Astrocyte Elevated Gene-1 siRNA Research Articles

AEG-1 as a Novel Therapeutic Target in Colon Cancer: A Study from Silencing AEG-1 in BALB/c Mice to Large Data Analysis.

Astrocyte elevated gene-1 (AEG-1) is overexpressed in various malignancies. Exostosin-1 (EXT-1), a tumor suppressor, is an intermediate for malignant tumors. Understanding the mechanism behind the interaction between AEG-1 and EXT-1 may provide insights into colon cancer metastasis. AOM/DSS was used to induce tumor in BALB/c mice. Using an in vivo-jetPEI transfection reagent, transient transfection of AEG-1 and EXT-1 siRNAs were achieved. Histological scoring, immunohistochemical staining, and gene expression studies were performed from excised tissues. Data from the Cancer Genomic Atlas and GEO databases were obtained to identify the expression status of AEG-1 and itsassociation with the survival. In BALB/c mice, the AOM+DSS treated mice developed necrotic, inflammatory and dysplastic changes in the colon with definite clinical symptoms such as loss of goblet cells, colon shortening, and collagen deposition. Administration of AEG-1 siRNA resulted in a substantial decrease in the disease activity index. Mice treated with EXT-1 siRNA showed diffusely reduced goblet cells. In vivo investigations revealed that PTCH-1 activity was influenced by upstream gene AEG-1, which in turn may affect EXT-1 activity. Data from The Cancer Genomic Atlas and GEO databases confirmed the upregulation of AEG-1 and downregulation of EXT-1 in cancer patients. This study revealed that AEG-1 silencing might alter EXT-1 expression indirectly through PTCH-1, influencing cell-ECM interactions, and decreasing dysplastic changes, proliferation and invasion.

Inhibition of osteosarcoma growth and metastasis using a polysaccharide derivative of Amy-g-PLLD for the delivery of AEG-1 siRNA

Osteosarcoma is the most common primary malignant neoplasm of the bone in children and adolescents and has a high risk of relapse and metastasis. Of the various methods to treat osteosarcoma, the use of genetic approaches to inhibit the rapid growth of osteosarcoma while limiting tumor metastasis has presented a challenge in its implementation. Here, we successfully synthesized a polysaccharide derivative (Amy-g-PLLD) for delivery of astrocyte elevated gene-1 (AEG-1) small-interfering RNA (siRNA) (siAEG-1), and used it for the first time to suppress osteosarcoma tumors in vitro and in vivo. Amy-g-PLLD/ siAEG-1 complexes were delivered into 143B human osteosarcoma cells with low resultant cytotoxicity. Osteosarcoma tumor proliferation and invasion were inhibited in vitro. Intratumoral injection of Amy-g-PLLD/siAEG-1 complexes markedly inhibited tumor growth and lung metastasis in 143B tumor-bearing mice. This biocompatible and effective approach employing a natural material-siRNA complex should pave the way for more genetic research in treating osteosarcoma.

Astrocyte elevated gene-1 participates in the production of pro-inflammatory cytokines in dental pulp cells via NF-κB signalling pathway.

To identify the role of astrocyte elevated gene-1 (AEG-1) and the mechanism underlying the inflammatory response in dental pulp cells. Astrocyte elevated gene-1 expression was detected at different stages of pulpitis in a rat model using immunohistochemistry. RT-qPCR and Western blot were used to assess AEG-1 expression in human dental pulp cells stimulated by lipopolysaccharide (LPS). The LPS-induced expression of inflammatory cytokine genes was quantified by RT-qPCR in cells transfected with AEG-1 or negative control (NC) siRNA. Immunofluorescence and Western blot were utilized to evaluate the activation of NF-κB signalling in AEG-1 knockdown cells. AEG-1 expression was also investigated by Western blot in dental pulp cells pre-treated with inhibitors of NF-κB and PI-3K signalling before the addition of LPS. Data were analysed statistically using one-way anova. The exposure of dental pulp tissue to LPS resulted in acute inflammation with necrosis and AEG-1 expression in the pulp tissue beneath the perforation. In LPS-stimulated dental pulp cells, AEG-1 mRNA and protein were significantly up-regulated (P<0 .05) in a time- and dose-dependent manner. In AEG-1 knockdown cells, the synthesis of IL-1, IL-6 and TNF-α mRNA was suppressed significantly (P<0.05) upon LPS induction. AEG-1 knockdown also inhibited nuclear translocation of p65. Suppression of NF-κB and PI-3K abrogated the LPS-induced up-regulation of AEG-1. Astrocyte elevated gene-1 participated in inflammatory cytokine synthesis via NF-κB signalling in the dental pulp. Both NF-κB and PI-3K signalling are involved in LPS-induced AEG-1 expression in dental pulp cells.

Nanoscale polysaccharide derivative as an AEG-1 siRNA carrier for effective osteosarcoma therapy.

BackgroundNanomedicine, which is the application of nanotechnology in medicine to make medical diagnosis and treatment more accurate, has great potential for precision medicine. Despite some improvements in nanomedicine, the lack of efficient and low-toxic vectors remains a major obstacle.ObjectiveThe aim of this study was to prepare an efficient and low-toxic vector which could deliver astrocyte elevated gene-1 (AEG-1) small interfering RNA (siRNA; siAEG-1) into osteosarcoma cells effectively and silence the targeted gene both in vitro and in vivo.Materials and methodsWe prepared a novel polysaccharide derivative by click conjugation of azidized chitosan with propargyl focal point poly (L-lysine) dendrons (PLLD) and subsequent coupling with folic acid (FA; Cs-g-PLLD-FA). We confirmed the complexation of siAEG-1and Cs-g-PLLD or Cs-g-PLLD-FA by gel retardation assay. We examined the cell cytotoxicity, cell uptake, cell proliferation and invasion abilities of Cs-g-PLLD-FA/siAEG-1 in osteosarcoma cells. In osteosarcoma 143B cells tumor-bearing mice models, we established the therapeutic efficacy and safety of Cs-g-PLLD-FA/siAEG-1.ResultsCs-g-PLLD-FA could completely encapsulate siAEG-1 and showed low cytotoxicity in osteosarcoma cells and tumour-bearing mice. The Cs-g-PLLD-FA/siAEG-1 nanocomplexes were capable of transferring siAEG-1 into osteosarcoma cells efficiently, and the knockdown of AEG-1 resulted in the inhibition of tumour cell proliferation and invasion. In addition, caudal vein injecting of Cs-g-PLLD-FA/siAEG-1 complexes inhibited tumor growth and lung metastasis in tumor-bearing mice by silencing AEG-1 and regulating MMP-2/9.ConclusionIn summary, Cs-g-PLLD-FA nanoparticles are a promising system for the effective delivery of AEG-1 siRNA for treating osteosarcoma.

AEG-1 mRNA expression in non-small cell lung cancer is associated with increased tumor angiogenesis

Astrocyte-elevated gene-1 (AEG-1) is implicated in the oncogenesis and angiogenesis of various types of human malignant disease. However, the angiogenesis roles of AEG-1 in non-small cell lung cancer (NSCLC) remain to be further elucidated. In the present study, the expression level of AEG-1 mRNA in seven human lung cell lines and 89 paired tissue samples (tumor tissues (TTs) and pair-matched normal adjacent tissues (PMNATs)) from NSCLC patients was detected by real-time PCR. Staining of vascular endothelial growth factor (VEGF) and intratumoral microvessel density (iMVD, labeled by CD105) were assessed by immunohistochemistry. Furthermore, cell migration and invasion were evaluated by wound healing assay and transwell assays. AEG-1 mRNA level was significantly higher in human lung cancer cells and TTs than that in human normal bronchial epithelial cell line 16HBE and PMNATs, respectively (P<0.001). Higher AEG-1 mRNA level in patients with NSCLC was correlated with clinical stages (P=0.028), differentiation (P=0.042), and lymph node metastasis (P=0.004). Moreover, Upregulated AEG-1 mRNA expression level was associated with higher tumor angiogenesis, reflected by the increase of VEGF expression and iMVD counting (P=0.021, P<0.001). However, 95D cell line transfected with AEG-1 siRNA oligos (siAEG-1) exhibited no significant decrease of cell invasion or migration capacities when compared with the control cells (P>0.05).These results suggested that AEG-1 may play important roles at the transcription level in malignant transformation and tumor angiogenesis in NSCLC, and anti-AEG-1 mRNA expression may be a novel potential strategy for anti-angiogenic therapy of NSCLC.

Astrocyte-elevated gene-1 confers resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase expression.

Previous studies have suggested that astrocyte-elevated gene-1 (AEG-1) contributes to the mechanisms of resistance to various chemotherapeutics. In this study, we investigated whether AEG-1 expression level correlated with that of thymidylate synthase (TS), as higher TS expression is known to be associated with the resistance to pemetrexed chemotherapy in patients with advanced lung adenocarcinoma. Using pemetrexed-resistant lung adenocarcinoma PC-9 cell line, we demonstrated that transfection of AEG-1 siRNA lowered TS expression and decreased pemetrexed IC50 value. In contrast, overexpression of AEG-1 was associated with increased expression of TS and higher pemetrexed IC50 value. Immunohistochemical staining of clinical biopsy samples showed that patients with lower AEG-1 expression had longer overall survival time. Moreover, analysis of repeated biopsy samples revealed that an increase in the TS level from baseline to disease progression was significantly associated with the elevation of AEG-1 expression. In conclusion, our data demonstrated that TS expression might be regulated by AEG-1 and that increased expression of these proteins contributes to lung cancer disease progression and may be associated with the development of resistance to pemetrexed.

Knockdown of astrocyte elevated gene-1 inhibited cell growth and induced apoptosis and suppressed invasion in ovarian cancer cells

Emerging evidence has demonstrated that AEG-1 (astrocyte elevated gene-1) plays a pivotal oncogenic role in tumorigenesis. However, the molecular mechanism by which AEG-1 exerts its oncogenic function is elusive in ovarian cancer. To explore the role and molecular insight on AEG-1-mediated tumorigenesis in ovarian cancer, multiple approaches are performed including MTT assay, flow cytometry for apoptosis and cell cycle assay, gene transfection, real-time RT-PCR, Western blotting, and Transwell assay. Our MTT assay showed that knockdown of AEG-1 by its siRNA significantly inhibited cell growth in ovarian cancer cells. Moreover, AEG-1 siRNA treatment induced G0/G1 cell cycle arrest and triggered cell apoptosis in ovarian cancer cells. Notably, inhibition of AEG-1 suppressed cell migration and invasion in ovarian cancer cells. Intriguingly, we identified that knockdown of AEG-1 remarkably inhibited the activation of Akt pathway. Our results also validated that knockdown of AEG-1 inhibited the expression of MMP-2 and VEGF, which could lead to inhibition of cell migration and invasion. These data suggest that AEG-1 could be a potential therapeutic target for the treatment of ovarian cancer.

Mechanisms of astrocyte elevated gene-1 promoting cancer metastasis through regulation of the micro-RNA-885-5p/ matrix metalloproteinase 9 signaling pathway in liver cancer

Objective To investigate the relationship between astrocyte elevated gene-1 (AEG-1) and microRNA-885-5p, observe the influence of microRNA-885-5p on MHCC-97H migration and invasion, identify the target gene of microRNA-885-5p and uncover the mechanisms of AEG-1 promoting cancer metastasis. Methods The experimental study was adopted. The AEG-1 over-expressed plasmid constructed and the siRNA of silent AEG-1 synthesized were transiently transfected into MHCC-97H cells, respectively. For plasmid trans-fection, MHCC-97H cells tranfected with empty Vector NC were divided into group A as the control, and MHCC-97H cells tranfected with AEG-1 over-expressed plasmid were divided into group B. For AEG-1 siRNA transfection, MHCC-97H cells tranfected with negative siRNA were divided into group C as the control, and MHCC-97H cells tranfected with AEG-1 siRNA were divided into group D. The relative expressions of microRNA-885-5p in MHCC-97H cells of group A, B, C, D were measured by fluorescent quantitative polymerase chain reaction(PCR). For microRNA-885-5p simulator transfection, MHCC-97H cells tranfected with negative microRNA simulator were divided into group E as the control, and tranfected with microRNA-885-5p simulator were divided into group F. Transwell assay was used to evaluate MHCC-97H migration and invasion. Targetscan software was used to predict the target gene of microRNA-885-5p. MHCC-97H cells co-transfected with reporter plasmid of target gene and negative control siRNA were divided into group G as the control, and MHCC-97H cells co-transfected with reporter plasmid of target gene and microRNA-885-5p simulator were divided into group H. MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and negative control siRNA were divided into group I as the control, MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and microRNA-885-5p simulator were divided into group J. The luciferase activities of MHCC-97H cells in group G, H, I, J were tested. MHCC-97H cells transfected with negative control siRNA were divided into group K, and tranfected with microRNA-885-5p simulator were divided into group L. Western blot test was used to detect the relative expression of target gene of microRNA-885-5p. MHCC-97H cells transfected with siRNA of target gene of microRNA-885-5p were divided into group M, and transfected with negative control siRNA were divided into group N. Transwell assay was used to evaluate MHCC-97H migration and invasion in group M and group N. Measurement data with normal distribution were presented as ±s and comparison between groups was done using the t test. Results The relative expressions of microRNA-885-5p in MHCC-97H cells of group A and group B were (10.68±1.32)×10-4 and (5.02± 0.20)×10-4, respectively, showing significant difference between the 2 groups (t=7.357, P 0.05) . The relative expressions of MMP9 in MHCC-97H cells of group K and group L were 0.75±0.03 and 0.25±0.03, respectively, showing significant difference between the 2 groups (t=19.086, P<0.05). The results of MHCC-97H migration and invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate were 1 210±163 and 1 192±170 in group M, 537±16 and 374±55 in group N, showing significant differences between the 2 groups (t=7.111, 7.916, P<0.05). Conclusions AEG-1 downregulates the expression of microRNA-885-5p, the target gene of which is MMP9. MicroRNA-885-5p inhabits the migration and invasion of hepatoma cells by negative regulation of MMP9. Key words: Liver neoplasms; Metastases; Astrocyte elevated gene-1; MicroRNA-885-5p; Matrix metalloproteinase 9

AEG-1 participates in high glucose-induced activation of Rho kinase and epithelial-mesenchymal transition in proximal tubular epithelial cells.

To prove whether astrocyte elevated gene-1 (AEG-1) plays a role in high glucose-stimulated Rho kinase activation and epithelial-mesenchymal transition (EMT) in human renal tubular epithelial (HK-2) cells. The protein levels of AEG-1, alpha-smooth muscle actin, E-cadherin and MYPT1 were determined by Western blot. AEG-1 protein level was upregulated in HK-2 cells stimulated with high glucose. AEG-1 siRNA downregulated Rho kinase protein expression and blocked high glucose-induced EMT. Our results show that AEG-1 acts a key role in high glucose-induced activation of Rho kinase and EMT in HK-2 cells.

Abstract 5400: A novel combinatorial therapy for hepatocellular carcinoma (HCC)

Abstract The incidence of hepatocellular carcinoma (HCC) is rising in the US with parallel increase in mortality rate. Lack of effective therapy for advanced HCC mandates development of novel targeted therapies to counteract this fatal malady. Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC, plays a pivotal role in hepatocarcinogenesis and serves as an ideal target for anti-HCC therapy. AEG-1 interacts with retinoid X receptor (RXR) and inhibits retinoic acid-induced gene expression and cell death. Retinoic acid has been evaluated for HCC treatment without promising result. Overexpression of AEG-1 might underlie the poor performance of retinoic acid in HCC clinical trials. We documented that combination of a lentivirus expressing AEG-1 shRNA (lenti.shAEG-1) and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We now have developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). PAMAM has a net positive charge that complexes with the net negative charge of the siRNA backbone, PEG increases stability of the nanoplexes and increases circulation time and the galactose binds to asialoglycoprotein receptors that are upregulated on liver cells. The polymer conjugate was characterized by 1H-NMR and optimal nanoplex formulations were characterized for surface charge and size using a zetasizer Nano ZS. We established orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) in the livers of athymic nude mice and monitored tumor development by bioluminescence imaging (BLI). One week after tumor establishment mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si and PAMAM-AEG-1si+ATRA by 8 i.v. injections over 4 weeks, and sacrificed 2 weeks after the last injection. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. Measurement of liver weight at the end of the experiment corroborated these findings. Analysis of AEG-1 mRNA in tumor samples showed a marked decrease in AEG-1 level by PAMAM-AEG-1si indicating efficacy of in vivo knockdown. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anti-cancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC and need to be evaluated stringently in endogenous mouse models of HCC for a potential Phase I/II clinical trial. Citation Format: Jyoti Srivastava, Devaraja Rajasekaran, Ayesha Siddiq, Rachel Gredler, Chadia L. Robertson, Maaged A. Akiel, Xue-Ning Shen, Kareem A.N. Ebeid, Aliasger K. Salem, Paul B. Fisher, Devanand Sarkar. A novel combinatorial therapy for hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2015-5400

Combination of Nanoparticle-Delivered siRNA for Astrocyte Elevated Gene-1 (AEG-1) and All-trans Retinoic Acid (ATRA): An Effective Therapeutic Strategy for Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a fatal cancer with no effective therapy. Astrocyte elevated gene-1 (AEG-1) plays a pivotal role in hepatocarcinogenesis and inhibits retinoic acid-induced gene expression and cell death. The combination of a lentivirus expressing AEG-1 shRNA and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We have now developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (Gal) (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). The polymer conjugate was characterized by (1)H-NMR, MALDI, and mass spectrometry; and optimal nanoplex formulations were characterized for surface charge, size, and morphology. Orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) were established in the livers of athymic nude mice and tumor development was monitored by bioluminescence imaging (BLI). Tumor-bearing mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si, and PAMAM-AEG-1si+ATRA. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth, and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. A marked decrease in AEG-1 level was observed in tumor samples treated with PAMAM-AEG-1si. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation, and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anticancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC.

Effect of down-regulation of astrocyte elevated gene-1 expression by small interfering RNA inhibits cell proliferation and induces apoptosis in human hepatocellular carcinoma HepG2 cells

Objective To investigate the effect of small interfering RNA (siRNA)-induced astro-cyte elevated gene-1 (AEG-1) down-regulation on the proliferation,apoptosis and cell cycle of human hep-atocellular carcinoma cells and its mechanism.Methods siRNA and AEG-1 siRNA were transfected to Human hepatocellular carcinoma HepG2 cells,respectively.The expression of AEG-1 mRNA was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR).The cell proliferation was detected by methyl thiazol tetrazolium (MTT) method.The apoptosis and cell cycle were analyzed by flow cytometry.Results Expression of AEG-1 mRNA in AEG-1 siRNA group (0.43 ±0.04)was significantly lower than that in control siRNA group (0.94 ± 0.08) and control group (0.96 ± 0.09)(P ＜0.01),respectively.After treated with AEG-1 siRNA for 72 h,the absorbance values of AEG-1 siRNA group (1.25 ± 0.08) was significantly lower than that in control siRNA group (2.49 ± 0.14) and control group (2.64 ± 0.14) (P ＜ 0.01),respectively.The cell rate in G0/G1 phase (61.38 ± 2.31) ％ was significantly higher than that in control siRNA group (42.15 ± 1.64)％ and control group (41.24 ± 1.97)％(P ＜ 0.01),and the cell rates in S phase (25.36 ± 1.21) ％,G2/M phase (13.26 ± 0.97) ％ were lower than those in control siRNA group (32.52 ± 1.23) ％,(25.33 ± 0.86) ％ and control group (33.60 ±1.24) ％,(25.16 ± 0.79) ％ (P ＜ 0.01),respectively.Conclusion The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in human hepatocellular carcinoma cells.Knockdown of AEG-1 expres-sion in human hepatocellular carcinoma cells significantly inhibits cell proliferation and apoptosis,and induces cell arrest in G0/G1 phase of the cell cycle. Key words: Astrocyte elevated gene-1 ; Cancer, hepatocellular; Small interfering RNA ; Apoptotic

Expression of astrocyte elevated gene-1 closely correlates with the angiogenesis of gastric cancer.

Previous studies have demonstrated that astrocyte elevated gene-1 (AEG-1) is overexpressed in several cancer types and that its upregulation may promote cell proliferation, cell transformation and tumor progression. The present study investigated the expression and prognostic value of AEG-1 in primary gastric cancer (GC) as well as its role in angiogenesis. The results obtained from real-time reverse transcription polymerase chain reaction and western blotting revealed the upregulation of AEG-1 mRNA (P=0.007) and protein expression (P<0.001) in the majority of cancerous tissues compared with matched adjacent non-cancerous gastric tissues. To further investigate the clinicopathological and prognostic roles of AEG-1, immunohistochemical analysis of 216 GC tissue blocks was performed. The results showed that high AEG-1 expression closely correlated with differentiation degree (P<0.001 ), T stage (P<0.001), N stage (P=0.003) and M stage (P=0.013). Consistent with the abovementioned results, AEG-1 upregulation was also found to significantly correlate with poor survival in GC patients (P<0.001). Furthermore, carcinomas with elevated AEG-1 expression demonstrated high vascular endothelial growth factor (VEGF) expression and microvessel density, which was labeled by cluster of differentiation 34. In addition, an AEG-1 siRNA assay in MGC-803 cells showed that the AEG-1 gene may promote VEGF and hypoxia-inducible factor-1α protein and mRNA expression. The results of the current study indicated that AEG-1 may serve as a valuable prognostic marker for GC and may be involved in regulating tumor angiogenesis.

AEG-1 expression characteristics in human non-small cell lung cancer and its relationship with apoptosis

Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, has been shown to promote cell growth and inhibit apoptosis, but the underlying molecular mechanisms and its functional significance in non-small cell lung cancer (NSCLC) remain to be elucidated. In the present study, statistical analysis displayed a significant correlation of AEG-1 expression with clinical staging (P = 0.048), differentiation (P = 0.019) and lymph node metastasis (P = 0.032). Simultaneously, the overall survival time in patients with higher AEG-1 expression was obviously shorter than that in patients with lower expression of AEG-1 (P < 0.001). Furthermore, we found that AEG-1 could inhibit apoptotic cell death in L-78 cells, as assessed by MTT, TUNEL and flow cytometry assay. After treating L-78 cells with AEG-1 siRNA, caspase-3 protein was significantly up-regulated and Bcl-2 protein was markedly decreased in L-78 cells, which was verified by the immunohistochemistry results about AEG-1, caspase-3 and Bcl-2. Furthermore, PI3K p110 protein and phosphorylated Akt were also largely attenuated by the treatment of AEG-1 siRNA. In conclusion, our results indicated that AEG-1 played a crucial role in the carcinogenesis of NSCLC and could inhibit apoptosis via activating cell survival signaling (enhancing the level of anti-apoptotic protein Bcl-2 and the activation of PI3K/Akt pathway).

Effect of silencing AEG-1 with small interfering RNA on the proliferation and cell cycle of gastric carcinoma SGC-7901 cells

To explore the effect of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell proliferation and cell cycle of gastric carcinoma cells, and its possible molecular mechanism. Control siRNA and AEG-1 siRNA were transfected into gastric carcinoma SGC-7901 cells. 48 h after transfection, the cells were divided into 3 groups including untransfected, siRNA control and AEG-1 siRNA transfection groups. Expressions of AEG-1 mRNA and protein in the 3 group cells were detected by real-time quantitative PCR and Western blot. The changes of cell proliferation were examined using CCK-8 kit, and the cell cycle distribution was detected by flow cytometry. Finally, expressions of cell proliferation and cell cycle related proteins were detected by Western blot. Real-time quantitative PCR and Western blot demonstrated that compared with the untransfected and siRNA control groups, expressions of AEG-1 mRNA and protein were significantly down-regulated in the AEG-1 siRNA transfection group (P < 0.05), but there was no significant difference between the untransfected and siRNA control groups (P > 0.05). Furthermore, in vivo experiment confirmed a significant down-regulation of AEG-1 protein in the AEG-1 siRNA transfection group (P < 0.05). In addition, AEG-1 siRNA obviously inhibited the proliferation of SGC-7901 cells at different time points after transfection with AEG-1 siRNA. The percentage of cells in G0/G1 phase in the AEG-1 siRNA transfection group [(61.26 ± 1.25)%] was significantly higher than those in the untransfected group [(46.17 ± 1.91)%] and siRNA control group [(46.46 ± 1.96)%], and there was a significant difference between them (all P < 0.001). Furthermore, the result of Western blotting revealed that down-regulation of AEG-1 expression evoked the down-regulation of cdk2 and cyclin D1 expressions and elevation of p21 expression in vitro and in vivo. The inhibition of cell proliferation and cell cycle arrest mediated by down-regulation of AEG-1 expression may be closely associated with the changes of expression of cell cycle related proteins including cdk2, cyclin D1 and p21.

Abstract A83: Inhibition of hypoxia inducible factor alpha and astrocyte elevated gene-1 mediates cryptotanshionone induced anti-tumor activity in hypoxic PC-3 cells

Abstract Although cryptotanshinone (CT) from Salvia miltiorrhiza Bunge was known to exert antitumor activity in liver, breast and prostate cancers, its molecular mechanism under hypoxia still remain unclear. Thus, in the present study, the roles of astrocyte elevated gene 1 (AEG-1) and HIF-1α in crytotanshinone induced antitumor activity were investigated in PC-3 prostate cancer cells under hypoxia. Cryptotanshinone exerted cytotoxicity against prostate cancer cells and suppressed HIF-1α accumulation and transcriptional activity and AEG-1 expression in hypoxic PC-3 cells. Also, AGE-1 was overexpressed in prostate cancer cells and also upregulated along with HIF-1α in hypoxic PC-3 cells. Interestingly, HIF-1α siRNA transfection blocked AEG-1 expression and activated caspase 9/3 and PARP cleavage, while AEG-1 siRNA transfection did not affect HIF-1α activity in hypoxic PC-3 cells. Of note, HIF prolyl hydroxylase inhibitor, dimethyloxalylglycine enhanced the stability of AEG-1 and HIF-1α during hypoxia. In addition, CT also significantly reduced cellular levels of VEGF, a downstream of HIF-1α, and disturbed tube formation in human umbilical vein endothelial cells maintained in conditioned medium of hypoxic PC-3 cells. Consistently, ChIP assay revealed that CT inhibited the binding of HIF-1α to VEGF promoter. Furthermore, CT at 10 mg/kg suppressed the growth of PC-3 cells in BALB/c athymic nude mice by 46.4% compared to untreated control. Consistently, immunohistochemistry revealed decreased expression of Ki-67(proliferation), CD34 (blood vessel), VEGF (angiogenesis), carbonic anhydrase(CA)IX (hypoxic marker) and AEG-1 indices in CT-treated group compared to untreated control. Overall, our findings suggest that CT exerts antitumor activity via inhibition of HIF-1α, AEG1 and VEGF as a potent chemotherapeutic agent. Citation Format: Sung Hoon Kim, Hyojeong Lee, Hanna Hyun Kim, Deok Beom Jung, Eunjung Sohn. Inhibition of hypoxia inducible factor alpha and astrocyte elevated gene-1 mediates cryptotanshionone induced antitumor activity in hypoxic PC-3 cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A83.

Identification of genes conferring resistance to 5-fluorouracil

Astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) patients and plays a significant role in mediating aggressive progression of HCC. AEG-1 is known to augment invasion, metastasis, and angiogenesis, and we now demonstrate that AEG-1 directly contributes to another important hallmark of aggressive cancers, that is, resistance to chemotherapeutic drugs, such as 5-fluorouracil (5-FU). AEG-1 augments expression of the transcription factor LSF that regulates the expression of thymidylate synthase (TS), a target of 5-FU. In addition, AEG-1 enhances the expression of dihydropyrimidine dehydrogenase (DPYD) that catalyzes the initial and rate-limiting step in the catabolism of 5-FU. siRNA-mediated inhibition of AEG-1, LSF, or DPYD significantly increased the sensitivity of HCC cells to 5-FU in vitro and a lentivirus delivering AEG-1 siRNA in combination with 5-FU markedly inhibited growth of HCC cells xenotransplanted in athymic nude mice when compared to either agent alone. The present studies highlight 2 previously unidentified genes, AEG-1 and LSF, contributing to chemoresistance. Inhibition of AEG-1 might be exploited as a therapeutic strategy along with 5-FU-based combinatorial chemotherapy for HCC, a highly fatal cancer with currently very limited therapeutic options.

Astrocyte elevated gene-1 activates cell survival pathways through PI3K-Akt signaling

Astrocyte elevated gene-1 (AEG-1) displays oncogenic properties. Its expression is elevated in diverse neoplastic states and it cooperates with Ha-ras to promote cellular transformation. Overexpression of AEG-1 augments invasion and anchorage-independent growth of transformed cells, while AEG-1 siRNA inhibits Ha-ras-mediated colony formation, supporting a potential functional role in tumorigenesis. Additionally, oncogenic Ha-ras induces AEG-1 expression through the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In the present study, we investigated whether AEG-1 could induce serum-independent cell growth, another property of oncogenes. Overexpression of AEG-1 inhibited serum starvation-induced apoptosis through activation of PI3K-Akt signaling, one of the effector pathways induced by activated Ras. AEG-1 also affected the phosphorylation state of Akt substrates that are implicated in apoptosis suppression, including glycogen synthase kinase 3beta, c-Myc, murine double minute 2, p53, p21/mda-6 and Bad. Additionally, AEG-1 blocked the activity of serum starvation-induced caspases. Taken together, these observations provide evidence that AEG-1 is an oncogene cooperating with Ha-ras as well as functioning as a downstream target gene of Ha-ras and may perform a central role in Ha-ras-mediated carcinogenesis. Activation of survival pathways may be one mechanism by which AEG-1 exerts its oncogenic properties.