets of genes by glargine may lead to stimulation of specific pathways or, alternatively, may provide additive effects, different from those classically induced by insulin. Given that metastases are probably the main factor contributing to tumor invasiveness, the identification of FLNA as a downstream target for insulin-like hormones may be of translational relevance in oncology. Clinical studies in endometrial cancer may add further relevant information regarding the possible differential actions of insulin analogs with respect to native insulin.

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.

Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis

BackgroundClinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. We previously demonstrated that overexpression of insulin receptor isoform A (IRA) and insulin-like growth factor-I receptor (IGF-IR) confers a proliferative and migratory advantage to vascular smooth muscle cells (VSMCs) promoting plaque growth in early stages of atherosclerosis. However, the role of insulin receptor (IR) isoforms, IGF-IR or insulin-like growth factor-II receptor (IGF-IIR) in VSMCs apoptosis during advanced atherosclerosis remains unclear.MethodsWe evaluated IR isoforms expression in human carotid atherosclerotic plaques by consecutive immunoprecipitations of insulin receptor isoform B (IRB) and IRA. Western blot analysis was performed to measure IGF-IR, IGF-IIR, and α-smooth muscle actin (α-SMA) expression in human plaques. The expression of those proteins, as well as the presence of apoptotic cells, was analyzed by immunohistochemistry in experimental atherosclerosis using BATIRKO; ApoE−/− mice, a model showing more aggravated vascular damage than ApoE−/− mice. Finally, apoptosis of VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR−/− VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs), was assessed by Western blot against cleaved caspase 3.ResultsWe observed a significant decrease of IRA/IRB ratio in human complicated plaques as compared to non-complicated regions. Moreover, complicated plaques showed a reduced IGF-IR expression, an increased IGF-IIR expression, and lower levels of α-SMA indicating a loss of VSMCs. In experimental atherosclerosis, we found a significant decrease of IRA with an increased IRB expression in aorta from 24-week-old BATIRKO; ApoE−/− mice. Furthermore, atherosclerotic plaques from BATIRKO; ApoE−/− mice had less VSMCs content and higher number of apoptotic cells. In vitro experiments showed that IGF-IR inhibition by picropodophyllin induced apoptosis in VSMCs. Apoptosis induced by thapsigargin was lower in IR−/− VSMCs expressing higher IGF-IR levels as compared to IRLoxP+/+ VSMCs. Finally, IRB VSMCs are more prone to thapsigargin-induced apoptosis than IRA or IRLoxP+/+ VSMCs.ConclusionsIn advanced human atherosclerosis, a reduction of IRA/IRB ratio, decreased IGF-IR expression, or increased IGF-IIR may contribute to VSMCs apoptosis, promoting plaque instability and increasing the risk of plaque rupture and its clinical consequences.

Fucoidan downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells.

Fucoidan, a sulfated polysaccharide present in brown seaweed, has demonstrated anticancer activity in lung, breast, liver and colon cells. The insulin-like growth factor(IGF) signaling pathway regulates growth in HT-29 cells through the insulin receptor substrate-1 (IRS-1)/phosphatidylinositol3-kinase (PI3K)/protein kinaseB (AKT) and Ras/Raf/extracellular signal-regulated kinase (ERK) pathways. The aim of the present study was to investigate whether fucoidan downregulates the IGF-IR signaling pathway in HT-29human colon cancer cells. Fucoidan treatment (0-1,000µg/ml) was administered for 24h in HT-29cells. First, we investigated IRS-1/PI3K/AKT pathway-related protein expression levels following treatment with fucoidan (0-500µg/ml) using western blot analysis. Fucoidan significantly inhibited the expression of IGF-IR, PTEN, PI3K andAKT as well as their phosphorylated forms (p-IRS-1, p-PI3K and p-AKT). Next, we investigated the effects of fucoidan on Ras/Raf/ERK pathway‑related protein expression levels in HT-29cells. Fucoidan significantly inhibited the expression of IGF-IR, Shc, Ras, SOS, Raf andMEK. HT-29cells were then incubated in the presence of fucoidan (0 or250µg/ml), and IGF-I (10nM) was added for 0to60min. Immunoprecipitation (IP) experiments showed that fucoidan inhibited IGF-I-induced phosphorylation of IGF-IR, PI3K, Shc (IP,IGF-IR), and phosphorylated IRS-1 and PI3K (IP,IRS-1) compared to the control group. Western blot analysis showed that fucoidan inhibited the expression of IGF-I-induced p-IGF-IR/IGF-IR and p-AKT/AKT, but not p-ERK/ERK. In conclusion, the inhibition of cell viability by fucoidan in HT-29cells may be due to the downregulation of IGF-IR signaling through the main IRS-1/PI3K/AKTpathway. Fucoidan also partially impacted Ras/Raf signaling in the Ras/Raf/ERK pathway. Therefore, we suggest that fucoidan may be a suitable candidate chemopreventive agent in HT-29colon cancer cells.

Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells

Background/Aims: Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. Methods: Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. Results: Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II–induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. Conclusion: Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms underlying HBV-related HCC and may lead to the development of effective therapies.

Effects of insulin-like growth factor-1 on the in vitro maturation of canine oocytes.

The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 μg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.

Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol.

Alcohol (ALC) causes suppressed secretion of prepubertal luteinizing hormone-releasing hormone (LHRH). Insulin-like growth factor-1 (IGF-1) and kisspeptin (Kp) are major regulators of LHRH and are critical for puberty. IGF-1 may be an upstream mediator of Kp in the preoptic area and rostral hypothalamic area (POA/RHA) of the rat brain, a region containing both Kp and LHRH neurons. We investigated the ability of IGF-1 to stimulate prepubertal Kp synthesis and release in POA/RHA, and the potential inhibitory effects of ALC. Immature female rats were administered either ALC (3g/kg) or water via gastric gavage at 0730hours. At 0900hours, both groups were subdivided where half received either saline or IGF-1 into the brain third ventricle. A second dose of ALC (2g/kg) or water was administered at 1130hours. Rats were killed 6hours after injection and POA/RHA region collected. IGF-1 stimulated Kp, an action blocked by ALC. Upstream to Kp, IGF-1 receptor (IGF-1R) activation, as demonstrated by the increase in insulin receptor substrate 1, resulted in activation of Akt, tuberous sclerosis 2, ras homologue enriched in brain, and mammalian target of rapamycin (mTOR). ALC blocked the central action of IGF-1 to induce their respective phosphorylation. IGF-1 specificity and ALC specificity for the Akt-activated mTOR pathway were demonstrated by the absence of effects on PRAS40. Furthermore, IGF-1 stimulated Kp release from POA/RHA incubated invitro. IGF-1 stimulates prepubertal Kp synthesis and release following activation of a mTOR signaling pathway, and ALC blocks this pathway at the level of IGF-1R.

Insulin-like growth factor-I induces chemoresistence to docetaxel by inhibiting miR-143 in human prostate cancer.

Elevated levels of insulin-like growth factor-I (IGF-I) are associated with carcinogenesis and cancer progression. However, the molecular mechanisms by which IGF-I promotes prostate cancer development remain to be elucidated. Docetaxel chemotherapy is an important therapeutic strategy in many types of human cancers including prostate cancer. In this study, we showed that IGF-I rendered PC-3 and DU145 cells more resistant to docetaxel treatment. IGF-I treatment decreased miR-143 expression, but increased the expression levels of IGF-I receptor (IGF-IR) and insulin receptor substrate 1 (IRS1), direct targets of miR-143. Overexpression of miR-143 abolished IGF-I-induced chemoresistance to docetaxel treatment, decreased expression levels of IGF-I, IRS1, and vascular endothelial growth factor (VEGF) in prostate cancer cell lines. Furthermore, docetaxel treatment significantly inhibited VEGF transcriptional activation, whereas IGF-I treatment induced VEGF transcriptional activation in a dose-dependent manner. Forced expression of IGF-IR and IRS1 cDNAs without the 3’ UTR regions restored miR-143-inhibited VEGF transcriptional activation. Finally, miR-143 inhibited tumor growth and made cells more sensitive to docetaxel treatment for decreasing tumor growth in vivo. Taken together, our data demonstrates that IGF-I induces docetaxel resistance and upregulates IGF-IR and IRS1 expression through miR-143 downregulation, whereas miR-143 acts as a tumor suppressor by targeting its targets IGF-IR and IRS1.

Design and Applications of Nanoparticles in Biomedical Imaging

173 Objectives: Insulin-like growth factor-1 receptor (IGF-1R) is an oncogenic protein associated with multiple cancer types. FPX-01 is a novel IGF-1R directed radioimmunoconjugate containing the targeting antibody FPI-1175 (formerly AVE1642). Potential imaging (In-111) and therapeutic (Ac-225) forms of FPX-01 were subjected to biological evaluation in pre-clinical cancer models. Methods: The antibody was conjugated with a lysine directed bifunctional chelate containing a DOTA and radiolabeled with Ac-225, In-111, or Lu-177. In vitro binding and residualization studies (with [177Lu]-FPX-01) were conducted using a panel of cancer cell lines that express different levels of IGF-1R and was validated by immunoblotting. Comparative biodistribution studies were carried out using Colo-205 (colorectal cancer) xenograft bearing athymic nude mice. Results: Binding studies demonstrated high affinity (Kd of 0.7 - 3.0 nM) of [177Lu]-FPX-01 across 8 different IGF-1R expressing cell lines (from lung, pancreatic, prostate cancers) and the Bmax values correlated with IGF-1R expression levels determined by immunoblotting. Residualization assays revealed strong retention of internalized [177Lu]-FPX-01. [225Ac]-FPX-01 biodistribution data demonstrated high levels of uptake in Colo-205 xenografts (35.5 ± 4.2 %ID/g @ 168 h) and biphasic blood clearance with low radioimmunoconjugate uptake in normal organs (less than 5.2 %ID/g @ 168 h). Further studies showed that [111In] and [177Lu]-FPX-01 had equivalent biodistribution to [225Ac]-FPX-01 demonstrating comparability across multiple isotopes. Tumor uptake at 168 h for [111In] and [177Lu]-FPX-01 was 50.7 ± 8.7 and 45.3 ± 6.7 %ID/g respectively. Tumor uptake of [177Lu]-FPX-01 was blocked by excess native FPI-1175 mAb demonstrating the specificity of FPX-01 for its biological target. Conclusion: The utility of FPX-01 as a radiopharmaceutical with application for oncologic imaging and radiotherapy was demonstrated in a series of preclinical studies. [111In]-FPX-01 had equivalent biodistribution to [225Ac]-FPX-01 and is suitable for use as an imaging surrogate.

Implication of connexin30 on the stemness of glioma: connexin30 reverses the malignant phenotype of glioma by modulating IGF-1R, CD133 and cMyc.

Gap-junctional intercellular communication (GJIC) plays a major role in the malignant growth of glioma. Although the mechanistic aspects of GJIC have been extensively studied, the role of connexins in the regulation of the malignant behavior of glioma stem cells (GSCs) remains unclear. In our previous studies, we have shown that connexin30 can interfere with the insulin-like growth factor 1 receptor (IGF-1R), which is known for self-renewal and pluripotency. Following our earlier in vitro observation, in this work, we aimed to study the consequence of this influence of Cx30 on IGF-1R by evaluating the marker of GSCs, CD133 and oncoprotein, cMyc. We strengthened our basis by examining human glioma samples of different grades as well as rat C6 xenografts (Cx30-transfected and -non-transfected C6 cells) along with the sphere formation assays in vitro. Investigation of stemness-related CD133 and cMyc in human samples and rat xenografts exhibited a reciprocal relationship between Cx30 and IGF-1R in the low and high grades (HG) of glioma. Cx30 was completely abolished in HG; levels of IGF-1R, CD133 and cMyc expression were positively correlated with HG. Cx30 transfection could attenuate the malignant burden of glioma in rat xenografts. Cx30 transfection also altered the tumor sphere formation of C6 glioma cells in vitro, an important property of GSCs, and there was a significant reduction of CD133 and cMyc expression by Cx30 both in vitro and in vivo. These factors indicate that dysfunction of Cx30 plays a crucial role in the prevention of the stemness of glioma, and the exploitation of this feature will help in the management of glioma.

Muscle Conditional Medium Reduces Intramuscular Adipocyte Differentiation and Lipid Accumulation through Regulating Insulin Signaling.

Due to the paracrine effects of skeletal muscle, the lipid metabolism of porcine intramuscular (i.m.) preadipocytes was different from that of subcutaneous (s.c.) preadipocytes. To investigate the development of i.m. preadipocytes in vivo, the s.c. preadipocytes were cultured with muscle conditional cultured medium (MCM) for approximating extracellular micro-environment of the i.m. preadipocytes. Insulin signaling plays a fundamental role in porcine adipocyte differentiation. The expression levels of insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF-1R) in i.m. Preadipocytes were higher than that in s.c. preadipocytes. The effects of MCM on adipocyte differentiation, lipid metabolism and insulin signaling transdution were verified. MCM induced the apoptosis of s.c. preadipocytes but not of s.c. adipocytes. Moreover, MCM inhibited adipocyte differentiation at pre-differentiation and early stages of differentiation, while the expression levels of INSR and IGF-1R were increased. Furthermore, MCM treatment increased adipocyte lipolysis and fatty acid oxidation through induction of genes involved in lipolysis, thermogenesis, and fatty acid oxidation in mitochondria. Consistent with the above, treatment of s.c. adipocytes with MCM upregulated mitochondrial biogenesis. Taken together, MCM can approximate the muscle micro-environment and reduce intramuscular adipocyte differentiation and lipid accumulation via regulating insulin signaling.

Systemic analysis of different colorectal cancer cell lines and TCGA datasets identified IGF-1R/EGFR-PPAR-CASPASE axis as important indicator for radiotherapy sensitivity

Insulin-like growth factor 1 receptor (IGF-1R) is proved to contribute the development of many types of cancers. But, little is known about its roles in radio-resistance of colorectal cancer (CRC). Here, we demonstrated that low IGF-1R expression value was associated with the better radiotherapy sensitivity of CRC. Besides, through Quantitative Real-time PCR (qRT-PCR), the elevated expression value of epidermal growth factor receptor (EGFR) was observed in CRC cell lines (HT29, RKO) with high radio-sensitivity compared with those with low sensitivity (SW480, LOVO). The irradiation induced apoptosis rates of wild type and EGFR agonist (EGF) or IGF-1R inhibitor (NVP-ADW742) treated HT29 and SW480 cells were quantified by flow cytometry. As a result, the apoptosis rate of EGF and NVP-ADW742 treated HT29 cells was significantly higher than that of those wild type ones, which indicated that high EGFR and low IGF-1R expression level in CRC was associated with the high sensitivity to radiotherapy. We next conducted systemic bioinformatics analysis of genome-wide expression profiles of CRC samples from the Cancer Genome Atlas (TCGA). Differential expression analysis between IGF-1R and EGFR abnormal CRC samples, i.e. CRC samples with higher IGF-1R and lower EGFR expression levels based on their median expression values, and the rest of CRC samples identified potential genes contribute to radiotherapy sensitivity. Functional enrichment of analysis of those differential expression genes (DEGs) in the Database for Annotation, Visualization and Integrated Discovery (DAVID) indicated PPAR signaling pathway as an important pathway for the radio-resistance of CRC. Our study identified the potential biomarkers for the rational selection of radiotherapy for CRC patients.

Abstract 5739: Nicotine promotes stemness-related properties and cell migration/metastasis through IGF-1R regulation in triple negative breast cancer

Abstract Purpose: To determine the role of insulin-like growth factor (IGF)-I receptor (IGF-IR) in alpha 9 nicotinic acetylcholine receptor (α9-nAChR)-induced stemness-related properties and to assess the therapeutic potential for preventing early tumor relapse and metastasis of TNBCs. Experimental Design: Expression levels of stemness-related genes, α9-nAChR and IGF-IR in tissues (n = 67) were analyzed by real-time Q-PCR and the correlation of different gene levels were calculated by the Pearson correlation analysis. The effects of either α9-nAChR activation or IGF-1R transactivation on stemness expression in TNBC were examined using ALDEFLUOR assay with flow cytometry, RNA interference, wound closure/migration/invasion assay, and animal models. The α9-nAChR, OCT4 and IGF-1R protein, p-IGF-1R in tissues were detected by immunohistochemical staining. Results: A high positive correlation between the gene expression levels of OCT4/NANOG/CD24/CD44 and α9-nAChR/IGF-IR in human breast cancer tissues was observed. Nicotine induced α9-nAChR activation trans-activates an IGF-IR expression and a phosphorylation of STAT3, which stimulated stemness-related properties in both the cell lines and the xenografted mouse tumors. The inhibition of either α9-nAChR or IGF-IR activation by RNA interference significantly suppressed the nicotine-induced stemness-related properties and cell metastasis both in vitro and in vivo. Conclusions: IGF-1R signal regulates the nicotine/α9-nAChR-induced stemness properties and cell metastasis therefore convene poor prognosis in TNBCs. Citation Format: Yung-Che Kuo, Jan-Show Chu, Kha-Liang Lee, Victor James Drew, Wei-Zhan Zhuang, Chi-Long Chen, Yuan-Soon Ho, Yen-Hua Huang. Nicotine promotes stemness-related properties and cell migration/metastasis through IGF-1R regulation in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5739. doi:10.1158/1538-7445.AM2017-5739

Abstract 2131: Tanshinone IIA can decrease growth factor receptors expression and dural-block both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways to inhibit human breast cancer BT-20 cells

Abstract Background : Tanshinone IIA (Tan-IIA, C19H18O3) with anti-inflammatory activities and antioxidant properties, is one of the diterpine quinones extracted from Salviae miltiorrhizae radix (Danshen). Tan-IIA can inhibit many human cancer cell lines through different molecular mechanisms. The phosphoinositide-3-kinase (PI3K)/AKT/ mammalian target of rapamycin (mTOR) and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer. Transmembrane tyrosine kinase has been strongly implicated in the proliferation, survival, and metastasis of human tumors. Both pathways represent important signal transduction mechanisms that facilitate the proliferation and survival of cancers driven by growth factor receptors, such as vascular endothelial growth factor receptor (VEGFR), insulin-like growth factor-I receptor (IGF-IR), or epidermal growth factor receptor (EGFR). Targeting both the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR Pathways for suppressing inhibitor resistant cells is necessary because the individual downstream components of these signaling cascades either through epigenetic modification or somatic mutation are also known to be frequently altered in cancer, thus contributing to resistance to anticancer therapies and tumorigenesis. Material and methods: In the present study, the human breast cancer BT-20 cells were treated with Tan-IIA in vitro. The cytotoxicity of Tan-IIA was evaluated by MTT assay. The effects of Tan-IIA on the protein expressions of EGFR, IGF-IR, VEGFR, PI3K, AKT, mTOR, Ras, Raf, MEK and ERK and β-actin in the BT-20 cells were examined by western blot analysis. Results: The results showed that Tan-IIA can induce the proliferation inhibition with time and dose dependent and inhibit the activity of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. In addition, it was showed that Tan-IIA treatment inhibited the protein expression levels of EGFR, VEGFR and IGF-IR significantly. Conclusions: These findings indicated that one of the molecular mechanisms for Tan-IIA to inhibit BT-20 cells maybe through inhibiting the protein expression levels of EGFR, VEGFR, IGF-IR and both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways. The use of Tan-IIA for breast cancer may become a feasible novel therapy option. Further studies are warranted to elucidate its mechanisms fully. Citation Format: Chin Cheng Su. Tanshinone IIA can decrease growth factor receptors expression and dural-block both Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR pathways to inhibit human breast cancer BT-20 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2017-2131

MicroRNA-503 suppresses cell proliferation and invasion in osteosarcoma via targeting insulin-like growth factor 1 receptor.

MicroRNAs (miRs) are a class of small non-coding RNAs and have key roles in various cancer types. Recently, miR-503 has been reported to act as a tumor suppressor in osteosarcoma. However, the detailed mechanism of the regulatory role of miR-503 in osteosarcoma cell proliferation and invasion has largely remained elusive. The present study found that miR-503 was significantly downregulated in osteosarcoma tissues compared to that in matched adjacent non-tumorous tissues. In addition, the expression of miR-503 in osteosarcoma of T3-T4 stage was significantly lower when compared with that in T1-T2 stage samples. miR-503 was also downregulated in osteosarcoma cell lines (Saos-2, MG63, U2OS and SW1353), when compared with that in the normal osteoblast cell line hFOB. Overexpression of miR-503 significantly inhibited the proliferation and invasion of U2OS cells and decreased the protein levels of insulin-like growth factor 1 receptor (IGF-1R), which was further identified as a novel target of miR-503 by a luciferase reporter assay. Moreover, overexpression of IGF-1R eliminated the suppressive effects of miR-503 on the proliferation and invasion of U2OS cells, suggesting that miR-503 inhibits osteosarcoma cell proliferation and invasion by directly targeting IGF-1R. Furthermore, IGF-1R was significantly upregulated in osteosarcoma tissues compared with that in adjacent non-tumor tissues, as well as in osteosarcoma cell lines compared with that in hFOB cells. In addition, the expression levels of IGF-1R were inversely correlated to the miR-503 levels in osteosarcoma tissues, suggesting that the increased IGF-1R expression may be caused by the reduced expression of miR-503. In conclusion, the present study demonstrated that miR-503 suppresses cell proliferation and invasion in osteosarcoma via targeting IGF-1R and thus highlights the importance of miR-503/IGF-1R signaling in the malignant progression of osteosarcoma.

Clinicopathological significance and impact on outcomes of the gene expression levels of IGF-1, IGF-2 and IGF-1R, IGFBP-3 in patients with colorectal cancer: Overexpression of the IGFBP-3 gene is an effective predictor of outcomes in patients with colorectal cancer.

The insulin-like growth factors (IGF) system is involved in tumor proliferation, invasion and metastasis in cancer. The current study investigated the association of IGF-1, IGF-2 and IGF-1 receptor (IGF-1R), IGF binding proteins type 3 (IGFBP-3) mRNA expression levels with clinicopathological characteristics and outcomes of 202 patients with untreated colorectal cancer (CRC). IGF-1, IGF-2, IGF-1R and IGFBP-3 mRNA expression levels were analyzed in surgical specimens of cancer tissues and adjacent normal mucosa cells using reverse transcription-quantitative polymerase chain reaction. The IGF-1R gene expression level was significantly higher in cancer tissue compared with adjacent normal mucosa. By contrast, IGF-1 gene expression levels were reduced in cancer tissue compared with normal mucosa. IGF-2 and IGFBP-3 gene expression levels did not differ significantly between cancer tissue and adjacent normal mucosa. As for the association of gene expression and clinicopathological characteristics, IGFBP-3 gene expression was significantly associated with lymph node metastasis. High IGFBP-3 gene expression was associated with poor 5-year overall survival compared with patients with low IGFBP-3 expression. Furthermore, IGFBP-3 gene expression was identified as an independent prognostic factor using multivariate analysis. Overexpression of the IGFBP-3 gene is considered an effective independent predictor of outcomes in patients with CRC.

Expression characteristics of proteins of IGF-1R, p-Akt, and survivin in papillary thyroid carcinoma patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is related to increased risk of papillary thyroid carcinoma (PTC). Insulin-like growth factor-1 receptor (IGF-1R) is increased in patients with T2DM. The increased IGF-1R may be responsible for the development of PTC. In this study, we investigated the expression of phosphorylation of Akt (p-Akt)/survivin pathway activated by IGF-1R in PTC subjects with and without diabetes.Clinicopathological data of 20 PTC patients with T2DM were retrospectively analyzed and compared with those of 21 PTC subjects without diabetes. Meanwhile, IGF-1R, p-Akt, and survivin expressions of PTC tissues were detected by immunohistochemical staining.The immunohistochemical results found that the expression level of IGF-1R was significantly higher in diabetic PTC patients than that in nondiabetic PTC patients (P < 0.05). However, no significant differences of p-Akt and survivin expression were found between PTC patients with T2DM and PTC patients without T2DM. In addition, among 20 PTC patients with T2DM, subgroup analysis showed that the ratio of tumor size >10 mm was significantly higher in IGF-1R moderate to strong expression group than that in IGF-1R negative to weak expression group (P < 0.05).IGF-1R expression level was higher in PTC patients with T2DM, and the increased IGF-1R expression was associated with lager tumor size. IGF-1R may play an important role in carcinogenesis and tumor growth in PTC patients with T2DM.

Embryonic-only arsenic exposure in killifish (Fundulus heteroclitus) reduces growth and alters muscle IGF levels one year later

Arsenic is a contaminant of drinking water and crops in many parts of the world. Epidemiological studies have shown that arsenic exposure is linked to decreased birth weight, weight gain, and proper skeletal muscle function. The goal of this study was to use killifish (Fundulus heteroclitus) as a model to determine the long-term effects of embryonic-only arsenic exposure on muscle growth and the insulin-like growth factor (IGF) pathway. Killifish embryos were exposed to 0, 50, 200 or 800ppb AsIII from fertilization until hatching. Juvenile fish were reared in clean water and muscle samples were collected at 16, 28, 40 and 52 weeks of age. There were significant reductions in condition factors, ranging from 12 to 17%, in the fish exposed to arsenic at 16, 28 and 40 weeks of age. However, by 52 weeks, no significant changes in condition factors were seen. Alterations in IGF-1R and IGF-1 levels were assessed as a potential mechanism by which growth was reduced. While there no changes in hepatic IGF-1 transcripts, skeletal muscle cells can also produce their own IGF-1 and/or alter IGF-1 receptor levels to help enhance growth. After a 200 and 800ppb embryonic exposure, fish grown in clean water for 16 weeks had IGF-1R transcripts that were 2.8-fold and 2-fold greater, respectively, than unexposed fish. Through 40 weeks of age, IGF1-R remained elevated in the 200ppb and 800ppb embryonic exposure groups by 1.8–3.9-fold, while at 52 weeks of age, IGF-1R levels were still significantly increased in the 800ppb exposure group. Skeletal muscle IGF-1 transcripts were also significantly increased by 1.9–5.1 fold through the 52 weeks of grow-out in clean by water in the 800ppb embryonic exposure group. Based on these results, embryonic arsenic exposure has long-term effects in that it reduces growth and increases both IGF-1 and IGF-1R levels in skeletal muscle even 1year after the exposure has ended.

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 IMP3 as a marker for predicting poor outcome in gastroenteropancreatic neuroendocrine neoplasms.

The aim of the present study was to investigate the expression and clinical significance of oncofetal protein insulin-like growth factor (IGF) II mRNA-binding protein 3 (IMP3) in the differentiation of gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). A total of 162 patients who were diagnosed with GEP-NEN, and who underwent surgical or endoscopic resection from January 2006 to March 2013, were enrolled in the study, including 85 cases of grade (G)1 neuroendocrine tumors, 40 cases of G2 neuroendocrine tumors, 28 cases of G3 neuroendocrine carcinomas and 9 cases of mixed stage adenoneuroendocrine carcinomas. The clinical and pathological data were recorded for analysis. The expression of IMP3, cluster of differentiation (CD)44, IGF1 receptor (IGF1R) and matrix metalloproteinase (MMP)2 was determined by immunohistochemistry. SPSS 13.0 software was used for data processing and analyses, and P<0.05 was used to determine significance. Oncofetal protein IMP3 exhibited a high expression rate (74.69%) in GEP-NEN. IMP3-positive cases demonstrated significantly decreased overall and disease-free survival times, as compared with IMP3-negative cases (P=0.012). Overexpression of IMP3 was correlated with tumor grade, clinical stage, tumor size and poor prognosis (all P<0.05). Therefore, patients with overexpressed IMP3 had a poorer prognosis (P<0.01); COX regression analysis revealed that the overexpression of IMP3, the tumor grade, tumor size and metastasis of GEP-NEN were each associated with the clinical outcomes. The results also indicated that the expression rates of CD44, IGF1R and MMP2 in GEP-NEN were 19.75, 53.7 and 55.56%, respectively. While it was negatively associated with the expression of CD44 (r=-0.131; P=0.096), the expression of IMP3 was positively correlated with the expression of IGF1R and MMP2 (r=0.288, P<0.01; r=0.208, P=0.008). In addition, the expression levels of IGF1R and MMP2 were positively associated (r=0.687; P<0.01). In conclusion, high IMP3 expression levels were determined to be associated with a high disease stage in patients with GEP-NEN, thus it may serve as a predictor for metastasis and poor clinical outcomes in GEP-NEN.