Cancer Research Annual Meeting 2010 Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Cancer Research 68, 1478, March 1, 2008. doi: 10.1158/0008-5472.CAN-07-6164
© 2008 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sarkar, D.
Right arrow Articles by Fisher, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sarkar, D.
Right arrow Articles by Fisher, P. B.

Cell, Tumor, and Stem Cell Biology

Molecular Basis of Nuclear Factor-{kappa}B Activation by Astrocyte Elevated Gene-1

Devanand Sarkar1,2,4, Eun Sook Park1, Luni Emdad1,3,4, Seok-Geun Lee1,4, Zao-zhong Su1,4 and Paul B. Fisher1,2,3,4

Departments of 1 Urology, 2 Pathology, and 3 Neurosurgery, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York; and 4 Department of Human Genetics, Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

Requests for reprints: Devanand Sarkar, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298-0037. E-mail: dsarkar{at}vcu.edu and Paul B. Fisher, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298-0037. E-mail: pbfisher{at}vcu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Malignant glioma is a consistently fatal brain cancer. The tumor invades the surrounding tissue, limiting complete surgical removal and thereby initiating recurrence. Identifying molecules critical for glioma invasion is essential to develop targeted, effective therapies. The expression of astrocyte elevated gene-1 (AEG-1) increases in malignant glioma and AEG-1 regulates in vitro invasion and migration of malignant glioma cells by activating the nuclear factor-{kappa}B (NF-{kappa}B) signaling pathway. The present studies elucidate the domains of AEG-1 important for mediating its function. Serial NH2-terminal and COOH-terminal deletion mutants were constructed and functional analysis revealed that the NH2-terminal 71 amino acids were essential for invasion, migration, and NF-{kappa}B–activating properties of AEG-1. The p65-interaction domain was identified between amino acids 101 to 205, indicating that p65 interaction alone is not sufficient to mediate AEG-1 function. Coimmunoprecipitation assays revealed that AEG-1 interacts with cyclic AMP-responsive element binding protein–binding protein (CBP), indicating that it might act as a bridging factor between NF-{kappa}B, CBP, and the basal transcription machinery. Chromatin immunoprecipitation assays showed that AEG-1 is associated with the NF-{kappa}B binding element in the interleukin-8 promoter. Thus, AEG-1 might function as a coactivator for NF-{kappa}B, consequently augmenting expression of genes necessary for invasion of glioma cells. In these contexts, AEG-1 represents a viable potential target for the therapy of malignant glioma. [Cancer Res 2008;68(5):1478–84]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumors of the central nervous system are the most prevalent solid neoplasms of childhood and the second leading cancer-related cause of death in adults between the ages of 15 to 34 years (1, 2). Glioblastoma multiforme (GBM) is the most aggressive malignant glioma, arising from cells of astrocyte lineage, characterized by a median survival of 10 to 12 months (1). Extensive surgical resection is not curative due to the highly invasive nature of GBM into normal brain parenchyma. Moreover, GBM is largely resistant to current treatments based on cytotoxic approaches targeting replicating DNA, such as chemotherapy or radiotherapy (3–5). As such, understanding the molecular mechanism of GBM pathogenesis is vital for developing a targeted therapy that can eradicate this aggressive cancer and promote enhanced disease-free patient survival.

Astrocyte elevated gene-1 (AEG-1) was cloned in our laboratory as a novel HIV-1– and tumor necrosis factor-{alpha}–induced transcript from primary human fetal astrocytes (PHFA; refs. 6–10). AEG-1 mRNA encodes a single-pass transmembrane protein of predicted molecular mass of ~64 kDa and pI 9.3 (10). Genomic BLAST search revealed that the AEG-1 gene is located at 8q22 where cytogenetic analyses of human gliomas confirm recurrent amplifications (11). Analysis of its expression pattern revealed that AEG-1 is overexpressed at both mRNA and protein levels in GBM compared with PHFA (8, 10). Additionally, AEG-1 expression is elevated in adult astrocytes displaying an aggressive glioma-like phenotype when injected into nude mice, resulting from the sequential expression of SV40 T antigen, telomerase (hTERT), and T24 Ha-ras (8, 10). A detailed expression analysis using immunocytochemistry and Western blotting in multiple normal adult human brains versus various brain tumor samples confirmed the in vivo overexpression of AEG-1 in >95% of diverse human brain tumors (10).

Overexpression of AEG-1 via an adenoviral vector augments the anchorage-independent growth of human glioma cell lines and increases their migration and invasion properties (9). Conversely, inhibition of AEG-1 by small interfering RNA (siRNA) in malignant glioma cell lines significantly inhibits migration and invasion. AEG-1 can synergize with Ha-ras to augment the transformed phenotype in immortal SV40 T antigen–expressing human melanocytes (FM516-SV) and AEG-1 is a Ha-ras downstream gene mediating its growth-promoting properties (8, 12). Additional studies by another group, who used the name metadherin, show the involvement of AEG-1 in tumor metastasis especially to lungs (13). In total, these findings implicate overexpression of AEG-1 in the development and progression of malignant gliomas.

A molecular mechanism by which AEG-1 increases migration and invasion of malignant glioma cells is by activating the nuclear factor-{kappa}B (NF-{kappa}B) pathway (9). Inhibition of NF-{kappa}B nullifies AEG-1–induced augmentation of anchorage-independent growth, invasion, and migration. AEG-1 is a transmembrane protein located predominantly in the endoplasmic reticulum and perinuclear space (8, 14). However, AEG-1 contains three putative nuclear localization signals (NLS) and upon treatment with TNF-{alpha} or when overexpressed, AEG-1 translocates into the nucleus where it interacts with the p65 subunit of NF-{kappa}B (9, 10). The importance of nuclear translocation and p65 interaction in mediating the tumor-promoting function of AEG-1 remains to be elucidated. The present studies provide detailed insights into the structural domains of AEG-1 mediating its actions and decipher the molecular mechanism by which AEG-1 activates the NF-{kappa}B pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. PHFA, isolated as described, and H4 [American Type Culture Collection (ATCC)] and U87MG (ATCC) malignant glioma cells were cultured as described (7, 8, 15).

Construction of plasmids and transfection. The COOH-terminal HA-tagged AEG-1 expression plasmid in the backbone of pcDNA3.1-Hygro has been described previously (8). This plasmid was used as template to construct different mutant constructs by PCR and cloned into the NheI/XhoI sites of pcDNA3.1-Hygro plasmid. All constructs have a COOH-terminal HA tag. The NH2-terminal deletion mutants were constructed using the common antisense primer 5'-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTACGTTTCTCGTCTGGC-3'. All the sense primers have a consensus Kozak sequence (GCCACC) preceding an ATG codon. The sense primers are, for N1, 5'-GCTAGCGCCACCATGTGGGCCGCGGCTTGCGCCGGC-3'; for N2, 5'-GCTAGCGCCACCATGGACGACCTGGCCTTGCTGAAG-3'; for N3, 5'-GCTAGCGCCACCATGCGTAAACGTGATAAGGTGCTG-3'; for N4, 5'-GCTAGCGCCACCATGACCGAGCAACTTACAACCGCA-3'; for N5, 5'-GCTAGCGCCACCATGTCTGGAAAAGGAGATTCTACA-3'; for N6, 5'-GCTAGCGCCACCATGTCTGTAAAACTCTCCTCACAG-3'. All COOH-terminal deletion mutants were constructed using the common sense primer 5'-GCTAGCGCCACCATGGCTGCACGGAGCTGG-3'. The antisense primers are for C1, 5'-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTAAAGAGTCTTGATAGGCTGGCT-3'; for C2, 5'-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTACCAATTGCCCCACTCTTC-3'; for C3, 5'-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTAAACTGGCTCAGCAGTAGA-3'; and for C4, 5'-CTCGAGTCAAGCGTAATCTGGAACATCGTATGGGTACTTTTCATTCCAGCCTCC-3'. NLSmut construct was created using site-directed mutagenesis using a kit from Stratagene according to the manufacturer's protocol with primers, sense 5'-TGCGCCGGCGCCGCTGTTGTAGTTGCTAGCCCGCCCCGC-3' and antisense 5'-GCGGGGCGGGCTAGCAACTACAACAGCGGCGCCGGCGCA-3'. The underlined bases are mutated converting RKKRR sequence (amino acids 79–83) to AVVVA sequence. N1-NLSmut was created using the same primers using N1 construct as template. Swapping NheI/HaeI–digested NH2-terminal fragment from N1 into C1 and C2 plasmids, respectively, created N1 + C1 and N1 + C2 constructs. The authenticity of all constructs was confirmed by sequencing. Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. For luciferase assay, cells were plated into 24-well plates and the next day transfected with AEG-1 or its different mutants along with 3{kappa}B-luc (luciferase reporter plasmid containing three tandem repeats of NF-{kappa}B binding site) and Renilla luciferase expression plasmid for transfection control. Luciferase assays were measured using Dual Luciferase Reporter Assay kit (Promega) according to the manufacturer's protocol.

Invasion and migration assays. Invasion assays were performed using modified Boyden chambers with a polycarbonate Nucleopore membrane (BD Bioscience) according to the manufacturer's protocol. For migration (wound healing) assays, a wound was introduced by scratching the confluent monolayer of cells with a pipette tip (time 0). Plates were washed twice with PBS to remove detached cells and incubated with complete growth medium, and cell migration into the wounded (scraped) empty space was followed over 18 h.

Anchorage-independent growth assay in soft agar. Anchorage-independent growth assays were performed by seeding 1 x 105 cells in 0.4% Noble agar on a 0.8% agar base layer both of which contained growth medium. Colonies were counted 2 weeks after seeding and the data from triplicate determinations were expressed as mean ± SD.

Total RNA extraction and reverse transcription-PCR. Total RNA was extracted from cells using Qiagen RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Two micrograms of total RNA were used for reverse transcription-PCR using Superscript II reverse transcriptase (Invitrogen) according to standard methods (9). Interleukin-8 (IL-8) sense, 5'-GGTGCAGAGGGTTGTGGAGAA-3'; IL-8 antisense, 5'-GCAGACTAGGGTTGCCAGATT-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-ATGGGGAAGGTGAAGGTCGGAGTC-3'; GAPDH antisense, 5'-GCTGATGATCTTGAGGCTGTTGTC-3'.

Preparation of whole-cell lysates, coimmunoprecipitation, and Western blot analyses. Preparation of whole-cell lysates, coimmunoprecipitation, and Western blot analyses were performed as described (9). The primary antibodies used were anti-HA (1:1,000; mouse monoclonal; Covance), anti-CBP (1:200; rabbit polyclonal, Santa Cruz Biotechnology), anti-AEG-1 (1:1,000; chicken polyclonal), and anti-EF1{alpha} (1:1,000; mouse monoclonal; Upstate).

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation (ChIP) assays were performed using a commercially available kit from Active Motif. H4 cells (5 x 107) were fixed with formaldehyde for 10 min at room temperature. Cells were harvested and the nuclei were isolated using a Dounce homogenizer. The nuclear pellet containing chromatin was sheared with enzyme Shearing cocktail solution; the chromatin was precleared with protein G beads; and anti-p65, anti–AEG-1, or anti-CBP antibody was added to the precleared chromatin. The DNA-protein complex was precipitated using protein G beads, washed thoroughly, and DNA was eluted from the beads. The eluted DNA was treated with RNase A and proteinase K, purified, and used as template for PCR using IL-8 promoter–specific primers, sense 5'-ATGTCAGCTCTCGACGAAAATAGA-3' and antisense 5'-GGAGGGATTGCAAGGTTTAGC-3'. The PCR products were analyzed by agarose gel electrophoresis.

Statistical analysis. Statistical analysis was performed using one-way ANOVA, followed by Fisher's protected least significant difference analysis. A P value of <0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AEG-1 is a 582-amino-acid residue protein (8). It has three putative NLS, between 79 and 91 amino acid residues, 432 and 451 amino acid residues, and 561 and 580 amino acid residues (Fig. 1A ; ref. 9). There is a putative transmembrane domain (TMD) between 51 and 72 amino acid residues. A lung homing domain has been identified in AEG-1 between 381 and 443 amino acid residues (13). Serial NH2-terminal and COOH-terminal deletion mutants were generated by PCR using AEG-1 cDNA as template (Fig. 1A). The NH2-terminal deletion mutants are N1 (amino acids 71–582 removing TMD), N2 (amino acids 101–582 removing TMD and first NLS), N3 (amino acids 205–582), N4 (amino acids 232–582), N5 (amino acids 262–582), and N6 (amino acids 290–582). The COOH-terminal deletion mutants are C1 (amino acids 1–513, removing the third NLS), C2 (amino acids 1–404, removing the second and third NLS), C3 (amino acids 1–356, removing the NLS and lung homing domain), and C4 (amino acids 1–289). Constructs containing mutations in NLS1 (NLSmut); mutations in NLS1 and lacking TMD (N1-NLSmut); lacking TMD and NLS3 (N1 + C1); and lacking TMD, NLS2, and NLS3 (N1 + C2) were also created. All the constructs have a COOH-terminal HA tag. The authenticity of these constructs was confirmed by sequencing and Western blot analysis using anti-HA antibody (Fig. 1B). All constructs produced expected sized products except N6, which could not be detected possibly caused by structural instability resulting in degradation.


Figure 1
View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Construction of AEG-1 deletion and mutant constructs. A, schematic of deletion and mutant constructs of AEG-1. Numbers, amino acid positions. Gray boxes, putative NLS. Black box, TMD. B, determination of authenticity of the generated constructs. H4 cells were transfected with the indicated constructs and the expression of the protein products was analyzed in the cell lysates by Western blotting using anti-HA antibody.

 
These deletion and mutant constructs were transfected into PHFA and H4 human malignant glioma cells along with a NF-{kappa}B reporter luciferase plasmid and a Renilla luciferase expression plasmid. Forty-eight hours after transfection, the cells were treated with TNF-{alpha} (10 ng/mL) and luciferase activity was measured by a luminometer. The firefly luciferase activity was normalized by Renilla luciferase activity. TNF-{alpha} treatment or AEG-1 transfection augmented the NF-{kappa}B reporter luciferase activity by 2.5- to 3-fold. A combination of TNF-{alpha} and AEG-1 increased the fold induction by ~6-fold (Fig. 2 ). All NH2-terminal deletion mutants lost the induction property, indicating that the first 71 amino acids are crucial for AEG-1–mediated induction of NF-{kappa}B reporter activity. In contrast, removal of the COOH-terminal regions significantly augmented AEG-1 activity, especially with the C2 construct, indicating the presence of a repression domain in this region. Mutation of NLS1 in NLSmut still retained the induction activity, indicating that this particular NLS is not essential for mediating AEG-1 function.


Figure 2
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. NH2-terminal region of AEG-1 is required for activation of NF-{kappa}B. H4 cells (A) and PHFA (B) were transfected with the indicated AEG-1 deletion or mutant constructs, NF-{kappa}B reporter luciferase plasmid, and Renilla luciferase expression plasmid. Forty-eight hours after transfection, cells were treated with TNF-{alpha} for 6 h and firefly luciferase activity was measured and normalized by Renilla luciferase activity. Columns, mean; bars, SD.

 
Analyzing the expression of IL-8, a NF-{kappa}B downstream gene, reinforced the results of the NF-{kappa}B reporter analysis. H4 cells were transfected with the different deletion and mutant AEG-1 constructs and IL-8 mRNA expression was analyzed by RT-PCR 48 h later (Fig. 3 ). AEG-1 (lane 2), the COOH-terminal deletion mutants (lanes 9–12), and NLSmut (lane 13) induced IL-8 mRNA expression, whereas all NH2-terminal deletion mutants (lanes 3–8; lanes 14–16) lost this induction property, indicating that the NH2-terminal 71 amino acids are crucial for activation of NF-{kappa}B and its downstream signaling.


Figure 3
View larger version (49K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. NH2-terminal region of AEG-1 is required for induction of IL-8, a NF-{kappa}B downstream gene. H4 cells were transfected with the indicated constructs and 48 h later cells were harvested. Total RNA was extracted and used for RT-PCR for IL-8 and GAPDH as control.

 
AEG-1 plays a crucial role in augmenting migration, invasion, and anchorage-independent growth of malignant glioma cells. Experiments were performed to define the domains of AEG-1 mediating these biological effects in H4 and U87MG cells. Migration was evaluated by a wound-healing assay, invasion by passage through a Matrigel-coated filter using a modified Boyden's chamber, and anchorage-independent growth was assayed by growth in soft agar. In agreement with the findings of NF-{kappa}B reporter assays, whereas AEG-1, its COOH-terminal deletion mutants, and NLSmut retained migration (Fig. 4A ), invasion (Fig. 4B), and anchorage-independent growth-promoting properties (Fig. 4C), all of the NH2-terminal deletion mutants lost these activities.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. NH2-terminal region of AEG-1 is required for functional activity of AEG-1. H4 and U87MG cells were transfected with the indicated constructs and 48 h later cells were analyzed for migration by would-healing assay (A), invasion by Matrigel invasion assay using a modified Boyden's chamber (B), and anchorage-independent growth in soft agar (C). Columns, mean; bars, SD.

 
Considering that AEG-1 interacts with the p65 subunit of NF-{kappa}B, experiments were designed to elucidate the domains of AEG-1 involved in mediating the p65 interaction. H4 cells were transfected with the different deletion and mutant AEG-1 constructs and 48 h later immunoprecipitation assays were performed using rabbit polyclonal anti-p65 antibody. The immunoprecipitates were washed stringently and subjected to SDS-PAGE, and immunoblotting was performed using anti-HA antibody. AEG-1, the NH2-terminal deletion mutants N1 and N2, and all the COOH-terminal deletion mutants could be precipitated by anti-p65 antibody (Fig. 5 ). However, the N3 to N6 NH2-terminal deletion mutants did not interact with p65, indicating that amino acids 101 to 205 mediate interaction of AEG-1 with p65.


Figure 5
View larger version (47K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Amino acids 101 to 205 of AEG-1 are required for interaction with p65. H4 cells were transfected with the indicated constructs and cell lysates were prepared 48 h later. Immunoprecipitation (IP) was performed by anti-p65 antibody and immunoblotting was performed by anti-HA antibody (top). As a control, both immunoprecipitation and immunoblotting (IB) were performed by anti-HA antibody (bottom). *, nonspecific band.

 
TNF-{alpha} treatment causes translocation of the p50-p65 subunit of NF-{kappa}B into the nucleus with subsequent binding to the NF-{kappa}B consensus site in the IL-8 promoter and enhanced gene transcription. AEG-1 is induced by TNF-{alpha} and overexpression of AEG-1 induces IL-8 transcription. Accordingly, we determined if AEG-1 is associated with p50-p65 complex bound to the IL-8 promoter. ChiP assays were performed using H4 cell lysates, untreated or treated with TNF-{alpha}. The cell lysates were subjected to immunoprecipitation using either control rabbit IgG or rabbit polyclonal anti-p65 or anti–AEG-1 antibodies. DNA was eluted from the immunoprecipitates and was subjected to PCR using primers flanking the NF-{kappa}B consensus site in the IL-8 promoter. Control IgG did not immunoprecipitate any DNA (Fig. 6A ). On the contrary, upon TNF-{alpha} treatment, but not under basal conditions, both p65 and AEG-1 associated with the IL-8 promoter.


Figure 6
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. AEG-1 associates with NF-{kappa}B DNA binding element in the IL-8 promoter and interacts with CBP. A, cells were either untreated or treated with TNF-{alpha} and ChIP assays were performed using the indicated antibodies with the IL-8 promoter as the target for PCR amplification. B, left, immunoprecipitation was performed with either normal IgY or chicken anti–AEG-1 antibody (Ab) and immunoblotting was performed by anti-CBP antibody. Right, immunoprecipitation was performed with either normal IgG or rabbit anti-CBP antibody and immunoblotting was performed by anti–AEG-1 antibody. *, nonspecific band. C, left, H4 cells were either mock-transfected (control) or transfected with control siRNA or AEG-1 siRNA and Western blots were performed using anti–AEG-1 and anti–EF-1{alpha} antibodies. Right, H4 cells were transfected with control siRNA or AEG-1 siRNA and treated with TNF-{alpha}, and ChIP assays were performed using the indicated antibodies with the IL-8 promoter as the target for PCR amplification. D, schematic representation of a potential molecular mechanism of NF-{kappa}B activation by AEG-1. BTM, basal transcription machinery.

 
Because sequence analysis failed to identify any DNA binding elements in AEG-1, we investigated whether AEG-1 might interact with other coactivators of transcription, thereby linking it to the basal transcription machinery. Cyclic AMP-responsive element binding protein (CREB)–binding protein (CBP) is a ubiquitous coactivator for NF-{kappa}B (16, 17). Immunoprecipitation was performed using either chicken anti–AEG-1 or rabbit anti-CBP antibodies followed by immunoblotting. Rabbit IgG and chicken IgY were used as controls for immunoprecipitation. Anti–AEG-1 and anti-CBP antibodies effectively immunoprecipitated CBP and AEG-1, respectively, whereas no specific band was observed with control antibodies (Fig. 6B). To further validate these associations, H4 cells were transfected with either control siRNA or AEG-1 siRNA and treated with TNF-{alpha}, and ChIP assays were performed using either control rabbit IgG or rabbit polyclonal anti-p65, anti–AEG-1, or anti-CBP antibodies. DNA was eluted from the immunoprecipitates and was subjected to PCR using primers flanking the NF-{kappa}B consensus site in the IL-8 promoter. AEG-1 siRNA, not control siRNA, could significantly inhibit AEG-1 protein level (Fig. 6C, left). In the presence of control siRNA p65, AEG-1 and CBP associated with the IL-8 promoter (Fig. 6C, right). When AEG-1 was inhibited by siRNA, p65 could associate with the IL-8 promoter, whereas the association of AEG-1 as well as CBP was significantly reduced (Fig. 6C, right). These findings indicate that AEG-1 might function as a bridging molecule between p65 NF-{kappa}B and CBP and the basal transcription machinery, thus facilitating transcriptional activation of NF-{kappa}B downstream genes necessary for migration and invasion (Fig. 6D). In these contexts, inhibiting AEG-1 expression or blocking AEG-1 interactions with NF-{kappa}B or other targets might be used as a strategy for abrogating the pathogenicity of malignant gliomas.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A key feature of malignant glioma is local invasion and infiltration accompanied by increased angiogenesis, remodeling of the vasculature, and destruction of surrounding normal tissue (18, 19). These properties of the tumor preclude its complete surgical removal, thus instigating recurrence from residual tumor cells. Molecules regulating adhesion, invasion, metastasis, and angiogenesis, such as urokinase-type plasminogen activator (uPA), uPAR, matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), and IL-8, are overexpressed in human malignant gliomas and the NF-{kappa}B complex transcriptionally regulates all of these genes (20–25). Aberrant or constitutive activation of NF-{kappa}B has also been documented in high-grade human gliomas (25–27). Indeed, interference with the uPA-uPAR system, MMP, and VEGF pathways, and more importantly, inhibition of NF-{kappa}B itself, inhibit tumor growth and neovascularization of glioma cells in preclinical studies and clinical trials (20, 21, 28, 29). Our observation that AEG-1 plays a key role in regulating NF-{kappa}B activation in malignant glioma and is overexpressed in human glioma samples indicates that targeting AEG-1 might be a potential approach for effective inhibition of malignant glioma pathogenicity.

The active NF-{kappa}B complex, p50-p65, is sequestered in the cytoplasm by I{kappa}B and upon receipt of an appropriate signal that leads to phosphorylation and ubiquitin-proteosome–mediated degradation of I{kappa}B, the p50-p65 NF-{kappa}B translocates into the nucleus where it binds to consensus NF-{kappa}B sequences in the promoter of diverse target genes, thereby augmenting their transcription (30, 31). Activation of transcription by NF-{kappa}B requires transcriptional coactivator proteins, such as those possessing histone acetyltransferase (HAT) activity (32–35). HAT plays a key role in altering chromatin structure, allowing recruitment of the basal transcription factors and RNA polymerase II to initiate transcription (16). NF-{kappa}B interacts with several HATs, such as CBP and its homologue p300, p300/CBP-associated factor, and members of the SRC/p160 family (32–35). These HATs acetylate core histone proteins as well as p50-p65 NF-{kappa}B to stimulate NF-{kappa}B–dependent gene expression (36). Our ChIP experiments reveal that AEG-1 is located on the consensus NF-{kappa}B binding element in the IL-8 promoter together with p50-p65. Additionally, we also document that in addition to interacting with p65 NF-{kappa}B, AEG-1 also interacts with CBP. In these contexts, AEG-1 might function as a bridging factor facilitating interaction among p50-p65 NF-{kappa}B, CBP, and the basal transcription machinery and therefore functions as a coactivator in regulating NF-{kappa}B–mediated transcription. AEG-1 is a highly basic protein that is rich in lysine, amino acid targets for acetylation. The rodent homologue of AEG-1 is LYRIC (Lysine Rich CEACAM1 co-isolated; ref. 14). As such, AEG-1 might itself be acetylated upon TNF-{alpha} treatment or in a state of constitutive overexpression and activation, as observed in glioma cells, to regulate transcription. Current studies are addressing this hypothesis.

AEG-1 translocates into the nucleus upon TNF-{alpha} treatment and when overexpressed interacts with p65 NF-{kappa}B (9). In this context, it was surmised that nuclear translocation is important in mediating NF-{kappa}B activation by AEG-1. There are three putative NLS in AEG-1 and deletion and mutation of either of these regions did not interfere with the functional activity of AEG-1. These findings indicate that there might either be a cryptic NLS in the amino-terminal end of AEG-1 or AEG-1 interacts with a chaperone protein that facilitates its nuclear import. A similar situation is evident for another coactivator SRC-3 that upon TNF-{alpha} treatment is phosphorylated by I{kappa}B kinase, which facilitates its nuclear translocation and participation in transcriptional regulation by NF-{kappa}B (33). In these contexts, the importance of posttranslational regulation, such as phosphorylation and acetylation, might be central in mediating AEG-1 function, an issue actively being pursued.

In summary, the present studies elucidate a novel mechanism of NF-{kappa}B regulation by AEG-1. The observation that in many glioma cells NF-{kappa}B is constitutively active emphasizes a vital role of overexpressed AEG-1 in the induction of constitutively active NF-{kappa}B and regulation of aggressive stages of malignant glioma. In these contexts, targeting AEG-1 by lentivirus-based short hairpin RNA approaches or by small-molecule inhibitors, in combination with radiotherapy and/or chemotherapy, might produce prolonged survival benefits in malignant glioma patients and significantly ameliorate the aggressiveness of this invariably fatal neoplasm.


   Acknowledgments
 
Grant support: Goldhirsh Foundation research grant (D. Sarkar), NIH grant P01 NS31492 (P.B. Fisher), and the Samuel Waxman Cancer Research Foundation (P.B. Fisher).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

D. Sarkar is a Harrison Scholar in Cancer Research. P.B. Fisher holds the Thelma Neumeyer Corman Chair in Cancer Research and is a Samuel Waxman Cancer Research Foundation Investigator.

Received 11/ 8/07. Revised 12/14/07. Accepted 12/31/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DeAngelis LM. Brain tumors. N Engl J Med 2001;344:114–23.[Free Full Text]
  2. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43–66.[Abstract/Free Full Text]
  3. Brandes AA, Turazzi S, Basso U, et al. A multidrug combination designed for reversing resistance to BCNU in glioblastoma multiforme. Neurology 2002;58:1759–64.[Abstract/Free Full Text]
  4. Hofer S, Herrmann R. Chemotherapy for malignant brain tumors of astrocytic and oligodendroglial lineage. J Cancer Res Clin Oncol 2001;127:91–5.[CrossRef][Medline]
  5. Kappelle AC, Postma TJ, Taphoorn MJ, et al. PCV chemotherapy for recurrent glioblastoma multiforme. Neurology 2001;56:118–20.[Abstract/Free Full Text]
  6. Su ZZ, Kang DC, Chen Y, et al. Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene 2002;21:3592–602.[CrossRef][Medline]
  7. Su Z-Z, Chen Y, Kang DC, et al. Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-{alpha} treatment. Gene 2003;306:67–78.[CrossRef][Medline]
  8. Kang D-C, Su Z-Z, Sarkar D, Emdad L, Volsky P, Fisher PB. Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene 2005;353:8–15.[CrossRef][Medline]
  9. Emdad L, Sarkar D, Su Z-Z, et al. Activation of the nuclear factor {kappa}B pathway by astrocyte elevated gene-1: implications for tumor progression and metastasis. Cancer Res 2006;66:1509–16.[Abstract/Free Full Text]
  10. Emdad L, Sarkar D, Su Z-Z, et al. Astrocyte elevated gene (AEG)-1: recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration. Pharmacol Ther 2007;114:155–70.[CrossRef][Medline]
  11. Warr T, Ward S, Burrows J, et al. Identification of extensive genomic loss and gain by comparative genomic hybridisation in malignant astrocytoma in children and young adults. Genes Chromosomes Cancer 2001;31:15–22.[CrossRef][Medline]
  12. Lee SG, Su ZZ, Emdad L, Sarkar D, Fisher PB. Astrocyte elevated gene-1 is a target of oncogenic Harvery-ras requiring phosphatidylinositol 3-kinase and c-myc. Proc Natl Acad Sci U S A 2006;103:17390–5.[Abstract/Free Full Text]
  13. Brown DM, Ruoslahti E. Metadherin, a cell surface protein in breast tumors that mediates lung metastasis. Cancer Cell 2004;5:365–74.[CrossRef][Medline]
  14. Sutherland HG, Lam YW, Briers S, Lamond AI, Bickmore WA. 3D3/lyric: a novel transmembrane protein of the endoplasmic reticulum and nuclear envelope, which is also present in the nucleolus. Exp Cell Res 2004;294:94–105.[CrossRef][Medline]
  15. Su ZZ, Lebedeva IV, Sarkar D, et al. Melanoma differentiation associated gene-7, mda-7/IL-24, selectively induces growth suppression, apoptosis and radiosensitization in malignant gliomas in p53-independent manner. Oncogene 2003;22:1164–80.[CrossRef][Medline]
  16. Huang WC, Ju TK, Hung MC, Chen CC. Phosphorylation of CBP by IKK{alpha} promotes cell growth by switching the binding preference of CBP from p53 to NF-{kappa}B. Mol Cell 2007;26:75–87.[CrossRef][Medline]
  17. Kalkhoven E. CBP and p300: HATs for different occasions. Biochem Pharmacol 2004;68:1145–55.[CrossRef][Medline]
  18. Rao JS. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer 2003;3:489–501.[CrossRef][Medline]
  19. Kleihues P, Louis DB, Scheithauer BW, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61:215–25.[Medline]
  20. Gondi CS, Lakka SS, Yanamandra N, et al. Expression of antisense uPAR and antisense uPA from a bicistronic adenoviral construct inhibits glioma cell invasion, tumor growth, and angiogenesis. Oncogene 2003;22:5967–75.[CrossRef][Medline]
  21. Lakka SS, Gondi CS, Yanamandra N, et al. Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angio!genesis, and tumor growth. Cancer Res 2003;63:2454–61.[Abstract/Free Full Text]
  22. Hu B, Jarzynka MJ, Guo P, Imanishi Y, Schlaepfer DD, Cheng SY. Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 expression through the {alpha}vβ1 integrin and focal adhesion kinase signaling pathway. Cancer Res 2006;66:775–83.[Abstract/Free Full Text]
  23. Jain RK, di Tomaso E, Duda DG, Loeffler JS, Sorensen AG, Batchelor TT. Angiogenesis in brain tumours. Nat Rev Neurosci 2007;8:610–22.[Medline]
  24. Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro-oncol 2005;7:122–33.[Abstract]
  25. Raychaudhuri B, Han Y, Lu T, Vogelbaum MA. Aberrant constitutive activation of nuclear factor {kappa}B in glioblastoma multiforme drives invasive phenotype. J Neurooncol 2007;85:39–47.[CrossRef][Medline]
  26. Wang H, Wang H, Zhang W, Huang HJ, Liao WS, Fuller GN. Analysis of the activation status of Akt, NF-{kappa}B, and Stat3 in human diffuse gliomas. Lab Invest 2004;84:941–51.[CrossRef][Medline]
  27. Kondo Y, Hollingsworth EF, Kondo S. Molecular targeting for malignant gliomas [review]. Int J Oncol 2004;24:1101–9.[Medline]
  28. Li L, Gondi CS, Dinh DH, Olivero WC, Gujrati M, Rao JS. Transfection with anti-p65 intrabody suppresses invasion and angiogenesis in glioma cells by blocking nuclear factor-{kappa}B transcriptional activity. Clin Cancer Res 2007;13:2178–90.[Abstract/Free Full Text]
  29. Robe PA, Martin D, Albert A, Deprez M, Chariot A, Bours V. A phase 1-2, prospective, double blind, randomized study of the safety and efficacy of Sulfasalazine for the treatment of progressing malignant gliomas: study protocol of [ISRCTN45828668]. BMC Cancer 2006;6:29–32.[CrossRef][Medline]
  30. Baldwin AS, Jr. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 1996;14:649–83.[CrossRef][Medline]
  31. Karin M, Cao Y, Greten FR, Li ZW. NF-{kappa}B in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002;2:301–10.[CrossRef][Medline]
  32. Zhong H, May MJ, Jimi E, Ghosh S. The phosphorylation status of nuclear NF-{kappa}B determines its association with CBP/p300 or HDAC-1. Mol Cell 2002;9:625–36.[CrossRef][Medline]
  33. Wu RC, Qin J, Hashimoto Y, et al. Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) coactivator activity by I{kappa}B kinase. Mol Cell Biol 2002;22:3549–61.[Abstract/Free Full Text]
  34. Furia B, Deng L, Wu K, et al. Enhancement of nuclear factor-{kappa}B acetylation by coactivator p300 and HIV-1 Tat proteins. J Biol Chem 2002;277:4973–80.[Abstract/Free Full Text]
  35. Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. The nuclear factor-{kappa}B engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem 1999;274:32091–8.[Abstract/Free Full Text]
  36. Chen LF, Greene WC. Regulation of distinct biological activities of the NF-{kappa}B transcription factor complex by acetylation. J Mol Med 2003;81:549–57.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
D. Sarkar, L. Emdad, S.-G. Lee, B. K. Yoo, Z.-z. Su, and P. B. Fisher
Astrocyte Elevated Gene-1: Far More Than Just a Gene Regulated in Astrocytes
Cancer Res., November 15, 2009; 69(22): 8529 - 8535.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
G. Hu, Y. Wei, and Y. Kang
The Multifaceted Role of MTDH/AEG-1 in Cancer Progression
Clin. Cancer Res., September 15, 2009; 15(18): 5615 - 5620.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. K. Yoo, R. Gredler, N. Vozhilla, Z.-z. Su, D. Chen, T. Forcier, K. Shah, U. Saxena, U. Hansen, P. B. Fisher, et al.
Identification of genes conferring resistance to 5-fluorouracil
PNAS, August 4, 2009; 106(31): 12938 - 12943.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
C. Yu, K. Chen, H. Zheng, X. Guo, W. Jia, M. Li, M. Zeng, J. Li, and L. Song
Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis
Carcinogenesis, May 1, 2009; 30(5): 894 - 901.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Z.-z. Su, S.-G. Lee, L. Emdad, I. V. Lebdeva, P. Gupta, K. Valerie, D. Sarkar, and P. B. Fisher
Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN)
PNAS, December 30, 2008; 105(52): 20906 - 20911.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
N. de la Iglesia, G. Konopka, K.-L. Lim, C. L. Nutt, J. F. Bromberg, D. A. Frank, P. S. Mischel, D. N. Louis, and A. Bonni
Deregulation of a STAT3-Interleukin 8 Signaling Pathway Promotes Human Glioblastoma Cell Proliferation and Invasiveness
J. Neurosci., June 4, 2008; 28(23): 5870 - 5878.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sarkar, D.
Right arrow Articles by Fisher, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sarkar, D.
Right arrow Articles by Fisher, P. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online