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WO2006093494A1 - Gène 1 élevé astrocyte et son promoteur dans les traitements de neurotoxicité et de maladies malignes - Google Patents

Gène 1 élevé astrocyte et son promoteur dans les traitements de neurotoxicité et de maladies malignes Download PDF

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WO2006093494A1
WO2006093494A1 PCT/US2005/006639 US2005006639W WO2006093494A1 WO 2006093494 A1 WO2006093494 A1 WO 2006093494A1 US 2005006639 W US2005006639 W US 2005006639W WO 2006093494 A1 WO2006093494 A1 WO 2006093494A1
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aeg
gene
cell
promoter
expression
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Paul Fisher
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Columbia University in the City of New York
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Columbia University in the City of New York
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Priority to US11/781,512 priority patent/US20080200412A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention is based, at least in part, on the discovery that Astrocyte Elevated Gene-1 (“AEG-I”) expression (i) suppresses the Excitatory Amino Acid Transporter -2 ("EAAT-2") promoter, thereby inhibiting glutamate transport; (ii) supports anchorage independent colony formation of cells, in which it is synergistic with the RAS oncogene; and (iii) is increased in a number of different malignancies. It is also based, in part, on the discoveries of the AEG-I promoter and its increased activity in the presence of activated mutant RAS.
  • AEG-I Astrocyte Elevated Gene-1
  • EAAT-2 Excitatory Amino Acid Transporter -2
  • the invention in various embodiments, provides for methods of treatment of malignancies and neurodegenerative disorders using inhibitors of AEG-I activity or gene therapy constructs in which the AEG-I promoter drives expression of a therapeutic gene, and provides for screening assays for identifying other compounds that have therapeutic benefit.
  • HIV-I infection of astrocytes contributes to Human Immunodeficiency Virus (HIV) Associated Dementia ("HAD”) by diverse mechanisms, including astrogliosis and glutamate excitotoxicity (7).
  • HIV Human Immunodeficiency Virus
  • Astrogliosis resulting from HIV infection involves the activation of astrocyte proliferation followed by apoptosis (8).
  • Perturbation of astroglial -neuronal interactions and secretion of neurotoxic cytokines and chemokmes in astrogliosis can also contribute to neuronal cell atrophy (9, 10).
  • EAAT2 Excitatory Amino Acid Transported, or "EAAT2”
  • EAAT2 Excitatory Amino Acid Transported
  • astrocytes infected by HIV-I or by gpl20 envelope protein treatment which might promote neuronal death by glutamate excitotoxicity (11, 12).
  • HIV-I infection or gpl20 treatment of human astrocytes alters gene expression patterns in vivo and in vitro (13-16).
  • a rapid subtraction hybridization i (“RaSH”) approach identified 15 astrocyte elevated genes (“AEGs”) and 10 astrocyte suppressed genes (“ASGs”) in primary human fetal astrocytes ("PHFA”) upon HIV infection (13-15).
  • AEGs 13 were known genes and two, AEG-I and AEG-11, were unknown at the time of cloning, while 8 of the ASGs were known and two, ASG-I and ASG-8, were unrecognized (13-15, and International Patent
  • the present invention is based, at least in part, on the discovery that Astrocyte Elevated Gene-1 (“AEG-I”) expression (i) suppresses the Excitatory Amino Acid Transporter -2 ("EAAT-2") promoter, thereby inhibiting glutamate transport; (ii) supports anchorage independent colony formation of cells, in which it is synergistic with the RAS oncogene; and (iii) is increased in a number of different malignancies. It is also based, in part, on the discoveries of the AEG-I promoter and its increased activity in the presence of activated mutant RAS.
  • AEG-I Astrocyte Elevated Gene-1
  • EAAT-2 Excitatory Amino Acid Transporter -2
  • the present invention in a first set of embodiments, provides for inhibitors of AEG-I.
  • the present invention provides for the AEG-I promoter, and for expression constructs comprising the AEG-I promoter operably linked to, and controlling expression of, a gene of interest.
  • the gene of interest may be a therapeutic gene or an antiviral gene, so that the expression construct may be used in the therapy or prevention of HAD or malignancy, or may be a reporter gene, so that the expression construct may be used in screening systems to detect agents that inhibit AEG-I expression.
  • the present invention provides for methods of inhibiting glutamate toxicity, comprising inhibiting the expression of AEG-I.
  • the present invention provides for methods of preventing or inhibiting growth or survival of malignant cells, comprising inhibiting the expression of AEG-I, optionally with concurrent or sequential inhibition of RAS activity.
  • the present invention provides for methods of treating a neurodegenerative condition in a subject, comprising administering, to the subject, an agent that inhibits expression of AEG-I.
  • the present invention provides for methods of treating HAD in a subject, comprising administering, to the subject, an agent that inhibits expression of AEG-I.
  • the present invention provides for methods of treating a malignancy in a subject, comprising administering, to the subject, an agent that inhibits expression of AEG-I.
  • the present invention provides for methods of identifying therapeutic agents that may be used to treat neurodegeneration, HAD, or malignancy, comprising determining whether a test agent prevents the suppression of the EAAT2 promoter by AEG-I, wherein inhibition of suppression has a positive correlation with therapeutic benefit. 4.
  • FIG. 1 Complete open reading frame technique.
  • A Schematic diagram of C-ORF (details in text).
  • B Representative C-ORF applications. C-ORF products of ISG-56, m ⁇ -9/syntenin and mda-5 are resolved in 1% agarose gel (lane 2). The size of C-ORF products is compared with RT-PCR product of each gene with common 3 ' nested primer and 5 ' primer from reported sequence (lane 1). Nested PCR of C-ORF with anchor primer only (lane 3).
  • C Application of C-ORF to AEG-I cloning. First round AEG-I C-ORF product is resolved as above (lane 2: GSP and lane 3: anchor primer only).
  • FIG. 2 A baculoviral tag- free recombinant protein production system.
  • A Schematic diagram of AEG-I fusion protein with chitin binding/intein domain.
  • B Protein electrophoretogram in 10% SDS-PAGE of input (lane 1) and elution (lanes 2 and 3) from chitin affinity column followed by DTT-induced self-cleavage.
  • C Immunoreactivity of ⁇ AEG-I antibody. Protein samples (20 ⁇ g) from transiently transfected 293 cells with pcDNA3.1- AEG-I -HA were resolved in 10% SDS-PAGE and analyzed by Western blotting. Blots were probed with either ⁇ HA antibody or ⁇ AEG-I antibody and detected by chemiluminescence.
  • FIG. 3 Expression pattern of AEG-I.
  • A AEG-I expression in different organs. Multiple tissue Northern blots (ClonTech) were probed with 32 P-labelled 1.6 kb AEG-I and ⁇ -actin cDNA probes.
  • B AEG-I expression in various cancer cell lines. Northern blots of the indicated RNA samples were probed with AEG-I and GAPDH probes. HMEC, human mammary epithelial cells; NC, normal cerebellum cells.
  • C Western blot analysis of AEG-I expression in selected cells with ⁇ AEG-1 antibody.
  • FIG. 4 Immunofluorescence microscopy of AEG-I. Subcellular localization of AEG-I was determined in IM-PHFA as described in Materials & Methods. Calreticulin and Mito-tracker were used for specific ER and mitochondria markers, respectively.
  • A. A luciferase reporter vector containing full EAAT2 promoter or EAATl promoter was co-transfected with either pcDNA3.1 or pcDNA3.1-AEG-l-HA. Two days later, cells were harvested and luciferase activity of the protein extracts was measured. Data shown is a representative of multiple experiments.
  • B A luciferase reporter vector containing the full EAAT2 promoter or the EAATl promoter was co-transfected with the indicated expression vectors. Samples were obtained and analyzed as above. Data shown is a representative of multiple experiments.
  • Fig. 6. Soft agar colony formation assay.
  • A. Colony forming ability of FM516-SV cells transiently transfected with indicated expression vectors. Data shown is average + SD of three independent transfections.
  • B. Colony forming ability of FM516-SV cells stably transfected with AEG-I and T24 Ha-r ⁇ s. Data shown is average + SD of three independent transfections.
  • Fig. 7 Expression of AEG-I in normal brain and glioblastoma cells and tissue.
  • B Western blot analysis of protein extracts derived from the cells or tissues indicated. The blot was probed with a specific anti- AEG-I antibody. Equivalency of loading was determined by probing the membrane with an anti-EFl ⁇ antibody.
  • Fig. 9 Nucleic acid sequence of the ⁇ EG-7 Promoter (SEQ ID NO:2) indicating a putative RAS-activated region at position 474 to 491. Position 1 of the sequence corresponds to the base pair immediately upstream of the translational initiation ATG codon.
  • Fig. 10 Schematic representation of the AEG-I Promoter showing the location of putative transcription factor binding sites. The promoter sequence was searched against Matlnspector (www .genomatix.de) and Match (www, gene- regulation.com) transcription factor databases. The presence and location of predicted transcription factor binding sites identified by homology searches is indicated.
  • H-ras can activate the human AEG-I promoter.
  • Fig. 12 Identification of the AEG-I promoter region responsible for Ha-ras-mediated activation of AEG-I transcription. Luciferase reporter activity of nested deletion constructs of the AEG-I promoter deleted at positions 1146, 787, 515 and 350, compared to full length (2759 bp) is shown. Activity of these constructs is compared between FM 516 immortalized human melanocytes and a RAS overexpressing clone of FM 516 (FM516-ras-cl7). Data shown is an average + SD of three independent transfections. Fig. 13. Effect of PBK, MEK and p38 MAPK inhibitors on AEG-I promoter activity. A.
  • Immortalized (THV) and ras transformed (THR) human astrocytes were treated with indicated amount of inhibitors and luciferase activity determined.
  • B. CREF and CREF-ras rat embryo fibroblasts were treated with indicated amount of inhibitors and luciferase activity determined.
  • C. Immortalized (THV) and RAS transformed (THR) human astrocytes were treated with indicated amount of inhibitors and luciferase activity determined.
  • D. CREF and CREF-ras rat embryo fibroblasts were treated with indicated amount of inhibitors and luciferase activity determined.
  • E. The immortalized melanocyte cell line FM516 and a stable mutant RAS overexpressing clone FM516-ras7 were treated with indicated amount of inhibitors and luciferase activity determined.
  • Fig. 14 Activity of the AEG-I promoter in cell lines containing mutated RAS genes.
  • A. Human pancreatic cancer cells were transfected with the AEG-I luciferase reporter and luciferase activity was determined.
  • B. The human colorectal cell line HCTl 16 and two related progeny lines C2 and ClO were transfected with the AEG-I luciferase reporter and luciferase activity was determined.
  • C The human colorectal cell line HCTl 16 and two related progeny lines C2 and ClO were transfected with the AEG-I luciferase reporter and luciferase activity was determined in the presence and absence of inhibitors.
  • the AEG-I protein activates its own promoter.
  • the present invention relates to inhibitors of AEG-I.
  • Such inhibitors can inhibit AEG-I expression (e.g., transcription, inhibition of AEG-I promoter activity or translation) or function (e.g. inhibition of AEG-I protein activity). Screening methods described herein, for example as set forth in sections 5.8 and 5.10, below, may be used to identify compounds that may be used as AEG-I inhibitors according to the invention.
  • inhibitors of AEG-I are directed to inhibition of transcription of AEG-I.
  • Such agents inhibit activity of the AEG-I promoter, and may be identified using screening methods which employ the AEG-I promoter linked to a reporter gene, as described in sections 5.2 and 5.10, infra.
  • mutant activated RAS activates the AEG-I promoter
  • one non-limiting example of a class of inhibitors of AEG-I transcription is molecules that inhibit RAS activity, such as, for example, molecules that inhibit translation of RAS, such as ⁇ vXi-RAS antisense RNA, interfering RNA, ribozymes or "DNAzymes.”
  • Non-limiting examples of anti- RAS agents are set forth in International Patent Application No. PCT/US02/26454 by the Trustees of Columbia University in the City of New York, by Fisher, Published as WO03016499 on February 27, 2003.
  • translation of AEG-I -encoding mRNA is inhibited using a nucleic acid molecule that is, at least in part, complementary to and/or hybridizable to the AEG-I coding sequence, such as an antisense nucleic acid, an interfering RNA ("siRNA or RNAi”), a ribozyme, or a DNAzyme.
  • a nucleic acid molecule that is, at least in part, complementary to and/or hybridizable to the AEG-I coding sequence, such as an antisense nucleic acid, an interfering RNA ("siRNA or RNAi”), a ribozyme, or a DNAzyme.
  • the portion complementary to AEG-I mRNA may be between about 5- 10, 5-50, 5-100, 10-50, 10-100, 20-50, 20-100, 100-500, 500-1000, 1000-2000, or 2000-3611 nucleotides in length.
  • the nucleic acid sequence of AEG-I is set forth in
  • Complementary molecules are those which contain complementary bases to at least 90 percent, and preferably at least 95 percent, and, in non-limiting embodiments, 100 percent of the nucleotides in a subsequence of AEG-I.
  • “Hybridizable” nucleic acid molecules hybridize to the AEG-I gene or cDNA (sense strand) under stringent conditions.
  • Stringent conditions are hybridization to filter-bound DNA or RNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 niM EDTA at 65°C, and washing twice or more in O.lxSSC (15-30 mMNaCl, 1.5-3 mM sodium citrate, pH 7.0)/0.1% SDS at 68°C.
  • a stringent hybridization washing solution may alternatively be comprised of 40 mM NaPO4, pH 7.2, 1-2% SDS and 1 mM EDTA, for which a washing temperature of at least 65-68°C is recommended.
  • an siRNA comprises the sequence 5'
  • -AGCAGCCACCAGAGATTGA -3' (SEQ ID NO:3), or a sequence that is at least 90 or 95 percent homologous thereto, or that hybridizes under stringent conditions, and is up to 19, 25, 35, or 50 nucleotides in length.
  • an inhibitor of AEG-I may comprise an antibody which specifically recognizes the AEG-I protein and inhibits its activity.
  • the inhibition of AEG-I protein activity may be due to binding of the antibody to the AEG-I protein so as to prevent access of other molecules required for or mediating AEG-I activity.
  • the inhibition of AEG-I protein activity may be due to binding of the antibody to an active site in the AEG-I protein thus inhibiting its activity, even if access to other parts of the AEG-I protein is not blocked.
  • the antibody may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, an antibody fused to another protein which possesses additional catalytic activity etc.
  • an AEG-I inhibitor may comprise a single or mixture of several peptides corresponding to regions or functional subdomains of the AEG-I protein.
  • An AEG-I inhibitor may also comprise an AEG-I peptide containing one or several point mutations that impair or reverse the activity of the unmodified protein. The presence of such inhibitory peptides in proximity to the full length functional AEG-I protein causes partial or full inhibition of the activity of the AEG-I protein.
  • Such partial mutant forms or an independently expressed subdomain of a protein which interferes with the biological or catalytic activity of a full length protein has usually been designated the term "dominant- negative" in the published literature.
  • the term "dominant-negative AEG-I protein" as used herein will encompass the properties embodied by an altered form of AEG-I protein which interferes with the activity of the unmodified form of AEG-I protein.
  • the AEG-I derived inhibitory peptides may have a range of between 10 to 900 amino acid residues.
  • a dominant-negative AEG-I protein may be used in protein/peptide therapy of a subject in need of such treatment.
  • the dominant-negative AEG- 1 protein of the invention may be prepared by chemical synthesis or recombinant DNA techniques, purified by methods known in the art, and then administered to a subject in need of such treatment.
  • Dominant-negative AEG-I protein may be comprised, for example, in solution, in suspension, and/or in a carrier particle such as microparticles, nanoparticles, liposomes, or other protein-stabilizing formulations known in the art.
  • formulations of dominant- negative AEG-I protein may stabilized by addition of zinc and/or protamine stabilizers as in the case of certain types of insulin formulations.
  • dominant-negative AEG-I protein may be linked covalently or non-covalently, to a carrier protein which is preferably non- immunogenic.
  • the inhibitory peptide may be delivered to a cell in purified form, in a pharmaceutical formulation or by means of an expression vector.
  • expression vectors used to deliver inhibitory forms of AEG-I peptides include DNA plasmid vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors etc. 5.2 THE AEG-I PROMOTER AND EXPRESSION CONSTRUCTS
  • the present invention provides for the AEG-I promoter.
  • An "AEG-I promoter,” as defined herein, includes a nucleic acid molecule having a sequence as depicted in Fig. 7 (SEQ E) NO:2), as well as nucleic acid molecules that are at least 80, 85, 90 or 95 percent homologous to SEQ ID NO: 2 (as determined using standard homology-determining software, such as BLAST or FASTA), and molecules that hybridize to a nucleic acid molecule having SEQ E) NO:2 under stringent hybridization conditions, as defined above.
  • the present invention further provides for a U cove-AEG-1 promoter" which retains RAS sensitivity, and which comprises up to 2300, 2500 or 2600 nucleotides of SEQ E) NO:2 and at least nucleotides 1 to 515 of SEQ E) NO:2.
  • the present invention yet further provides for the nucleic acids that are at least 80, 85, 90 or 95 percent homologous to said core AEG-I promoter, or hybridize thereto under stringent conditions, and to vectors comprising the core AEG-I promoter optionally linked to a gene of interest.
  • An AEG-I promoter of the invention may be comprised in a vector molecule, and may be operably linked to any gene of interest. Such linkage may be used to effect selective expression of the gene of interest in astrocytes infected with HIV and in certain types of cancer cells, such as glioblastoma multiforme, astrocytoma, breast cancer, colon cancer, pancreatic cancer, and melanoma cells, and cells of cancers having an activating mutation in RAS.
  • Vectors comprising an AEG-I promoter operably linked to any gene of interest include but are not limited to DNA plasmid vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors etc.
  • an AEG-I promoter may be operably linked to the El A and ElB genes in a replicative adenoviral vector. Such linkage is expected to enhance the replication and cytotoxicity of the said adenovirus since AEG-I promoter activity is increased in the presence of AEG-I gene product. Thus enhance efficacy and specificity may be achieved in killing cancer cells expressing high amounts of AEG-I protein.
  • the AEG-I promoter driven replicating adenovirus amy also contain an additional therapeutic gene in the E3 region.
  • Suitable genes of interest include therapeutic genes, antiviral genes, as well as reporter genes.
  • a therapeutic gene is defined as a nucleic acid molecule that encodes a product, such as a protein, that confers a therapeutic benefit.
  • a reporter gene is defined as a nucleic acid molecule that encodes a detectable product.
  • Suitable therapeutic genes include, but are not limited to, a gene that augments immunity, such as EFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-2, IL-4, IL-12 etc., a gene that has an anti-cancer effect, including genes with anti-proliferative activity, anti- glutamate activity, anti-metastatic activity, anti-angiogenic activity, or pro-apoptotic activity, such as mda-7/IL-24 (Sarkar et al. (2002) Biotechniques Suppl: 30-39; Fisher et al. (2003) Cancer Biol Ther 2:S23-37), p53 (Haupt et al., Semin Cancer Biol. 2004 14(4):244-252; Haupt et al.
  • a gene that augments immunity such as EFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-2, IL-4, IL-12 etc.
  • a gene that has an anti-cancer effect including genes with anti-proliferative
  • RNAi or antisense-&4S Liu et al. Cancer Gene Ther (2004) 11(11):748-756.
  • RNAi or antisense VEGF Qui et al. Hepatobiliary Pancreat Dis hit (2004) 3(4):552-557
  • antisense or RNAi mda-9/syntenin Sarkar et al. Pharmacol Ther (2004) 104(2): 101-115) etc.
  • Suitable antiviral genes include, but are not limited to, a gene that augments immunity and/or inhibits viral replication, such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL- 2, IL-4, IL-12 etc.
  • Suitable reporter genes include, but are not limited to, luciferase, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, beta- galactosidase, beta-glucuronidase, etc. 5.3 METHODS OF INHIBITING GLUTAMATE TOXICITY
  • the present invention provides for a method of inhibiting glutamate toxicity, comprising inhibiting the expression of AEG- 1. It has been observed that ectopic expression of AEG-I specifically inhibited EAAT2 promoter activity ( ⁇ 3.5-fold), where EAAT2 functions to prevent an extracellular accumulation of toxic glutamate in proximity to neurons.
  • Conditions in which inhibition of glutamate toxicity may be useful include neurodegenerative conditions such as but are not limited to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and epilepsy, cerebral ischemia with or without infarction (including but not limited to cerebral infarction and transient ischemic attacks), and HAD.
  • Inhibitors of AEG-I in this embodiment include those which cause inhibition of AEG-I expression ⁇ e.g., transcription, inhibition of AEG-I promoter activity or translation) or AEG-I function ⁇ e.g. inhibition of protein activity) as described in section 5.1, supra, hi a further embodiment, screening methods described herein, for example as set forth in sections 5.8 and 5.10, below, may be used to identify compounds that may also be used as AEG-I inhibitors according to the invention.
  • the present invention provides for a method of preventing or inhibiting growth and/or proliferation of malignant cells, comprising inhibiting the expression of AEG-I, optionally with concurrent or sequential inhibition of RAS activity.
  • AEG-I expression was elevated in adult astrocytes transformed by sequential expression of SV40 T/t antigen, telomerase (hTERT) and T24 Ha-ra? (22) and, thereby, displaying an aggressive glioma-like phenotype; Expression of AEG-I promoted anchorage independent growth of
  • FM516-SV cells that could not normally form colonies in soft agar and, although expression of the T24-Ha-r ⁇ s oncogene in FM516-SV cells resulted in a higher rate in agar growth, co-expression of AEG-I and Ha-ras in FM516-SV cells resulted in a synergistic increase in colony formation in soft agar;
  • AEG-I exhibited elevated expression in many cancers, including malignant gliomas, melanomas, pilocytic astrocytoma, anaplastic astrocytoma, anaplastic meningioma, medulloblastoma, oligoastrocytoma, anaplactic oligodendroglioma and carcinomas of the breast, colon and pancreas; and
  • Northern blot analysis of AEG-I expression in human cancer cells identified a different set of transcripts (9 kb, 4 kb and 1.5 kb) that might result from alternative splicing.
  • the present invention provides for a method for preventing or inhibiting the survival and or/proliferation of a malignant cell comprising administering, to the cell, an effective amount of an AEG-I inhibitor as described above.
  • the cancer cell is a glioblastoma multiforme cell, a breast cancer cell, a melanoma cell, a colon cancer cell, or a pancreatic cancer cell.
  • such method may be combined, either concurrently or sequentially, with administration of an anti-RAS agent (as described above).
  • the present invention provides for a method of treating a neurodegenerative condition in a subject, comprising administering, to the subject, an effective amount of an agent that inhibits expression of AEG-I.
  • an agent that inhibits expression of AEG-I can inhibit AEG-I expression (e.g., transcription, inhibition of AEG-I promoter activity or translation) or function (e.g. inhibition of AEG-I protein activity) as set forth in sections 5.1.
  • Screening methods described herein, for example as set forth in sections 5.8 and 5.10, below, may be used to identify compounds that may be used as AEG-I inhibitors according to the invention.
  • Conditions which may be treated include, but are not limited to,
  • Alzheimer's disease Parkinson's disease, amyotrophic lateral sclerosis, and epilepsy, cerebral ischemia with or without infarction (including but not limited to cerebral infarction and transient ischemic attacks), and HAD.
  • the present invention provides for a method of treating HAD in a subject, comprising administering, to the subject, an agent that inhibits expression of AEG-I.
  • an agent that inhibits expression of AEG-I can inhibit AEG-I expression (e.g., transcription, inhibition of AEG-I promoter activity or translation) or function (e.g. inhibition of AEG-I protein activity) as set forth in sections 5.1.
  • Screening methods described herein, for example as set forth in sections 5.8 and 5.10, below, may be used to identify compounds that may be used as AEG-I inhibitors according to the invention.
  • the present invention provides for a method of treating a malignancy in a subject, comprising administering, to the subject, an agent that inhibits expression of AEG-I.
  • an agent that inhibits expression of AEG-I can inhibit AEG- 1 expression ⁇ e.g., transcription, inhibition of AEG-I promoter activity or translation) or function ⁇ e.g. inhibition of AEG-I protein activity) as set forth in sections 5.1 above.
  • Screening methods described herein, for example as set forth in sections 5.8 and 5.10, below, may be used to identify compounds that may be used as AEG-I inhibitors according to the invention.
  • Malignancies that may be treated include, but are not limited to, glioblastoma multiforme, melanoma, breast cancer, colon cancer, pancreatic cancer, and a cancer the cells of which exhibit a mutant activated form of RAS. 5.8 METHODS OF IDENTIFYING THERAPEUTIC AGENTS
  • the present invention provides for a method of identifying therapeutic agents that may be used to treat neurodegeneration, HAD, or malignancy, comprising determining whether a test agent prevents the suppression of the EAAT2 promoter by AEG-I, wherein inhibition of suppression has a positive correlation with therapeutic benefit.
  • the invention provides for an assay for identifying an agent capable of reversing or suppressing inhibition of EAAT-2 promoter activity by AEG-I, comprising (i) preparing a nucleic acid construct having an EAAT-2 promoter operably linked to a reporter gene (defined herein as a gene having a detectable product) for example, but not limited to, a luciferase gene; (ii) introducing the construct by methods known to those skilled in the art, in a transient or stable configuration, into a cell in the assay system, such as a eukaryotic cell, for example, but not limited to, an immortalized astrocyte cell; (iii) exposing the cell to an AEG-I gene product by methods including but not limited to exposing cells or tissues containing the EAAT-2 promoter to purified AEG-I protein or to vectors including DNA plasmids, adenoviruses, retroviruses, adeno-associated viruses, lentiviruses etc.
  • AEG-I gene product or providing the AEG-I gene product by transfecting a cell within the assay system (which may or may not also contain the EAAT2 promoter/reporter construct) with an AEG-I gene operably linked to a constitutively or inducibly active promoter; (iv) exposing the cell to a test agent; (v) measuring the amount of reporter gene product produced by a cell exposed to the test agent; and (vi) comparing the amount of reporter gene product produced by a cell containing the EAAT-2 promoter and AEG-I gene product which has been exposed to the test agent to the amount of reporter gene product produced by a cell which has not been exposed to the test agent.
  • the cell (generally in a cell culture or organism) exposed to the test agent and the cell not exposed to the test agent should otherwise be maintained under the same or similar conditions.
  • An increase in the level of reporter gene product in a cell exposed to the test agent has a positive correlation with an increase in EAAT-2 promoter activity via reduction or suppression of the inhibitory activity of the AEG-I gene product on the EAAT-2 promoter.
  • a test agent causing such increase in the level of reporter gene product is therefore identified as having potential therapeutic activity by inhibiting the activity of the AEG-I gene product.
  • the type of cell utilized in an assay to identify a therapeutic agent is a cancer cell.
  • the cancer cell is drawn from a group wherein the EAAT2 promoter may also be active, comprising of but not limited to a glioblastoma multiforme cell, a breast cancer cell, a melanoma cell, a colon cancer cell, pancreatic cancer cell or a cervical cancer cell e.g. HeLa.
  • such method to identify a therapeutic agent may also be combined, either concurrently or sequentially, with administration of an an ⁇ -RAS agent (as described above). 5.9 GENE THERAPY USING THE AEG-I PROMOTER
  • An AEG-I promoter construct operably linked to a therapeutic gene may be used to modulate cell proliferation in a subject, wherein a nucleic acid encoding the therapeutic gene driven by the AEG-I promoter, may be introduced into a cell of the subject.
  • AEG-I promoter construct operably linked to a therapeutic gene may be contained in an expression vector, hi preferred, non-limiting embodiments, such vectors may include DNA plasmid vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors etc. Examples of therapeutic genes include those set forth in 5.2.
  • a viral vector containing a nucleic acid encoding the AEG-I promoter construct operably linked to a therapeutic gene may be a non-replicating or replicating adenoviral vector which may be administered to a population of target cells at a multiplicity of infection (MOI) ranging from 10-100 MOI.
  • MOI multiplicity of infection
  • the amount of a non- replicating or replicating adenoviral vector viral vector administered to a subject may be lxl0 9 pfu to lxl0 12 pf ⁇ .
  • a vector containing a nucleic acid encoding the AEG-I promoter construct operably linked to a therapeutic gene may be introduced into a cell ex vivo and then the cell may be introduced into a subject.
  • a vector containing a nucleic acid encoding the AEG-I promoter construct operably linked to a therapeutic gene may be introduced into a cell of a subject (for example, an irradiated tumor cell, glial cell or fibroblast) ex vivo and then the cell containing the nucleic acid may be optionally propagated and then (with its progeny) introduced into the subject.
  • the present invention further encompasses the use of an AEG-I promoter construct operably linked to a therapeutic gene in combination with other forms of therapy.
  • an AEG-I promoter construct operably linked to a therapeutic gene in combination with other agents that have an anti-proliferative effect, including, but not limited to, anti-RAS agents, radiation therapy and chemotherapeutic agents.
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with a generator of free radicals (International Patent Application No. PCT/US03/28512, by Fisher et al, published as WO 04/060269 on July 22, 2004 by the Trustees of Columbia University and Virginia Commonwealth University).
  • free radical generators include, but are not limited to arsenic trioxide, NSC656240, 4-HPR, and cisplatin.
  • ROS include but are not limited to singlet oxygen, hydrogen peroxide, superoxide anion, hydroxyl radicals, peroxynitrite, and oxidants.
  • the free radical generators are arsenic trioxide, NSC656240 or 4-HPR.
  • the disrupter of mitochondrial membrane potential is PK 11195.
  • an. AEG-I promoter construct operably linked to a therapeutic gene may be administered together with a regimen of radiation therapy (International Patent Application No. PCT/US03/28512, by Fisher et al., published as WO 04/060269 on July 22, 2004 by the Trustees of Columbia University).
  • a regimen of radiation therapy International Patent Application No. PCT/US03/28512, by Fisher et al., published as WO 04/060269 on July 22, 2004 by the Trustees of Columbia University.
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with between 2 and 100 Gy of radiation, either as a single treatment or in multiple treatments.
  • one external treatment of 2 Gy may be administered each of 5 days a week for six weeks for a total of 60 Gy. If intraoperative radiation is administered, the amount administered may be between 3 and 15 Gy total, and preferably 6 Gy.
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with an anti-r ⁇ s agent (International Patent Application No. PCT/US02/26454, by Fisher et al., published as WO 03/016499 on February 27, 2003 by the Trustees of Columbia University); particularly in the treatment of a disorder of cell proliferation associated with a mutation in a r ⁇ s gene.
  • Suitable anti-ras agents include, but are not limited to, small interfering RNAs (RNAi), antisense RNA (including but not limited to oligonucleotides having phosphorothioate residues), or farnesyl transferase inhibitors.
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with a chemotherapy agent, including, but not limited to, interferon alpha, tamoxifen, cisplatin, daunorubicin, carmustine, dacarbazine, etoposide, fluorouracil, ifosfamide, methotrexate, mitomycin, mitoxanthrone HCl, vincristine, vinblastine, and adriamycin, to name a few.
  • a chemotherapy agent including, but not limited to, interferon alpha, tamoxifen, cisplatin, daunorubicin, carmustine, dacarbazine, etoposide, fluorouracil, ifosfamide, methotrexate, mitomycin, mitoxanthrone HCl, vincristine, vinblastine, and adriamycin, to name a few.
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with an anticancer antibody, such as, but not limited to, trastuzumab (Herceptin). Further, an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with more than one other anti-proliferative agent (e.g., free radical generator, radiation, anti-r ⁇ s agent, chemotherapeutic agent, anticancer antibody, etc.).
  • an anticancer antibody such as, but not limited to, trastuzumab (Herceptin).
  • an AEG-I promoter construct operably linked to a therapeutic gene may be administered together with more than one other anti-proliferative agent (e.g., free radical generator, radiation, anti-r ⁇ s agent, chemotherapeutic agent, anticancer antibody, etc.).
  • the amounts of anti-proliferative therapy added to the dose of an AEG- 1 promoter construct operably linked to a therapeutic gene may be those doses conventionally used for such therapy.
  • the combination of MDA-7 (expressed by an AEG-I promoter construct) with another form of antiproliferative therapy may allow for the use of lower doses of said antiproliferative therapy.
  • Expression of the therapeutic gene of interest driven by the AEG-I promoter in susceptible cells may achieve selective growth inhibition, selective reduction or arrest of cell proliferation or selective induction of apoptosis.
  • Susceptible cells are those which permit overexpression of the therapeutic gene of interest due to conditions including but not limited to HIV infection or expression of mutated RAS genes so as to cause selective and relatively enhanced expression of the AEG-I promoter compared to normal cells or cells not otherwise possessing such properties as described supra.
  • the type of cells or cancers treated by a therapeutic agent utilizing an AEG-I promoter is drawn from a group including but not limited to a glioblastoma multiforme cell, a breast cancer cell, a melanoma cell, a colon cancer cell, and a pancreatic cancer cell, hi an additional non- limiting embodiment an AEG-I promoter based therapeutic agent may also be combined, either concurrently or sequentially, with administration of an anti-RAS agent (as described above).
  • the present invention provides for drug discovery/screening assays utilizing expression constructs comprising a reporter gene operably linked to the AEG-I promoter (SEQ ID NO:2), for use in screening systems to detect agents that inhibit AEG-I expression.
  • the invention provides for an assay for identifying an inhibitor of AEG-I promoter activity, comprising (i) preparing a nucleic acid construct having an AEG-I promoter operably linked to a reporter gene (defined herein as a gene having a detectable product) for example, but not limited to, a luciferase gene; (ii) introducing the construct into a cell, such as a eukaryotic cell, for example, but not limited to, an immortalized astrocyte cell or a malignant cell; (iii) exposing the cell to a test agent; (iv) measuring the amount of reporter gene product produced by a cell exposed to the test agent; and (v) comparing the amount of reporter gene product produced by a cell exposed to the test agent to the amount of reporter gene product produced by a cell containing the AEG-I promoter carrying construct which has not been exposed to the test agent.
  • a reporter gene defined herein as a gene having a detectable product
  • a reporter gene defined herein as a gene
  • the cell (generally in a cell culture or organism) exposed to the test agent and the cell not exposed to the test agent should otherwise be maintained under the same or similar conditions.
  • a decrease in the level of reporter gene product in a cell exposed to the test agent has a positive correlation with a decrease in AEG-I promoter activity.
  • a test agent causing such decrease in the level of reporter gene product is therefore identified as an inhibitor of AEG-I promoter activity.
  • Such test agent is further identified as an inhibitor of transcription of an endogenous cellular AEG- 1 gene or inhibitor of transcription of an exogenously introduced AEG-I gene in a cell or tissue of a live organism.
  • the type of cells or cancers utilized in drug discovery using an AEG-I promoter is drawn from a group including but not limited to a glioblastoma multiforme cell, a breast cancer cell, a melanoma cell, a colon cancer cell, or a pancreatic cancer cell, hi an additional non-limiting embodiment an AEG-I promoter based screen may also be combined, either concurrently or sequentially, with administration of an zn ⁇ -RAS agent (as described above).
  • PHFA hTERT-immortalized PHFA
  • HMEC primary human mammary epithelial cells
  • RNA extraction and Northern blot analysis Total RNA was extracted and Northern blotting was performed as described (18).
  • the cDNA probes used were full-length human AEG-I, ⁇ -actin, Ha-ras and GAPDH.
  • Complete Open Reading Frame (C-ORF) Technique C-ORF consists of three steps, reverse transcription, second strand cDNA synthesis and PCR amplification of extended cDNA (Fig. IA).
  • RNA samples (2 ⁇ g) were reverse transcribed by Superscript RT II (Invitrogen) with minor modifications from the manufacturer's protocol using 5 mM DTT, 2 pmole gene specific RT primer (GSPl) and 5 U RNaseOut (Invitrogen) at 45 0 C.
  • First strand cDNA was purified with
  • the annealing mixture was incubated at 95 0 C for 1 min, gradually cooled at 5°C/min to 42 0 C where it was kept for 5 min, supplemented with 5 ⁇ l polymerase mixture consisting of 0.25 ⁇ l Advantage cDNA polymerase mix, 0.5 ⁇ l 10 mM dNTPs and 0.5 ⁇ l 1OX KlenTaq buffer and incubated for 30 min at 68 0 C.
  • Nested PCR reactions (50 ⁇ l) were performed with essentially the same parameters using 0.5 ⁇ l primary PCR product, 5 ⁇ l 1OX KlenTaq buffer, 0.2 mM dNTPs, 10 pmole nested GSP (GSP2), 10 pmole anchor primer and 0.5 ⁇ l Advantage cDNA polymerase mix.
  • GSP2 10 pmole nested GSP
  • anchor primer 10 pmole anchor primer
  • Single primer reaction with GSP2 only or anchor primer only was performed with primary PCR reactions to distinguish potential C-ORF artifacts.
  • PCR reactions were resolved in 1% agarose gels and bands were purified with a gel purification kit (Qiagen). Purified bands were sequenced either with anchor primer or GSP2.
  • AEG-I containing a C-terminal hemagglutinin (HA)-tag was amplified by RT-PCR using primers 5 ' CGGGATCCATGGCTGCACGGAGCTGGCAGGACGA 3 ' (SEQ ID NO:8) and 5 ' CGGGATCCCTCGAGTCACAGCGAAGCGTAGTCTGGGACGTCGTATGGGTA 3 ' (SEQ ID NO:9) and an AEG-I expression vector (pcDNA3.1- AEG- 1-HA) was constructed by cloning the RT-PCR product into Bam HI site of pcDNA3.1/Hygro (+) (Invitrogen).
  • EAAT2 and EAATl (GLAST) promoter-luciferase constructs were described previously (16, 17). Plasmid expressing PTEN was kindly provided by Dr. Ramon Parsons. Protein synthesis and antibody production.
  • a baculoviral transfer vector expressing intein/chitin binding domain fusion protein was constructed by substitution of GST moiety of a baculoviral vector (EcoNl/Pstl fragment of pAcGHLT-B, Phamingen) with intein/chitin binding domain of pTYB12 (NEB, Beverly, MA) obtained by PCR with 5 ' TCCCCTATACTAGGTAAAATCGAAGAAGGTAAACTGGTAA 3 ' and 5 ' CTTCCTTTCGGGCTTTGTTAGCAGCC 3 ' and digestion with EcoNl/Pstl (pAcINT, Fig.
  • AEG-J Full ORF of AEG-J was cloned into EcoRl/XhoI site of pAcINT (pAcINT-AEG) and transfected into Sf9 cells with BaculoGold ® DNA using the manufacturer's protocol (Pharmingen). Baculovirus expressing AEG-I was generated, and amplified in Sf9 cells. The infected insect cell extract was subjected to chitin- affinity column (NEB) and AEG-I protein devoid of chitin binding domain was obtained by incubation of the column in 50 mM DTT that induced intein-mediated protein self-cleavage as suggested by the manufacturer (Fig. 2B, NEB). An anti- AEG- 1 antibody was generated by immunization of chicken with purified AEG-I protein (Genetel Laboratories, Madison, WI).
  • PHFA, IM-PHFA or H4 cells were plated on 12-well plate a day prior to transfection. Cells were co- transfected using the calcium phosphate method (17). Luciferase activity was measured and normalized against protein amount as described (17).
  • FM-516SV cells (2X10 5 cells/6-cm plate) plated the previous day were transfected with either pcDNA3.1, pcDNA3.1 AEG-I, T24 Ha-ras (20 ⁇ g each), or AEG-1/T24 Ha-ras (10 +
  • AEG-I (AS) Adenoviral vector The recombinant replication-defective Ad.AEG-l(AS) was created by cloning Nhel-Xbal fragments from the pcDNA 3.1 vectors into pZeroTg vector in an antisense orientation. Production of infectious virus in 293 cells, analysis of recombinant virus genomes to confirm the recombinant structure, plaque purification, and titration of virus were performed by standard procedures.
  • AEG-I siRNA Constuction of AEG-I siRNA.
  • the sequence (5' AGCAGCCACCAGAGATTGA 3') (SEQ ID NO:3)used for AEG-I silencing corresponds to the 449-467 nucleotides of the AEG-I ORF.
  • the siRNA was constructed with SilencerTM siRNA kit according to manufacturer's protocol. A control scrambled siRNA was used to determine specificity of the inhibition and the sequence 5' GGGTCGTCTA TAGGGATCGAT 3' (SEQ ID NO: 10)
  • C-ORF Complete open reading frame technology
  • C- ORF has been successfully applied to cloning previously unknown genes, including mda-5 (Fig. IB), hPNPase M ⁇ 35 and 5 ' extension and identification of splice variants of PCTA-I in a single step (19-21), which further confirms the utility of this approach.
  • cDNAs cloned by single round C-ORF contained full open reading frame, they ended short by 34 nucleotides (nts) for ISG-56, 27 nts for mda- 9/syntenin and 26 nts for mda-5, respectively.
  • Premature ending of cDNA clone could be ascribed to incomplete reverse transcription reaction caused by RNA secondary structure and annealing preference of degenerate sequence in dSLAP to specific cDNA sequences such as GC rich regions.
  • GC content of dSLAP-annealed sequences in ten C-ORF products was averaged as 70.8%, which implied certain but mild preference for GC-rich regions.
  • the fact that the C-ORF products were almost full-length cDNAs suggested that second strand cDNA synthesis in the procedure preferred starting from the near-end of a cDNA rather than from the middle GC-rich sequence.
  • the stem-loop structural motif of dSLAP that was factored in the primer design might be responsible for the preferential starting of second strand synthesis from the end of a cDNA.
  • Full length cloning of AEG-L Full length AEG-I was cloned by application of C-ORF and bioinformatics. Initially, C-ORF produced from the EST sequence corresponding to 1106-1386 nts ended up to 464 nt whose sequence matched with EST AW978779, AI816426, BF206322 and BE314632 (Fig. 1C).
  • Chitin-binding domain/intein module of the fusion protein was cleaved by treatment with 50 mM DTT after immobilization of the fusion protein on a chitin-agarose matrix (Fig 2B). Contamination of released chitin binding domain/intein in electrophoretogram was removed by simply passing through a chitin- affinity column a second time.
  • the electrophoretic mobility of the tag- free AEG-I protein (M,- 86) was much slower than expected from the predicted molecular mass (64 kDa), but comi grated with HA-tagged AEG-I protein transiently expressed in HEK 293 cells.
  • AEG-I protein The slow mobility of AEG-I protein is probably due to unidentified posttranslational modification or simply to the strong positive charge predicted from high p/ value (9.33).
  • Antibody against AEG-I was raised with the purified recombinant protein in chicken and used for immunodetection (Fig. 2C). Both endogenous and transfected AEG-I-HA proteins detected by anti-HA antibody were also recognized by chicken anti- AEG-I antibody. However, the chicken anti-AEG-1 antibody recognized an additional band of lower intensity, suggesting potential alternative splicing of the endogenous gene.
  • AEG-I Molecular structure of AEG-I.
  • the full-length AEG-I cDNA consists of 3611-b ⁇ , excluding the poly A tail.
  • the ORF from 220 to 1968 nts encodes a putative 582 amino acid protein with a calculated molecular mass of 64-KDa with a p/ of 9.33.
  • Genomic BLAST search identified AEG -1 gene consisting of 12 exons/11 introns at 8q22 where cytogenetic analysis of human gliomas indicated recurrent amplification.
  • Protein motif analysis, such as SMART identified a transmembrane domain, which was supported by independent transmembrane protein prediction methods (PSORT II, TMpred and HMMTOP).
  • Three transcripts of 4, 6 and 9 kb that could be alternative splice variants or unprocessed/partially processed AEG-I transcripts were detected.
  • AEG-I expression was high in two categories of organs, muscle- dominating organs such as skeletal muscle, heart, tongue and small intestine, and endocrine glands including thyroid and adrenal gland, which might suggest a role of AEG-I in calcium-associated processes.
  • FIG. 3B shows the representative band of the 4 kb transcript and it is presently not clear whether the 1.5 kb transcript, that was also reported for mouse AEG-I (3D3/Lyric), is a specific variant in cultured cell lines (35).
  • AEG-I expression was up-regulated in diverse cancer cells including melanoma (HO- 1, C8161 and MeWo), breast cancer (MCF-7, MDA-MB-157, -231 and -453) and glioblastoma multiforme (T98G and Gl 8).
  • AEG-I expression was elevated in adult astrocytes transformed by sequential expression of SV40 T/t antigen, telomerase (hTERT) and T24 Ha-ras (22) and, thereby, displaying an aggressive glioma-like phenotype.
  • AEG-I expression is elevated in malignant glioma cell lines (such as U87 MG, U251 MG, T98G and others) as well as in human samples of brain tumors (Fig 7 A and 7B).
  • AEG-I expression is also elevated in an experimental glioma model in which SV40 T/t antigen, oncogenic ras and hTERT are sequentially overexpressed.
  • Intracellular localization of AEG-I Intracellular localization of AEG- 1 was examined in IM-PHFA by immunofluorescence microscopy with anti- AEG-I polyclonal antibody (Fig. 4). AEG-I was detected at the perinuclear region and in endoplasmic reticulum-like structures, but not at the plasma membrane, and co- localized with ER-specific protein calreticulin, but not with the mitochondrial marker MitoTracker. The observed ER localization of AEG-I favors type Ib membrane topology of the AEG-I protein. Similar localization was also observed when N- terminal or C-terminal HA-tagged AEG-I was overexpressed and immunocytochemistry was performed with anti-HA antibody.
  • AEG-I downregulates EAAT2 promoter activity.
  • Infection of PHFA with HIV-I treatment with g ⁇ l20 or TNF- ⁇ resulted in elevated AEG-I expression and decreased EAAT2 expression (11, 13, 17).
  • the effect of AEG-I expression on EAAT2 promoter activity was analyzed by transient co-transfection of an AEG-I expression vector with a luciferase reporter of the EAAT2 -promoter (Fig. 5A).
  • PI-3K-Akt signaling pathway was required for upregulation of EAAT2-Prom activity by EGF and cAMP (17).
  • high EAAT2 expression in H4 cells might result from constitutive activation of the PI3K-Akt pathway (23).
  • AEG-I expression induced after 3 and 7 days following HIV-I infection, TNF- ⁇ or gpl20 treatment in PHFA (13) may directly contribute to EAAT2 downregulation through a PI3K-Akt-independent pathway.
  • AEG-I promotes anchorage independent growth. Steady state mRNA levels of AEG-I are elevated in diverse cancers, including malignant gliomas, breast carcinomas and melanomas, suggesting an association between AEG-I expression and tumorigenesis (Fig. 3C). Thus, we determined whether AEG-I could alter tumor- associated phenotypes of nontransformed cells by soft agar colony formation assays (Fig. 6A). Expression of AEG-I promoted anchorage independent growth of FM516- SV cells that could not normally form colonies in soft agar.
  • C-ORF uses a structured annealing primer in second strand cDNA synthesis that apparently promotes the reaction to start near the 3 ' end of the first strand cDNA.
  • C-ORF although not completely free from sequence context of target genes, demonstrates successful applications in most applications using a single reaction thus making it a useful and attractive alternative in full-length cDNA cloning.
  • a tag-free recombinant protein production system utilizing self- cleavable intein was originally developed for use in bacterial systems (27).
  • EAAT2 which is primarily expressed in astrocytes, is a major HIV-modulated glutamate transporter in brain and its transcription is downregulated upon HFV infection and gpl20 treatment in astrocytes (16, 17).
  • EAAT2 leading to glutamate excitotoxicity has been implicated as a major determinant of the pathogenesis of HAD and also of various neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis and ischemic injury (31-33).
  • the inverse correlation of AEG-I and EAAT2 expression by HIV infection and AEG-I downregulation of EAAT2 promoter indicate an essential contribution of AEG-I to the generation of HAD.
  • Analysis of AEG-I expression in other neurodegenerative diseases will provide a global perspective of the role of AEG-I in the molecular pathogenesis of these disorders.
  • 3D3/lyric and AEG-I suggest type Ib topology based on ER location, detected by immunofluorescence microscopy, and clusters of basic amino acids at the C-terminal juxtaposition of its transmembrane domain (35). Sequence motif analysis equivocally supports both topologies depending on the algorithm employed.
  • ER, perinuclear or nuclear localization of metadherin is difficult to reconcile with its postulated role in metastasis (35) unless the localization might be cell type dependent. Therefore, the exact localization and membrane topology needs to be clarified by further analysis.
  • Tumorigenic potential of AEG-I was supported by two observations, elevated expression in subsets of cancer cell lines and promotion of anchorage independent growth of immortalized melanocytes and astrocytes. Substantiation of tumorigenic potential also comes from enhanced metastasis of metadherin ⁇ 4E(j-i expressing cells (34).
  • AEG-I may promote tumor progression indirectly, since overexpression of AEG-I did not promote cell proliferation. Synergy with oncogenic Ha-r ⁇ s that could stimulate cell proliferation further supports a more limited (potentially cooperative) role of AEG-I in tumor formation.
  • Anchorage-independent growth is a key component in tumor cell expansion, which might be stimulated by AEG-I upregulation by an unidentified mechanism.
  • THV and THR immortalized human astrocytes were cultured and are described in Rich et al. (22).
  • CREF and CREF-ras rat embryo fibroblasts and FM516 and FM516-ras human immortalized melanocytes are described in (17). All immortalized or cancer cell lines other than THV and THR were cultured as described (18).
  • the full-length AEG-I promoter DNA used in this analysis has a 3' boundary 1 base-pair 5' to the translation initiation site (ATG codon) of the AEG-I gene (designated as nucleotide 1 in Fig. 7; SEQ ID NO:2) and extends 2759 bp upstream.
  • genomic DNA region corresponding to the AEG-I promoter was isolated by PCR using the PCRx Enhancer System (Invitrogen, Carlsbad, CA) with forward primer: 5'- GGTACCCTTTAGTAATCCCTCCCTCTCT -3' (Kpnl site underlined) (SEQ ID NO:4) and reverse primer: 5'- CTCGAGATCTTCCCTCCCGTCAGAGGGACT -3' (Xhol site underlined) 9SEQ ID NO:5).
  • PCR conditions were as follows: Denaturation 95 0 C, 30 sec; annealing 55 0 C, 30 sec and extension 68 0 C, 3 min, repeated for 30 cycles.
  • This region of genomic DNA was cloned into the luciferase reporter pGL-3 basic (Promega, Madison, WI) using restriction sites in Kpnl and MoI in the plasmid polylinker.
  • the nested 5' deletion constructs of the ⁇ EG-i full length promoter were constructed as follows:
  • the clone with a 5' boundary at position 1146 was constructed by PCR using the forward primer 5'- GGTACCGAATTTTTGCAAACCCCTTT -3' (Kpnl site underlined) (SEQ ID NO: 13) and reverse primer 5'-
  • the clone with a 5' boundary at position 787 was constructed by isolating a Sacl-Xhol fragment from pGL3-AEGl full promoter and cloned into the Sacl/Xhol site of pGL3-basic polylinker.
  • the clone with a 5' boundary at position 515 was constructed by isolating a KpnI-PflMI fragment from pGL3-AEGl-787 clone.
  • the cohesive ends made by the restriction enzyme reaction were blunt ended with DNA polymerase I, Klenow fragment (NEB) and then ligated into pGL3-basic.
  • the clone with a 5' boundary at position 350 was constructed by isolating a Kpnl-BssHII fragment from the pGL3-AEGl-787 clone.
  • the cohesive ends made by the restriction enzyme reaction were blunt ended with DNA polymerase I, Klenow fragment (NEB) and then ligated into pGL3 -basic.
  • Putative binding sites for transcription factors shown in Fig. 8 were determined by doing a binding-site consensus search against two transcription factor databases i.e. Matlnspector (www.genomatix.de) and Match (www.gene- regulation.com).
  • CREF, CREF-ras, FM516, FM516-ras, THV and THR cells were plated on 24-well plates one day prior to transfection. Cells were transfected using Lipofectamine 2000 reagent under conditions recommended by the manufacturer (InVitrogen, Carlsbad, CA). Luciferase activity was measured and normalized against ⁇ -galactosidase activity produced by a co-transfected vector as described (17).
  • the PI3 kinase inhibitor LY294002, MEK inhibitor PD 98059 and p38 MAP Kinase inhibitor SB 203580 were used to treat cells at the indicated concentration and to determine the effect of inhibition of the respective pathway on AEG-I promoter activity using the AEG-I luciferase reporter.
  • a binding site for a i ⁇ S-responsive transcriptional element (RREB) (Ray et al., (2003) MoI Cell Biol 23(1): 259-271) was detected between positions 491 and 474 of the MlAEG-I promoter, Fig. 9 (SEQ ID NO:2).
  • the 2759 bp genomic DNA fragment was tested for promoter activity by transient transfection into human immortalized astrocytes (THV), immortalized human melanocytes (FM516) and immortalized rat embryonal fibroblasts (CREF) and in corresponding mutated RAS-gene overexpressing clones of these cell lines i.e. THR, FM516-ras and CREF-ras (Fig. 11).
  • Deletion analysis of the AEG-I promoter was also performed to determine the location of potentially important regulatory regions. Nested 5' deletions constructs of the full length 2759 bp promoter were made with cut-off points at nucleotide positions 1146, 787, 515 and 350 respectively (Fig. 10). These deletion constructs were analyzed for reporter activity compared to the full length promoter. Deletion of approximately 2400 of the 2759 bp contained in the full length promoter reduced the basal activity of the deleted promoter by approximately 2-fold. Deletions upto 515 bp from the 3' end did not significantly affect basal activity of the promoter (Fig. 12, compare activity of the 2759 versus 515 bp fragment).
  • deletion mutants were analyzed for RAS responsiveness in a FM-516-ras-cl7 background
  • deletion of approximately 1600 bp (Fig. 12, compare the 2759 bp construct to the 1146, 787 and 515 constructs) resulted in about 25 percent loss of activity.
  • Most 90% loss of RAS responsiveness was observed when the putative RREB element was deleted (Fig. 12, compare the activity of the 2759 versus the 350 bp reporter constructs).
  • the putative RAS responsive element is located between positions 491-474.
  • the AEG-I promoter shows strong RAS responsiveness.
  • the AEG-I promoter may comprise of a "core-promoter" region as defined by the construct with 5' boundary at position 515 and which retains responsiveness to RAS activation. Additional transcriptional enhancer elements may reside in the region between nucleotide positions 2759 and 1146. Other transcription factors with putative binding sites in the AEG-I promoter as shown in Fig. 10, or additional unknown factors could also play a role in the activity and responsiveness of the promoter.
  • FIG. 13 A-E Effect of inhibition of PD Kinase, MEK kinase and p38 MAP kinase on AEG-I promoter activity.
  • Signal transduction pathways impinging on or directly downstream of the RAS pathway were analyzed to determine their effect on AEG-I promoter activity (Figs. 13 A-E). These analyses were performed utilizing a concentration of 20 and 50 ⁇ M of LY294002 and PD 98059 inhibitors in THV/THR astrocytes (Fig 13. A), and CREF and CREF-ras fibroblasts (Fig. 13 B). A dose dependent inhibition of promoter activity was observed in the cell lines tested.
  • the p38 MAPK pathway was inhibited utilizing 5 ⁇ M of SB 203580 in THV/THR astrocytes (Fig. 13C) and CREF and CREF-ras fibroblasts (Fig. 13C). Again, inhibition of promoter activity was observed in the presence of the respecitve inhibitor.
  • a RAS overexpressing immortalized melanocyte line (FM516-ras7, Fig. 13E) was tested for the effect of inhibiting PI3K and p38 MAPK activity on AEG-I promoter activity. Again, inhibition of the AEG-I promoter was observed following treatment with inhibitor.
  • AEG-I promoter activity in human cancer cell lines Human cancer cell lines endogenously expressing mutated RAS genes were tested for the ability to support AEG-I promoter activity. This contrasts with the earlier series of cells where a mutated RAS gene was exogenously introduced into the cell.
  • the cell lines tested included the human pancreatic cancer derived cells PANC-I, AsPC-I, MiaPaCa-2 (all containing mutated k-RAS) and BxPC-3 (wild type k-RAS).
  • PANC-I and AsPC-I cells supported relatively high levels of AEG-I promoter activity while BxPC-3 and Mia PaCa-2 cells showed relatively lower transfected AEG-I luciferase reporter activity (Fig. 14A).
  • the colorectal cancer cell line HCTl 16 which is mutated in the k-RAS gene was also tested for AEG-I promoter activity in addition to two related cell lines.
  • C2 is a clone of HCTl 16 somatically knocked out for the k-RAS gene and the other, ClO is a clone derived from C2 wherein a mutated Ha-RAS gene has been reintroduced into C2 so that it now expresses a mutated Ha-RAS gene instead of a mutant k-RAS gene.
  • HCTl 16 cells support approximately 70-fold and 22-fold higher activity of the AEG-I promoter compared to C2 and ClO lines respectively (Fig. 14B).
  • AEG-I promoter activity in the parental HCTl 16 line was inhibited to a lesser extent than astrocytes and fibroblasts described in the earlier section. However some inhibition of promoter activity was observed with all inhibitors in this cell line (Fig. 14C).
  • the C2 and ClO cell lines were however not responsive to PD 98059 and showed some responsiveness with the other two inhibitors.
  • the AEG-I gene product can activate its own promoter.
  • Transfection of the full length AEG-I luciferase reporter construct into a FM516 melanocyte stably expressing AEG-I protein showed that a positive feedback loop may cause AEG-I gene product to enhance its own expression level (Fig 15A).
  • This responsiveness to AEG-I was shown by all deletion constructs described previously except the reporter deletion containing only nucleotides 1 to 350 of the AEG-I promoter (Fig. 15B).
  • the HIV- Tat (Simm et al Virology 2002 ;294(1):1-12) and Ne/(Lathi et al., Virology. 2003; 310(1):190-196) gene products were tested on the full length AEG-I promoter at a plasmid expression vector concentration range of 50 to 800 ng/transfection point (Fig. 16). While the Tat gene product had slight (1.5 -fold) stimulatory activity under the conditions tested, the Ne/ gene product showed approximately 5-fold activation over control at the highest amount (800 ng) of plasmid tested. Thus, the AEG-I promoter appears to be responsive to protein products produced by HIV infection of cells.
  • the AEG-I promoter showed a dose responsive stimulation of promoter activity when cells transfected with the luciferase reporter was treated with Bromo-cyclicAMP at a range of 0.1 to 100 ⁇ M (Fig. 17) when tested in primary human fetal astrocytes (PHFA).
  • PHFA primary human fetal astrocytes

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Abstract

La présente invention repose, au moins en partie, sur la découverte selon laquelle l’expression de Gène 1 élevé astrocyte ('AEG-1 ') (i) supprime le promoteur de Transporteur 2 d’acide aminé excitateur ('EAAT-2'), inhibant ainsi le transport de glutamate ; (ii) supporte la formation de cellules en colonie indépendante de l’ancrage, avec une synergie vis-à-vis de l’oncogène RAS ; et (iii) est augmentée dans un certain nombre de différentes maladies malignes. L’invention, selon divers modes de réalisation, concerne des procédés de traitement de maladies malignes et de troubles neurodégénératifs à l’aide d'inhibiteurs de l’activité AEG-1 et concerne des essais de criblage pour identifier d’autres composés ayant des effets bénéfiques thérapeutiques.
PCT/US2005/006639 2005-02-25 2005-02-25 Gène 1 élevé astrocyte et son promoteur dans les traitements de neurotoxicité et de maladies malignes Ceased WO2006093494A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090324596A1 (en) * 2008-06-30 2009-12-31 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers
US10745701B2 (en) 2007-06-28 2020-08-18 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BROWN D.M. ET AL.: "Metadherin, a cell surface protein in breast tumors that mediates lung metastasis", CANCER CELL, vol. 5, 2004, pages 365 - 374, XP002999405 *
SU Z.-Z. 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, vol. 306, 2003, pages 67 - 78, XP004416780 *
SU Z.-Z. ET AL.: "Insights into glutamate transport regulation in human astrocytes: Cloning of the promoter for excitatory amino acid transport 2 (EAAT2)", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 100, no. 4, 2003, pages 1955 - 1960, XP002968061 *
SUTHERLAND H.G.E. ET AL.: "3D3/lyric: a novel transmembrane protein of the endoplasmic reticulum and nuclear envelope, which is also present in the nucleolus", EXPERIMENTAL CELL RESEARCH, vol. 294, 2004, pages 94 - 105, XP002984409 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10202605B2 (en) 2007-06-28 2019-02-12 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers
US10745701B2 (en) 2007-06-28 2020-08-18 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers
US20090324596A1 (en) * 2008-06-30 2009-12-31 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers

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