MX2007004364A - E2-epf5, a novel therapeutic protein and target - Google Patents

Abstract

The present invention relates to novel uses for ubiquitin conjugating enzyme E2-EPF5. In particular, inhibition of E2-EPF5 activity is shown to reduce the production VEGF, as well as other proteins regulated the transcription factor HIF-1, in response to hypoxia. Based on these findings the present invention provides therapeutic methods and pharmaceutical compositions useful for the treatment of diseases related VEGF induced angiogenesis. In addition, E2-EPF5 is provided as target for the development of therapeutics. Accordingly, the invention provides screening methods for identifying candidate compounds that inhibit E2-EPF5 activity and may therefore be used to treat VEGF induced angiogenesis such as tumor related angiogenesis. Finally, the invention also provides methods for the inhibition of VEGF and other HIF-1 regulated proteins.

Description

E2-EPF5. A NOVELTY THERAPEUTIC PROTEIN AND DIANA METHODS OF CLASSIFICATION Field of the Invention The present invention pertains to therapeutic methods and pharmaceutical compositions for the inhibition of VEGF-dependent vascularization and, in particular, of vascularization related to tumors. Background of the Invention Vascular endothelial growth factor (VEGF) encodes a protein responsible for multiple of the vascular responses observed in human tumors, including the stimulation of growth of new blood vessels and the permeabilization thereof. VEGF is essential for the establishment of angiogenesis in most solid tumors and the constitutive upregulation of VEGF expression is seen as a major contributor to tumor angiogenesis. The expression of VEGF and various other angiogenic growth factors are critically regulated by hypoxia involving the transcriptional induction of the VEGF gene by the transcription factor HIF-1 (factor-1 inducible by hypoxia). Hypoxia is a reduction in normal tissue oxygen tension homeostasis levels and occurs during acute and vascular disease, lung disease and cancer. For example, tumor cells adapt to hypoxia by inducing HIF-1. The HIF-1 mediates adaptive responses to changes in tissue oxygenation and activates the transcription of a variety of genes involved in multiple processes that include cell proliferation, cell survival, invasion and metastasis, apoptosis, angiogenesis, glucose and iron metabolism. HIF-1 is overexpressed in numerous human cancers and is associated with poor prognosis and increased patient mortality. Recent studies have indicated that HIF-1 mediates resistance to chemotherapy and radiation due to hypoxia, activation and oncogene mutations. The inhibition of HIF-1 activity may therefore represent an important component of anti-angiogenesis therapies. E2-EPF5 is a member of the enzyme family (E2) of ubiquitin conjugation that conjugates ubiquitin to cellular proteins. Ubiquitin and ubiquitin-like modifiers are processed and bound to substrate proteins by a mechanistically conserved enzymatic cascade, which includes enzymes (E1) of ubiquitin activation, enzymes (E2 or UBCs) of conjugation of ubiquitin and ligases (E3) of ubiquitin Such modifications have different functions, the best characterized of them being the degradation of ubiquitin-dependent protein by the trajectory of the 26S proteosome. Both the E2s and the E3s cooperate to transfer ubiquitin to the target protein or target thus determining the substrate specificity. Although the availability of the human genome sequence has opened the possibility to complete the identification of the ubiquitin family members including E2s, E3s and desubicitination proteases, only a few substrates of ubiquitination and the understanding of the ubiquitin system have been identified. its implications in human diseases is still in its infancy. There is, however, growing evidence that the modification of cellular proteins by ubiquitin and ubiquitin-like molecules is an essential regulatory mechanism in multiple biological processes, especially in cell cycle progression, cell differentiation and DNA repair. The present invention now provides a new role for the E2-EPF5 enzyme conjugation of ubiquitin in hypoxia and, in particular, in the construction of proteins regulated by the transcription factor HIF-1 in response to hypoxia. Brief Description of the Invention In a first aspect, the invention provides a method for the treatment of diseases related to aberrant neovascularization comprising administering an effective amount of an agent that inhibits the expression of the gene encoding the conjugation E2-EPF5 enzyme. of ubiquitin or that inhibits the activity of the E2-EPF5 gene product. In a preferred embodiment, neovascularization is angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neovascularization as seen in diabetic retinopathy, skin angiogenesis in psoriasis, or hypoxia-induced angiogenesis in cirrhosis of the liver. In a particularly preferred embodiment, the neovascularization is VEGF-dependent tumor angiogenesis. In one aspect of the invention, the agent is an inhibitory nucleic acid capable of specifically inhibiting the E2-EPF5 enzyme of ubiquitin conjugation, preferably an antisense oligonucleotide compound, more preferably a siRNA compound. The other aspect of the invention, the agent is an antibody that binds specifically to the E2-EPF5 enzyme of ubiquitin conjugation. The other aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of an agent that inhibits the expression of the gene encoding the E2-EPF5 enzyme of ubiquitin conjugation or that inhibits the activity of the E2- gene product. EPF5. Preferably, the E2-EPF5 inhibitor is an antisense oligonucleotide or a siRNA or an antibody that specifically binds E2-EPF5. In a further aspect, the present invention relates to methods for identifying compounds useful for VEGF-dependent vascularization treatment comprising: (a) contacting an E2-EPF5 polypeptide with a test compound (b) that detects modulation of the biological activity of E2-EPF5. Preferably, the biological activity of E2-EPF5 is reduced. In still another aspect, the present invention relates to a method for reducing the amount or for inhibiting the activity of one or more polypeptides regulated by HIF-1 in response to hypoxia, which comprises inhibiting the expression of conjugation of ubiquitin related to the enzyme E2-EPF5. Preferably, the genes or proteins regulated by HIF-1 are selected from the group consisting of GLUT-1, GLUT-3, HK2, EPO, NOS2, VEGF, TGF-alpha, TGF-beta, VEGFR-2, C-Met , UPAR, CXCR4, carbonic anhydrase IX (CAIX). Detailed Description of the Invention The present invention is based on the surprising discovery that the inhibition of the E2-EPF5 enzyme from ubiquitin conjugation interferes with the expression of HIF-1-dependent protein in response to hypoxia. In particular, it was found that the inhibition of E2-EPF5 interferes with the accumulation of VEGF in response to hypoxia. Proangiogenic VEGF is required for the proliferation and migration of the endothelial cells that constitute the first blood vessels. VEGF is a 34 to 45 kDa glycoprotein with a wide range of activities that, in addition to the promotion of angiogenesis, also include increased vascular permeability, a crucial aspect of inflammation. VEGF not only acts as an endothelial cell growth factor, but also in several other cells including Kaposi sarcoma cells associated with HIV and other tumor cells and leukemia. Neoplastic cells often express high levels of VEGF, which are not only thought to be related to the stimulation of angiogenesis, but also to the activation of autocrine signaling pathways of proliferation stimulation. In this contrast, it is important to consider that in addition to a direct effect via the transcription trajectories of signals initiated in the VEGF receptors, VEGF also induces indirect effects. For example, the Notchl receptor is up-regulated by VEGF also allowing an increased response for the ligands of this cell surface receptor as well. Expression of VEGF has been associated with various pathological conditions, such as tumor angiogenesis, hypoxia-related angiogenesis, various forms of blindness (eg, diabetic retinopathy, proliferative diabetic retinopathy, age-related macular degeneration), rheumatoid arthritis, psoriasis and healing of wounds and others. A number of studies have shown that high levels of VEGF alone are sufficient to induce neovascularization. For example, it has been shown that a single injection of VEGF increased the development of collateral vessels in a rabbit ischemia model (Takashita et al., 1995 J; Clin. Invest. 93, 662). VEGF can also induce neovascularization when injected into the cornea. The present invention now provides methods for reducing the amount or activity of one or more polypeptides regulated by HIF, and in particular VEGF, which accumulates in response to hypoxia. A number of genes encoding proteins with a variety of functions are known to be regulated by HIF under hypoxia, including, but not limited to, the following genes: GLUT-1, GLUT-3, HK2, CAIX (involved for example in adaptation metabolic); EPO, NOS2 (involved for example in resistance to apoptosis); VEGF, TGF-alpha, TGF-beta, VEGFR-2 (involved for example in angiogenesis), C-Met, UPAR, CXCR4 (involved for example in invasion, tumor metastasis). The term "polypeptide regulated by HIF" in the context of the present invention includes any protein Or polypeptide, whose expression is controlled or influenced by the transcription factor HIF-1, for example by binding of HIF-1 to a transcriptional regulatory element of the gene encoding said protein or polypeptide. Preferably, protein expression is activated by the activity of the transcription factor HIF-1, i.e., more protein is expressed in response to activation of HIF-1. HIF-1 is a heterodimer consisting of one to three alpha subunits (HIF-1a, HIF-2a or HIF-3a) and a beta subunit (HIF-1β, also known as the Nuclear Translocator of Hydrocarbon Aryl, or ARNT ). HIF-1b is expressed constitutively, whereas the expression of alpha subunits is highly regulated. As for any other protein, the level of the alpha subunits is determined by the protein synthesis and protein degradation regimes. The synthesis of the HIF1 alpha subunits is regulated via oxygen-independent mechanisms, whereas degradation is regulated mainly via 02-dependent mechanisms. Thus, although the genes for the alpha subunits are transcribed and translated mostly continuously, the subunit proteins alpha are maintained at very low levels due to rapid destruction via proteosomal degradation. This destruction is inhibited under hypoxic conditions and this is the main induction mechanism of HIF1a and the genes dependent on this transcription factor. E2-EPF5 encodes a 25 kDa class II ubiquitin conjugation enzyme with a basic C-terminal extension of 65aa long of low sequence complexity. E2-EPF5 was first discovered in a patient suffering from a skin disease called follicular pemphigus endemic (EPF) (Liu et al., 1992, JBC 267, 15829). It was later postulated that E2-EPF5 is functionally distinct from other characterized E2 isoenzymes since it catalyzes the formation of multi-ubiquitin chain via the K11 residue and not through the K48 residue, which is a mechanism that is considered to average Proteolytic events (Bach and Ostendorff, Trends in Biochemical Sciences (2003), 28 (4), 189-195). It was also found that E2-EPF5 supports self-ubiquitination that suggests a possible self-regulatory model for E2-EPF5. Substrates for E2-EPF5 have not yet been reported, but their highly basic carboxy terminal extension domain, which is unique within members of the E2 family may indicate specificity for acidic proteins (Liu et al., J. Biol. Chem. 271, 2817-2822). The activity of the term (biological) E2-EPF5 includes, within the context of the present invention, in addition to its activities relative to ubiquitin also the interference with HIF-1-mediated induction of genes regulated by hypoxia. The sequence of the human E2-EPF5 is available from the public databases (GenBank Accession M91670, Gl: 181915, SwissProt entry Q16763). The cDNA sequence is set forth as SEQ ID NO: 1. The amino acid sequence is set forth as SEQ ID NO: 2. However, the term E2-EPF5 also includes any homologous or heterologous sequences, variants and fragments so long that they maintain the biological activity of E2-EPF5 described herein. The percentage of homology between the homologous sequence and the reference sequence is desirably at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferable at least 99%. The sequence comparisons are carried out using a Smith-Waterman sequence alignment algorithm (see for example http://www-to.usc.edu/software/seqaln/index.html). A "fragment" means any polypeptide molecule having at least 5, 10, 15 or optionally at least 25, 35 or 45 contiguous amino acids of E2-EPF5. Possible additional fragments include the site or catalytic domain including the recognition sites, ubiquitin binding sites, important sites for subunit interaction, and important sites to carry out the other functions of the ubiquitin conjugation enzyme. Such domains or motifs can be identified by means of computerized routine homology search procedures. Fragments, for example, may extend in one or both directions from the functional site to encompass 5, 10, 15, 20, 30, 40, 50 or up to 100 amino acids. Also included in the term fragment are, for example, epitopes of E2-EPF5. An epitope of E2-EPF5 represents a site in the polypeptide against which an antibody can be produced and to which the antibody binds. Therefore, polypeptides comprising the amino acid sequence of an E2-EPF5 epitope are useful for making antibodies to the E2-EPF5 polypeptide. Preferably, an epitope comprises a sequence of at least 5, more preferably at least 10, 15, 20, 25 or 50 amino acid residues in length. In one aspect, the invention provides methods for the treatment of aberrant neovascularization comprising administering an effective amount of an agent that inhibits the activity of E2-EPF5 enzyme relative to the conjugation of ubiquitin. In a related aspect, the present invention provides the use of an agent that inhibits the activity of E2-EPF5 for the manufacture of a medicament for the treatment of a pathological condition related to VEGF-dependent vascularization, in particular for tumor angiogenesis. The term "aberrant neovascularization" as used herein means a vascularization that does not normally occur in a healthy organism and is related to an abnormal state or disease. Aberrant neovascularization is controlled or is preferably influenced by VEGF activity, ie "VEGF-dependent vascularization". In a particular preferred embodiment the aberrant neovascularization is VEGF-dependent tumor angiogenesis. The term "VEGF-dependent vascularization" as used herein refers to any generation of new blood vessels by stimulation by VEGF and includes, without limitation, angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neovascularization as observed in diabetic retinopathy and some other diseases of the eye, skin angiogenesis in psoriasis, or hypoxia-induced angiogenesis in cirrhosis of the liver. The other aspect, the present invention provides a method for inhibiting a gene regulated by HIF-1 in a cell comprising inhibiting E2-EPF5 expression or activity of enzyme relative to the conjugation of ubiquitin. Inhibition of the gene regulated by HIF-1 can be achieved, for example, by decreasing the amount of HIF-1 by the degradation of ubiquitin-dependent protein, for example by interfering with the synthesis or stabilization of HIF-1a protein or the transactivation of HIF. -1a. In one embodiment, the present invention provides a method for reducing the amount of a polypeptide regulated by HIF-1 which comprises inhibiting the expression or activity of E2-EPF5 of enzyme relative to the conjugation of ubiquitin. In a particular preferred embodiment, the gene regulated by HIF-1 is VEGF. Accordingly, the present invention further provides anti-angiogenic methods. Thus, methods are provided for the inhibition of angiogenesis, including tumor angiogenesis, which comprise inhibiting E2-EPF5 expression or activity of enzyme relative to the conjugation of ubiquitin. In the context of gene expression or protein activity, the term "inhibition" means a reduction of gene expression or protein activity. Preferably, such reduction is at least 20%, more preferably at least 50%, 60%, 70%, 80%, 90% or 95% compared to the level of expression or activity without inhibition. The inhibition of genes or proteins can be achieved by any suitable technique. The skilled person knows a variety of methods and techniques of how to inhibit gene expression or protein activity. For example, E2-EPF5 can be inhibited by RNA or antisense interference technologies or by using LMW compounds that interfere with the E2-EPF5 function or by any agent that decreases E2-EPF5 enzyme relative to ubiquitin conjugation. An agent that inhibits the activity of E2-EPF5 of enzyme relative to the conjugation of ubiquitin can be any substance that reduces the biological activity of E2-EPF5. The agent can inhibit, for example, the expression of an E2-EPF5 gene or an enzymatic activity of E2-EPF5, can induce the degradation of E2-EPF5 polypeptides or can interfere with the biological activity of E2-EPF5 in any other way. In a preferred embodiment, the inhibitory agent is a low molecular weight compound or an inhibitory nucleic acid or an antibody. As contemplated herein, the term "inhibitory nucleic acid" refers to nucleic acid compounds capable of causing gene-specific inhibition or gene expression. Typical inhibitory nucleic acids include, but are not limited to, antisense oligonucleotides, DNA aptamers, triple helix RNA, ribozymes, and siRNA. For example, knowledge of a nucleotide sequence can be used to design siRNA or antisense molecules that potentially inhibit E2-EPF5 expression of enzyme relative to ubiquitin conjugation. Similarly, ribozymes can be synthesized to recognize specific nucleotide sequences of a gene and cleave it. Techniques for the design of such molecules for use in the inhibition for the purpose of gene expression by any person skilled in the art are well known. The inhibitory nucleic acid compounds of the present invention can be synthesized by conventional means in a commercially available automated DNA synthesizer, for example a DNA / RNA synthesizer model 380B, 392 or 394 from Applied Biosystems (Foster City, CA), or instrument Similary. Phosphoramidite chemistry can be used. The inhibitory nucleic acid compounds of the present invention can also be modified, for example, nuclease resistant structures such as, for example, phosphorothioate, phosphorodithioate, phosphoramidate or the like, described in many references, can be used. The length of the inhibitory nucleic acid must be sufficient to ensure that the biological activity is inhibited. Thus, for example in the case of antisense oligonucleotides, it has to be large enough to ensure that specific binding will take place only in the target polynucleotide and not in other random sites. The upper range of length is determined by several factors, including the inconvenience and expense of synthesizing and purifying oligomers greater than about 30 to 40 nucleotides in length, the greater tolerance of longer oligonucleotides for inequalities than shorter oligonucleotides and the like. Preferably, the antisense oligonucleotides of the invention have lengths in the range of about 15 to 40 nucleotides. More preferably, the oligonucleotide portions have lengths in the range from about 18 to 25 nucleotides. Double-stranded RNA molecules, ie sense-antisense RNA, also called low-interference RNA (siRNA), can also be used to inhibit the expression of nucleic acids for E2-EPF5. RNA interference is a method in which exogenous RNA duplexes are administered, short where one strand corresponds to the coding region of the target mRNA (Elbashir et al., Nature 2001, 411: 494-498). Upon entering cells, siRNA molecules cause not only the degradation of exogenous RNA duplexes, but also single-stranded RNAs that have identical sequences, including endogenous messenger RNAs. Consequently, the siRNA may be more potent and effective than traditional antisense RNA methodologies since it is believed that the technique acts through a catalytic mechanism. The preferred siRNA molecules are typically from 19 to 25 nucleotides long, preferably about 21 nucleotides in length and comprise the sequence of a nucleic acid for E2-EPF5. Effective strategies for delivering siRNA to target cells include, for example, transduction using physical or chemical transfection. Alternative siRNAs can be expressed in cells using, for example, several Pollll promoter expression cassettes that allow transcription of functional siRNA or precursors thereof. See, for example, Scherr et al., Curr. Med. Chem. 2003, 10 (3): 245-256; Turki et al., Hum. Gene Ther. 2002, 13 (18): 2197-2201; Cornell et al., Nat. Struct. Biol. 2003, 10 (2): 91-92. The invention also covers other small RNAs capable of mediating the interference of RNA (RNAi) such as for example micro-RNA (miRNA) and short-hair RNA (shRNA). In another preferred embodiment, the agent that inhibits E2-EPF5 activity of enzyme relative to the conjugation of ubiquitin is an antibody. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, fragments produced by a Fab expression library. , anti-idiotypic antibodies (anti-ld) and epitope binding fragments of any of the foregoing. Various methods known in the art can be used for the production of polyclonal antibodies. Various methods known in the art can be used for the production of antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target or target gene product, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals can be immunized by injection with E2-EPF5 polypeptides, derivatives or fragments, supplemented with suitable auxiliaries. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the Kohler and Milstein hybridoma technique, (1975, Nature 256: 495-497), the human B-cell hybridoma technique, and the Hybridoma-EBV technique. Such antibodies can be of any kind of immunoglobulin including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma that produces the mAbs of this invention can be cultured in vitro or in vivo. The production of high titers of mAbs in vivo makes this the currently preferred production method. In addition, techniques developed for the production of "chimeric antibodies" (ie, a molecule in which different portions of different species of animals, such as those that have a variable or hypervariable region derived from a mAb and a constant region of human immunoglobulin) by dividing the genes of a mouse antibody molecule of antigen-appropriate specificity together with genes from a human antibody molecule of appropriate biological activity. Alternatively, the techniques described for the production of single chain antibodies can be adapted to produce E2-EPF5 antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Such techniques are known in the art. In a preferred embodiment, the E2-EPF5 antibody is a "Humanized antibody". Techniques useful for the production of "humanized antibodies" can be adapted to produce antibodies to the polypeptides, fragments, E2-EPF5 derivatives and functional equivalents described herein. Such techniques are described in the patents of US Pat. No. 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,545,580; 5,661,016 and 5,770,429, whose descriptions of all are incorporated herein by reference in their entireties. Antibody fragments can be generated that recognize specific E2-EPF5 epitopes by known techniques. For example, such fragments include, but are not limited to: fragments of F (ab ') 2 that can be produced by digestion of pepsin and the antibody molecule and Fab fragments that can be generated by reducing disulphide bridges the fragments of F (ab ') 2. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Suitable antibodies for E2-EPF5 proteins can be obtained from a commercial source or produced according to conventional methods. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, single domain antibodies, Fv fragments, Fab fragments, F (ab ') fragments. 2 > fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-ld) and epitope binding fragments of any of the foregoing. A wide variety of antibody / immunoglobulin frameworks or scaffolds may be employed as long as the resulting polypeptide includes one or more binding regions that are specific for the E2-EPF5 protein. Such frameworks or scaffolds include the 5 major idiotypes of human immunoglobulins or fragments thereof (such as those described elsewhere herein) and include immunoglobulins from other animal species, preferably having humanized aspects. Single heavy chain antibodies such as those identified from camelids are of particular interest in this regard. It continues to discover and develop frameworks, scaffolding and novel fragments by those skilled in the art. Alternatively, non-immunoglobulin frameworks and scaffolds may be employed, as long as they encompass a specific binding region for the E2-EPF5 protein. Known non-immunoglobulin frameworks or scaffolds include Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA) maxicuerpos (Avidia, Inc., (Mountain View, CA)), Protein A (Affibody AG, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany). According to the present invention, the anti-E2-EPF5 antibody or fragments thereof, or the polypeptide comprising a specific binding region of E2-EPF5, independently of the framework or scaffolds employed, can be bound, in a manner either covalent or non-covalent, to an additional portion. The additional portion can be a polypeptide, an inert polymer such as PEG, small molecule, radioisotope, metal, ion, nucleic acid or other biologically relevant type of molecule. Such a construct, which may be known as an immunoconjugate, immunotoxin or the like, is also included in the meaning of antibody, fragment or antibody polypeptide comprising a specific binding region of E2-EPF5., as used in the present. In another embodiment of the present invention, the inhibitory agent is a small molecule (e.g., organic or inorganic molecules that are less than about 2 kDa in molecular weight, more preferable than less than about 1 kDa in molecular weight, and / or are able to cross the blood barrier of the brain or enter an appropriate cell) which affects the expression of E2-EPF5 or the polypeptide activity of E2-EPF5. Such a small molecule compound can be identified by the classification methods described below. One aspect of the present invention provides the gene and gene product of E2-EPF5 as a drug target for the development of therapeutics for use in the treatment of individuals suffering from a disease as described above. Provided herein, therefore, as part of the present invention, there are classification methods for identifying compounds that can be used to treat diseases such as those described herein. These methods comprise, in preferred embodiments, contacting a test compound with a reaction mixture containing an E2-EPF5 polypeptide. In preferred embodiments, E2-EPF5 is a polypeptide having an amino acid sequence which may comprise the sequence set forth in SwissProt entry Q16763. However, any homolog, ortholog, variant, etc. can be used. of E2-EPF5 in these methods, as fusion constructs of those polypeptides (e.g., a fusion construct as described in the examples, infra). For example, the E2-EPF5 polypeptide may have an amino acid sequence that is substantially homologous (eg, at least 75%, 80%, 85%, 90%, 95% or 99% identical) to the SwissProt entry Q16763 . In a particularly preferred embodiment, E2-EPF5 is provided as a novel target or target for the classification of therapeutics useful in the treatment of diseases to which VEGF-dependent angiogenesis plays a role and, in particular, in tumor angiogenesis or vascularization. ocular or hypoxia-induced angiogenesis. The present invention provides methods for identifying a compound useful for the inhibition of VEGF-dependent vascularization comprising (a) contacting an E2-EPF5 polypeptide with a test compound (b) that detects a modulation of the biological activity of E2 -EPF5. The modulation is usually detected with respect to a control reaction that the test compound does not have. Modulation, as used herein, refers to an increase or reduction in biological activity, preferably at least 10%, at least 20%, at least 30%, at least 50% or so less 100% In another embodiment, the present invention provides a method for identifying a compound useful for the treatment of a disease related to VEGF-dependent vascularization, eg, tumor vascularization, comprising: i) contacting a test compound with a polypeptide of E2-EPF5 under permissive sample conditions for biological activity of E2-EPF5; ii) determining the level of said at least one biological activity of E2-EPF5; iii) comparing said level with that of a control sample that lacks said test compound; and iv) selecting a test compound that causes said level to change for further testing as a compound for the prophylactic and / or therapeutic treatment of a disease related to VEGF-dependent vascularization. Classification assays of compounds can include cell-based or cell-free systems. The cell-based systems may be native, ie, cells that normally express the E2-EPF5 of ubiquitin conjugation enzyme, as a biopsy or expanded in cell culture. In another embodiment, however, the cell-based assays involve recombinant host cells that express the ubiquitin conjugation enzyme. Determining the ability of the test compound to interact with the E2-EPF5 of ubiquitin conjugation enzyme can also comprise determining the ability of the test compound to preferentially bind with the polypeptide as compared to the ability of a known binding molecule ( for example, ubiquitin) to bind to the polypeptide. The polypeptides can be used to identify compounds that modulate the conjugation activity of ubiquitin. Such compounds, for example, may increase or decrease the affinity for the ubiquitinated protein substrate, or remnants of the ubiquitinated protein substrate. Such compounds could also, for example, increase or decrease the rate of binding to these compounds. Such compounds could also compete with these components to bind to the ubiquitin conjugation enzyme or displace these components bound to the ubiquitin conjugation enzyme. Such compounds could also affect the interaction with other components, such as ATP, other subunits, such as E1 activating enzymes or E3 ligases. E2-EPF5, ubiquitin conjugation enzyme derivatives and fragments can be used in rapid classification methods, for example automated high-throughput (HTS) filters to assay candidate compounds for the ability to bind to the ubiquitin conjugation enzyme. Numerous rapid classification tests are known to be suitable by the skilled person. These compounds can be further classified against E2-EPF5 of functional ubiquitin conjugation enzyme to determine the effect of the compound on the E2-EPF5 of ubiquitin conjugation enzyme. Compounds that activate (agonist) or inactivate (antagonist) E2-EPF5 can be identified to a desired degree. Modulator methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The E2-EPF5 polypeptides of the present invention can be used to classify a compound for the ability to stimulate or inhibit the interaction between the E2-EPF5 protein and a target molecule that normally interacts with the E2-EPF5 protein. The target may be ubiquitin, ubiquitinated substrate, or polyubicuitin or another component of the pathway with which the ubiquitin conjugation enzyme protein (e.g., E1 or E3 proteins) normally interacts. The assay includes the steps of combining the E2-EPF5 protein with a candidate compound under conditions that allow the E2-EPF5 protein or fragment to interact with the target molecule, and to detect the formation of a complex between the E2-EPF5 protein and the target or to detect the biochemical consequence of the interaction with E2-EPF5 and the target. Any of the associated effects of the ubiquitin conjugation function can be tested. This includes the production of ubiquitinated substrates, proteolysis, decreased free polyubicuitin, substrate stability. Determining the ability of the ubiquitin conjugation enzyme to bind to a target molecule can also be performed using a technology such as Real Time Bimolecular Interaction Analysis (BIA). As used in this, "BIA" is a technology to study biospecific interactions in real time, without labeling any of the interactants (for example, BIAcoret®). Changes in surface plasmon resonance (SPR) in the optical phenomenon can be used as an indication of the real-time reactions between biological molecules. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: spatially treatable parallel phase or solid phase libraries in solution; library synthesis methods that require deconvolution; library method of "one pearl one compound"; and methods of library synthesis using chromatographic affinity selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or libraries of small molecule compounds (Lam, K S. (1997) Anticancer Drug Des. 12: 145) . Examples of methods for the synthesis of molecular libraries can be found in the art, for example in Proc. Nati Acad. Sci., DeWitt et al., (1993), E.U.A. 90: 6909; Proc. Nati Acad. Sci., E.U.A. Erb et al., (1994), 91: 11422; J. Med. Chem. Zuckermann et al., (1994), 37: 2678; Science 261: 1303, Cho et al., (1993); Angew. Chem. Int. Ed. Engl. 33: 2059, Carell et al., (1994); Angew. Chem. Int. Ed. Engl. 33: 2061, Carell et al., (1994); and J. Med. Chem. 37: 1233, Gallop et al., (1994). Libraries of compounds that can be presented in solution (for example, Houghten (1992) Biotechniques 13: 412-421), or in beads (Lam (1991) Nature 354: 82-84), chunks (Fodor (1993) Nature 364: 555 -556), bacteria (Ladner U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. '409), plasmids (Culi et al., (1992) Proc. Nati. Acad Sci. USA 89: 1865-1869 ) or phage (Scott and Smith (1990) Science 249: 386-390); (Devlin (1990) Science 249: 404-406); Cwirla et al., (1990) Proc. Nati Acad. Sci. 97: 6378-6382); Felici (1991) J. Mol. Biol. 222: 301-310); (Ladner supra). Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tail fusion peptides and members of random peptide libraries (see, for example, Lam et al., (1991) Nature 354: 82-84; Houhgten et al., (1991) Nature 354: 84-86) and molecular libraries derived from combinatorial chemistry made of amino acids of D- and / or L- configuration; 2) phosphopeptides (eg, members of random and partially degenerate direct phosphopeptide libraries, see, for example, Songyang et al (1993) Cell 72: 767-778); 3) antibodies (eg, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric and single chain antibodies as well as Fab, F (ab ') 2, fragments of Fab expression library, and epitope binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from libraries of combinatorial and natural products). Suitable assays for measuring the activity of the enzyme relative to ubiquitin conjugation are well known in the art. These assays include, but are not limited to, the appearance of the substrate, including the increase in the amount of protein from polyubicutin or ubiquitinated substrates or protein remaining, appearance of intermediates and end products, such as the disappearance of ubiquitin-free monomers, protein rotation in general, specific protein rotation, ubiquitin binding, protein binding of the ubiquitinated substrate, interaction of subunits, interaction with ATP, interaction with cellular components such as trans-acting regulatory factors, stabilization of specific proteins and the like. In one aspect, identified compounds are provided by the classification methods according to the present invention. Such compounds are preferably low molecular weight compounds or antibodies, in particular monoclonal antibodies, or inhibitory nucleic acids. The compounds preferably have anti-angiogenic activity, that is, they inhibit the growth of blood vessels in general and in particular the aberrant neovascularization. The anti-angiogenic activity of such can be determined by methods known in the art. Such methods include, for example, microscopic evaluation of angiogenesis in tumors, migration assays of matrigel and angiogenesis in vivo, angiogenesis assay of microparticle release of alginate, disk angiogenesis assay, angiogenesis sponge implant model, hollow fiber assay for tumor angiogenesis or corneal assay for angiogenesis. Such assays are described, for example, in "Angiogenesis Protocols" March 2001, ISBN: 0-89603-698-7, Series: Methods in Molecular Medicine, Volume #: 46. In order to develop angiogenic and anti-angiogenic strategies , concerted efforts have been made to provide animal models for further quantitative analysis of angiogenesis in vivo. In vivo techniques consist of implanting the cornea and iris pouch in the eye, the rabbit ear chamber, the dorsal skin fold chamber, the cranial window, the hamster cheek pouch window, the implant trial of sponge, fibrin clots, sodium alginate beads and Matrigel plugs, mesenteric rat window, chorioallantoic membrane of chicken embryos and the air sac in mice and rats (1). In this chapter we will discuss the avascular cornea assay, and the advantages and disadvantages of using this assay in different species. The corneal test is based on the placement of an angiogenic inducer (tumor tissue, cell suspension, growth factor) in a corneal pocket in order to evoke the vascular growth of peripherally placed limbal vasculature. Compared to other in vivo assays, this test has the advantage of measuring only new blood vessels, because the cornea is initially avascular. A further aspect of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions comprise an effective amount of an agent that inhibits the expression of the gene encoding the E2-EPF5 enzyme of ubiquitin conjugation or that inhibits the activity of the E2-EPF5 gene product. They may comprise, for example, antibodies, mimetics, agonists, antagonists, or nucleic acids inhibitors of E2-EPF5 ubiquitin conjugating enzyme according to the present invention. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose and water. . The compositions can be administered to a patient alone or in combination with other agents, drugs or hormones.

The pharmaceutical compositions encompassed by the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-articular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, Enteral, topical, sublingual or rectal. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. More details on formulation and administration techniques can be found in the latest edition of Remington's Pharmaceuticals Sciences (Maack Publishing Co., Easton, Pa.). Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, pastes, suspensions and the like, for ingestion by the patient. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. The salts tend to be more soluble in aqueous solvents or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1% -2.0% sucrose and 2-7% mannitol, at a pH range of 4.5 to 5.5, which is combined with shock absorber before use. After the pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration the labeling must include quantity, frequency and method of administration. Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is in the capacity of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate range of concentration and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient, fragments thereof, antibodies, agonists, antagonists or inhibitors of the E2-EPF5 ubiquitin conjugation enzyme, which improves the symptoms or condition. The therapeutic efficacy and toxicity can be determined by normal pharmaceutical procedures in cell cultures or experimental animals, for example, ED50 (the therapeutically effective dose in 50% of the population) and LD50 (the lethal dose for 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index, and can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The information obtained from cell culture assays and animal studies is used in the formulation of a dosage range for use in humans. The dosage contained in such compositions is preferably within a range of circulating concentrations that include ED50 with or without low toxicity. The dosage varies within this range depending on the dosage form used, the sensitivity of the patient and the route of administration. The exact dosage will be determined by the physician, in light of factors related to the subject that requires treatment. The dosage and administration are adjusted to provide sufficient levels of the active portion or to maintain the desired effect. Factors that can be taken into account include the severity of disease status, the general health of the subject, the age, weight and gender of the subject, diet, timing and frequency of administration, combination (s) es) of drugs, reaction sensitivity and tolerance / response to therapy. The long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week or once every two weeks depending on the half-life and elimination regime of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of approximately 1 g, depending on the route of administration. The guide as to the dosages and particular methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, the delivery of polynucleotides or polypeptides will be specific for cells, conditions, locations, etc., in particular. Pharmaceutical formulations suitable for oral administration of proteins are described, for example, in the patents of U.S. Nos. 5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; 5,976,569 and 6,051,561. Having generally described this invention, further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. Examples 1. Antisense Oliqonucleotides (ASO) and siRNA The siRNAs are available from a number of commercial suppliers, such as for example Qiagen, Dharmacon, Proligo. The oligonucleotides can be synthesized using TOM-phosphoramidite chemistry, as described by the manufacturer (Xeragon) and purified by RP-HPLC. The purity is evaluated by capillary gel electrophoresis. The quantification is carried out by UV according to the extinction coefficient at 260 nM. The double-stranded RNA recosido (dsRNA) is performed as described elsewhere (Elbashir et al., 2001). Modified antisense oligodeoxyribonucleotides are synthesized using phosphoramidite chemistry purified by HPLC, analyzed and characterized by electro-mass mass spectrometry and capillary gel electrophoresis. The quantification is carried out by UV according to the extinction coefficient at 260 nM. All oligonucleotides are designed against the human E2-EPF5 sequence (GenBank Accession M91670, Gl number, Gl: 181915, SwissProt entry Q16763) and verified by BLAST against the Refseq and UNIGENE databases to maximize the level of specificity. In order to identify the most potent oligonucleotides, a series of ASOs and target siRNAs from E2-EPF5 are classified in a dual color reporter test as previously described (Hüsken, D. et al., 2003). A vector containing the coding sequence of E2-EPF5 is recombined with a target vector containing the reporher sequences CFP- and YFP. The resulting dual replicer plasmid pNAS-X generates a fusion mRNA where the target sequence of E2-EPF5 is inserted into the 3'-UTR region of the expressed YFP reporter gene. The ASO activity of E2-EPF5 and siRNA is measured by the degree of inhibition of the YFP reporter protein. The expression of eCFP protein serves as a means to normalize the efficiency of plasmid transfection. The list of classified E2-EPF5 oligonucleotides is shown in Table 1 (ASOs pointing to E2-EPF5, N = DNA, n = DNA with 2'-0-methoxyethyl, s = phosphorothioate internucleotide bond, c = 2'- methoxyethyl 5-methyl cytidine) and Table 2 (target siRNAs E2-EPF5), respectively (only the antisense sequence is shown N (G, C, A, U) = ribonucleoside, n (g, a, t) = deoxyribonucleoside) . The majority of active oligonucleotides (8162, 17.828, 11.723) and corresponding controls are used in follow-up studies. Table 1 Table 2 2. Cell culture All reagents of the cell culture are from Gibco-Invitrogen AG (Basel, Switzerland). The basal culture medium used is Dulbecco Modified Eagle's Medium (DMEM) for HeLa and RPMI1640 cells for NCI-H1299 cells. The media is supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine plus optionally 50 micrograms / ml of antibiotic Gentamicin. For routine maintenance the cells are divided 1: 4 twice a week. On the day before transfection, the cells are trypsinized, resuspended in medium with FBS, but without antibiotics, and plated, either in 24-well culture plates at a density of 1.5 to 2 x 10 4 cells per 0.5 ml per well or in six-well plates at a density of 5 to 10 x 104 cells per 2 ml per well. The plates are incubated for 24 hours at 37 ° C in C025% at high humidity. 3. Transfection of siRNAs and deferrioxamine (DFO) and treatment of cobalt chloride Transfection of siRNAs is performed using Oligofectamine according to the protocols provided by the manufacturer (Invitrogen). Briefly, the siRNAs are diluted in Optimem up to 1.2 μ? of siRNA by μ ?. Separately, Oligofectamine is diluted to 0.25 μ? of Oligofectamine by μ? of Optimem. Equal amounts of these siRNA and oligogyctamine solutions are mixed and incubated for 20 to 25 minutes at room temperature to allow complex formation to proceed. Next, the siRNA / Oligofectamine complexes are diluted in standard medium with 10% FBS without antibiotics to the desired final concentration, typically 20 to 80 nM of siRNA. Subsequently, the old medium is removed from the cells and replaced with siRNA / Oligofectamine suspension: for 24-well plates 0.5 ml per well and for 6-well plates 0.5 ml per well. After 48 to 72 hours of incubation in a humidified C02 incubator, the media with the siRNAs are replaced with fresh media with 10% FBS either without inducer or containing 150 μ? of desferrioxamine (abbreviated: DFO) or CoCI2 (both from Sigma, from recently prepared aqueous buffer solutions). After 6 h at 37 ° C the cell medium is harvested for determination of secreted VEGF while cell monolayer total cell extracts are prepared for Western blotting or total RNA analysis. 4. Transfection of ASOs and DFO and treatment of cobalt chloride: The ASOs are stored at 1 mM TE concentration (10 mM Tris, pH 8.0, 1 mM EDTA). Before the experiment, a working solution 10 μ? in Optimem (Life Sciences Inc.). The ASOs are diluted 2 x to the final concentration in Optimem, mixed with Lipofectin or Effectene diluted also at 2 x the final concentration and left at room temperature for 30 minutes. The standard medium is removed from a confluent T24 cell culture of 30 to 50% and the cell monolayer once with serum free OptiMEMI (Gibco BRL). The mixture of ASO / Lipofectin is then added to the cell monolayer which is then incubated 4 h at 37 ° C in the CO 2 incubator, after which the medium is removed and replaced with standard medium with 10% FBS. After 24 to 72 hr of incubation in a humidified C02 incubator, the media is replaced with fresh media with 10% FBS either without inducer or containing 150 μ? of desferrioxamine (abbreviated: DFO) or CoCI2 (both from Sigma, from recently prepared aqueous buffer solutions). After 6 h at 37 ° C the cell medium is harvested for determination of secreted VEGF while the cell monolayer total RNA is prepared or global cell protein extract for Western blot analysis. 5. Real-time reverse transcriptase PCR Total RNA is prepared using the RNeasy 96 package (Qiagen # 74183) following the manufacturer's instructions. Primary pairs and TaqMan probes labeled FAM for real-time PCR are designed using the Primer Express v1.0 program (ABI PRISM, PE Biosystems) and purchased from Microsynth (Switzerland), Qiagen or Applied Biosystems ("Demand Tests"). The following primary sequences are used: E2-EPF5 (Acc. Nr Gl 181915): Reverse primed: 5'-AAAGACCTTGATGCCATCGG-3 '(SEQ ID NO: 27) Primary forward: 5'-TCCGCCTGGTGTACAAGGA-3' (SEQ ID NO. : 28) TaqMan probe: 5'-FAM-TGACGACACTGACCGCAGACCCA-TAMRA-3 '(SEQ ID NO: 29) HIF-1a (Acc. Nr): NM_18105 ABI Demand test: HsOCM 53153_ml VEGF (Acc. Nr): NM_003376 Reverse Primary: 5'-CACATTTGTTGTGCTGTAGGAAGC-3 '(SEQ ID NO: 30) Primary adelate: 5'-TGAGATCGAGTACATCTTCAAGCC-3' (SEQ ID NO: 31) TaqMan Probe: 5'-FAM- CCATGCAGATTATGCGGATCAACCTCA-TAMRA-3 '(SEQ ID NO: 32) OR ABI Test on demand: Hs00173626_ml GLUT-1 (Acc. Nr): NM_006516 ABI Test on demand: Hs00197884_m1 Hexokinase 2 (Acc. Nr). NM_000189. Reverse primed: 5'-TGTCTTGAGCCGCTCTGAGAT-3 '(SEQ ID NO: 33) Primary forward: 5'-TGTCCGTAACATTCTCATCGATTT-3' (SEQ ID NO: 34) TaqMan probe: 5'-FAM-CCAAGCGTGGACTGCTCTTCCGAG-TAMRA-3 '(SEQ ID NO: 35) Beta Actin (Acc, Nr): X00351 Reverse Primary: 5'-TAATGTCACGCACGATTTCCC-3 '(SEQ ID NO: 36) Primary forward: 5'-TCACCGAGCGCGGCT-3' (SEQ ID NO: 37) TaqMan Probe: 5'-FAM -CAGCTTCACCACCACGGCCGA-TAMRA-3 '(SEQ ID NO: 38) Bcl-XL (Acc. Nr): Z23115 Reverse Primary: 5'-GGTCGCATTGTGGCCTTT-3 '(SEQ ID NO: 39) Primary forward: 5'-TCCTTGTCTACGCTTTCCACG-3' (SEQ ID NO: 40) TaqMan Probe: 5'- FAM-ACAGTGCCCCGCCGAAGGAGA-TAMRA-3 '(SEQ ID NO: 41) CYPA (Cyclophilin-A, Acc. Nr): NM_021130 Reverse Primary: 5'-TCGAGTTGTCCACAGTCAGCA-3 '(SEQ ID NO: 42) Primary forward: 5'-GCGCTTTGGGTCCAGGA-3' (SEQ ID NO: 43) TaqMan Probe: 5 '-FAM-TGGCAAGACCAGCAAGAAGATCACCA-TAMRA-3' (SEQ ID NO: 44) For the real-time PCR reaction, 25 ng of total RNA are mixed in 6 μ? of water with 0.33 μ? of primary 5 'and 3' (10 μ? each), 0.33 μ? of TaqMan probe (5 μ?), 6.33 μ? RT PCR of Master Mix package (Eurogentec, # RT-QRT-032X) in a total volume of 13 μ? following the manufacturer's instructions. Reverse transcription and real-time PCR is performed on a 5700 or 7700 ABI PRISM sequence detector (Applied Biosystems) as follows: 30 minutes reverse transcription at 48 ° C, 10 minute denaturation at 95 ° C followed by 50 cycles of denaturation during 15 seconds at 95 ° C and annealing and elongation for 1 minute at 60 ° C. Relative quantification of gene expression is calculated using the delta-Ct method as described in ABI PRISM 5700 user bulletin # 2. Western blot Was prepared from cells in mammalian protein extraction reagent M-PER according to the protoCol provided by the manufacturer (Pierce, Rockford, IL). 10 μg of total protein extract are loaded in pre-processed acrylamide gels at 8% (NOVEX, San Diego, CA) and after electrophoretic separation are transferred into PVDF membranes. After saturation of the TBST-5% milk powder membrane, the proteins are immunostained with the relevant primary antibodies diluted in TBST-5% milk powder. Then, the membrane is rinsed 3 x with TBST and for 30 min incubated with the appropriate secondary antibody also dissolved in TBST-5% milk powder. Then the membrane is rinsed 3 x with TBST and Western ECL blot detection reagents (Amersham Biosciences) are used to visualize the immunostained according to the manufacturer's protocol. The dilutions used for primary antibodies are 1: 250 for mouse anti-HIF-1a (BD Biosciences / Pharmingen 610958), 1: 100,000 for anti-mouse p-actin (Sigma A5441), 1: 2000 for anti-tRNA ( Aryl Hydrocarbon Receptor Nuclear Translocator, Novus NB 730-H), 1: 500 for rabbit anti-GLUT1 (Glucose Transporter ABCAM ab652), 1: 1,000 for rabbit anti-HIF2a-EPAS1 (Novus ab199 Cat. No. 730 -H) As secondary antibodies are used: goat anti-mouse IgG (Fab-specific) -peroxidase antibody (Sigma A2304) that at a dilution of 1: 1: 5000-20,000 or goat polyclonal antibody to rabbit IgG (conjugated HRP) Novus catalog number NB 730-H at a dilution of 1: 20,000. 7. Quantification of VEGF secretion Secreted VEGF is determined using an enzyme-linked immunoassay package (ELISA) for human VEGF (R &D Systems) according to the manufacturer's instructions. 8. Characterization of VEGF secretion In order to characterize the specific gene inhibitors of E2-EPF5, NCI-H1299 and HeLa cells are exposed to E2-EPF5 siRNAs and the inhibition of E2-EPF5 mRNA was studied by TaqMan RT-PCR. The E2-EPF5 mRNA is reduced from 70 to 90% after 48 h, respectively 72 h of siRNA incubation in both cell lines. The unequal and non-relative control siRNAs do not affect the expression of the E2-EPF5 gene. In the same manner, NCI-H1299 cells expressing E2-EPF5 recombinant labeling with an HIS-FLAG epitope at the N terminus are exposed to E2-EPF5 siRNAs and controls. All the siRNAs that equalize decrease the E2-EPF5 protein levels compared specifically with the controls, with 17828 showing the strongest reduction. 9. Inhibition of E2-EPF5 suppresses VEGF expression in hypoxia NCI-H1299, HeLa and DU-145 cell lines, which have been reported to be operable for oligonucleotide transfection, are exposed to hypoxia-mimicking compounds: desferrioxamine (DFO) ), cobalt chloride (CoCl2) and N-Oxalylglycine. The results show that in NCI-H1299 and HeLa cells, DFO consistently induced VEGF mRNA 4-7 times and VEGF protein 2-3 times after 6 and 24 h of stimulation. The degree of induction by CoCl2 is similar, but less consistent, declining after prolonged stimulation. N-oxalylglycine significantly induces VEGF expression especially after 6 h. The basal level of expression of VEGF mRNA and protein (2 ng of VEGF protein secreted per h of cells per million cells) is found to be approximately 10 times higher (2160 ± 120 pg VEGF / h, 106 cells) in DU- 145 than in the other two cell lines tested (NCI-H1299: 318 ± 40 pg VEGF / h, 106 cells; HeLa: 301 ± 60 pg VEGF / h, 106 cells). The induction of VEGF of 2-3 times significant in RNA and 1.3-1.5 times at the protein level can be demonstrated only with all inducers, but only after 6 no after 24 h of stimulation. Notably, under the conditions applied, it was not found that the E2-EPF5 mRNA is significantly regulated in any of the cell lines investigated. To confirm the results of the reporter-HRE gene, the effect of siRNA-mediated inhibition of E2-EPF5 on the expression of target genes driven by endogenous HRE, especially vascular endothelial growth factor (VEGF) is examined. The NCI-H1299 cells are exposed for three days to E2-EPF5 siRNAs followed by a 6 h exposure with DFO and CoCl2, respectively. As shown in Table 4, all the E2-EPF5 siRNAs induce a marked reduction of VEGF secretion compared to cells transfected with corresponding unequal and non-relative control oligonucleotides. In alignment with the activities observed in the suppression of siRNA of E2-EPF5 17828 leads to the most significant suppression of VEGF. The same results are obtained using CoCl2 in place of DFO as a hypoxia inducing agent.

Table 4: Effect of inhibition of E2-EPF5 mediated with siRNA over VEGF secretion. The NCI-H1299 cells are transfected with 60 nM siRNAs against E2-EPF5, which corresponds to unequal controls and an unrelated control siRNA. 72 h later, the cells are incubated with DFO 150 μ? for 6 h compared to the untreated 8548 control. The VEGF protein concentrations in conditioned media are determined by ELISA and expressed as pg / ml by the total amount of protein in each well. A further analysis applying the more potent E2-EPF5 17828 siRNA reveals that in both NCO-H1299 and HeLa cell lines, the inhibition of E2-EPF5 reduces the induced expression of VEGF mRNA and the secretion of VEGF protein induced by hypoxia. The reversion of mRNA and hypoxic VEGF protein is almost complete at the levels observed in normoxically cultured cells as this is the case through a control siRNA that points to the transcription factor HIF-1a. In addition, as shown in Table 5, the reductive regulation of E2-EPF5 inhibits another endogenous Hif-1 target gene, namely GLUT-1 (glucose transporter 1) while the HIF-1 mRNA is not significantly changed. This suggests that the suppression of E2-EPF5 inhibits the Hif-1-mediated transcriptional activation of HRE-driven genes without affecting the transcription of Hif-1 itself.

Table 5: Inhibition of siRNA-mediated E2-EPF5 suppresses the hypoxia-induced expression of GLUT-1 mRNA to a degree similar to that of a siRNA targeting HIF-1. HeLa cells are transfected with 40 nM siRNAs against E2-EPF5 (17828), HIF-1a (25560), corresponding to unequal controls (E2-EPF5: 25296, Hif-1a: 25584) and a non-related control siRNA 8548. 72 h later, the cells are incubated with DFO 150 μ? for 6 h compared to the untreated 8548 control. The GLUT-1 mRNA is assayed by TaqMan RT-PCR. 10. Expression of E2-EPF5 and cell proliferation E2-EPF5 encodes an enzyme (E2) conjugation of ubiquitin. The E2s 1 in ubiquitin to cellular proteins thus converting them into targets for proteosomal degradation or modulating their function, similar to phosphorylation. The trajectories of ubiquitin play a key role in the regulation of cell growth and proliferation by controlling the abundance of cell cycle proteins (Bashier et al., 2003) and it has been found that many components of the ubiquitination machinery are deregulated, mutated or amplified in several cancers and / or correlated with a poor prognosis. The expression of E2-EPF5 in various human tissues is examined by TaqMan PCR in real time using specific primers for E2-EPF5. E2-EPF5 is upregulated slightly in the thymus and testes suggesting a potential relationship between cell proliferation and E2-EPF5 expression (Table 6). Tissue Units Arb STDEVP 1 brain 0.33 0.08 2 heart 0.38 0.35 3 kidney 0.04 0.04 4 liver 0.03 0.03 5 lung 0.10 0.01 6 trachea 0.13 0.03 7 bone marrow 0.94 0.20 8 colon 0.33 0.14 9 intestine 0.33 0.12 spleen 0.44 0.19 11 stomach 0.11 0.00 12 thymus 1.59 0.53 13 heart 0.71 0.03 14 mammary gland 0.15 0.00 prostate 0.84 0.47 16 skeletal muscle 0.03 0.01 17 testicles 3.78 0.01 18 uterus 0.27 0.02 19 brain 0.78 0.19 cerebellum 0.92 0.12 21 fetal brain 0.71 0.11 22 fetal liver 0.87 0.24 23 spinal cord 0.50 0.05 24 placenta 0.17 0.06 adrenal gland 0.32 0.07 26 liver 0.04 0.02 27 pancreas 0.01 0.00 28 prostate 0.39 0.01 29 salivary gland 0.03 0.01 30 thyroid 0.03 0.02 Table 6: Expression of E2-EPF5 mRNA in various tissues (Clontech). Tissue distribution is determined by real-time TaqMan PCR using specific primers for E2-EPF5. 11. Cloning of human E2-EPF5 cDNA in vector PDONR201 The E2-EPF5 cDNA is cloned by two sequential PCR reactions followed by the insertion of a Gateway ™ donor vector. The first DNA amplification is done in the presence of 1 ng of Quick-Clone ™ cDNA from human fetal liver tissue (Clontech) using 10 pmol each of the PCR primaries specific for E2-EPF5 (forward: ATC GAA GGT CGT ATG AAC TCC AAC GTG GAG AAC CTA CCC CCG (SEQ ID NO: 45), reverse: TCA CTT GTC GTC GTC GTC CTT GTA GTC CAG CCG CCG CAG CGC CCG CAG CGC CCG (SEQ ID NO: 46), 10 nmol each of the dNTPs, 2 mM of MgSO2, 5 U of DNA polymerase TaKaRa Ex Taq ™ in 50 μ? of Ex Taq ™ cushion, oversized with 50 μ? of mineral oil, in a thermocycler block. The PCR cycling conditions are as follows: 94 ° C for 10 min, [94 ° C for 1 min, 62 ° C for 1 min, 72 ° C for 1 min] 30 cycles, 72 ° C for 10 min, after 10 ° C sustained. The PCR products were analyzed by PAGE. The DNA is eluted from agarose by the Gene Clean II package. The weak DNA bands are reamplified by the same protocol. A typical yield of amplified PCR product is about 8 μg of DNA in 50 μ? of H20. The PCR product is composed of 5'-FXa site-specific sequence FLAG tag-3 '. The second DNA amplification is done in the presence of 100 mg of DNA template or template of the first PCR reaction, 100 pmol each of the primary PCR ATTB1FXA2 (GGG ACA AGT TTG TAC AAA AAA GCA GGC TTA GCT GGT ATC GAA GGT CGT ATG (SEQ ID NO: 47)) and ATTB2FLAG (GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TCA CTT GTC GTC GTC GTC CTT GTA GTC (SEQ ID NO: 48)), 20 nmol each of the dNTPs, 2 mM MgSO4, 10% DMSO, 2.5 U Pwo DNA polymerase in 100 μ? of buffer (Tris-HCl 10 mM pH 8.8, KCI 25 mM, (NH4) S04) 5 mM, treated with 100 μ? of mineral oil, in a thermocycler block. The PCR cycling conditions are as follows: 94 ° C 2 min, [94 ° C for 1 min, 62 ° C for 1 min, 72 ° C for 1 min] 2 cycles, [94 ° C for 1 min, 60 ° C for 1 min, 72 ° C for 1 min] 2 cycles, [94 ° C for 1 min, 55 ° C for 1 min, 72 ° C for 1 min] 30 cycles, 72 ° C for 2 min, then 10 ° C sharp. The att-PCR products are analyzed by PAGE. The DNA was purified for cloning with the PCR Purification package (Qiagen). Typically, the performance of att-PCR amplified products is 7 to 8 μ9 of DNA in 50 μ? of H20. The att-PCR product is composed of 5'-FXa E2 site-specific sequence FLAG tag-ATTB2-3 '. The att-PCR product is cloned into the Gateway ™ donor pDONR201 vector. The BP Clonasa ™ enzyme mixture catalyses a site-specific and site-specific recombination reaction in vitro via the attB1 / B2 (PCR product) and attP1 / P2 (vector) sites. The reaction mixture (20 μm) contains BP buffer (as provided), 200 ng of att-PCR DNA, 300 ng of pDONR201 vector and 4 μl of BP Clonasa ™ enzyme mixture. After 1 h at 25 ° C proteinase K (2 μg / μL) is added and the samples are incubated for 10 min at 37 ° C. 1 μ? _ Is used to transform competent E. coli DH5a cells. Transformants are selected on LB plates with kanamycin (50 mg / L). The clones are characterized using restriction enzymes and by DNA sequencing. A suitable clone is designated pBM2537 / NPL002981. 12. Cloning of E2-EPF5 cDNA into Gateway ™ expression vector pDEST12.2 Clonasa ™ LR enzyme mixture mediates the GATEWAY LR recombination reaction via the attL / L2 (entry clone pBM2537) and attR1 / R2 sites (vector pDEST12.2), The reaction mixture (20 μ?) contains LR buffer (as provided by the manufacturer), 200 ng of input clone DNA (pBM2537), 300 ng of expression vector (pDEST12.2 , GIBCO-BRL-Invitrogen Corp.) and 4 μ? of Clonasa ™ LR enzyme mixture. After 1 h at 25 ° C proteinase K (2 μg / μL) was added and the mixtures were incubated for 10 min at 37 ° C. 1 μl was used to transform the competent DH5a cells. Transformants are selected on LB plates with ampicillin (50 mg / L). The clones are characterized using restriction enzymes and by DNA sequencing. A suitable clone is designated pDEST-EPF5 / NPL006653. 13. Transfection of NCI-H1299 with pDEST type expression plasmids Media, fetal calf serum (FCS), Versene, Lipofectin, Geneticin are purchased from Life Sciences Inc. Petri dishes from cell culture (d = 8 cm) used are Falcon type 3003, 6-well, 24-well and 96-well multi-plates are obtained from Nunc (Life Sciences, Inc.). NCI-H1299 cells (CRL-5803) can be obtained from the American Type Culture Collection (ATCC). The cells are maintained at 37 ° C in a humidified atmosphere with 5% C02 in RP I1640 with 10% FCS and 60 μ? /? T? of Gentamicin. To propagate the culture, the cells are divided weekly: that is, they are rinsed twice with Versene, treated for 5 min with trypsin-EDTA solution, diluted in 15 x of the volume of the original medium and re-plaquena 10 ml / 8 cm of tissue culture box or 20 ml per 75 cm2 of flask T). After 3 to 4 days, 0.5 volume of medium is added to replenish the nutrients. The NCI-H1299 cells are transfected using Lipofectamine Plus reagent (Life Sciences-Invitrogen Corp.). Briefly, the cells are seeded in multi-boxes from 6 wells to 1 x 105 for 6 wells and grown for 1 day. In two wells of a 96-well plate, 2 μg of plasmid DNA (pDEST-12.2 or pDEST-EPF5) is mixed with 100 μ? of Optimem and 15 μ? of PLUS reagent, and incubated at room temperature for 15 minutes. In parallel, 10 μ? of Lipofectamine with 100 μ? of Optimem medium and also incubate for 15 minutes at room temperature. Subsequently, the DNA mixtures are added to the Lipofectamine solution, mixed well and incubated again at room temperature for 15 minutes. At halftime, the medium containing FCS is aspirated from the cells, the cells are rinsed with 1 ml / well of Optimem and then 1 ml of Optimem is added to each well. Then, 100 μ? of the plasmid / PLUS / Lipofectamine reagent complex to the medium and the cells (now in 1150 μl of medium) are incubated for 4 h at 37 ° C in a humidified incubator under 5% CO. At this point, the DNA-liposome mixture it is removed from the cells and 2 ml of fresh RPMI1640 medium is added (with 10% FCS and 60 μg / ml of Gentamicin) The next day the cells are trypsinized as described above, resuspended in 3 ml of RPMI1640 medium with FCS and 300, 150, 60 or 30 μ? of the cell suspension are coated in 8 cm Petri dishes with 10 ml of RPMI1640 with 10% FCS and 60 μg / ml of Gentamycin The next day 1.0 mg / ml is added of Gentamicin The selective medium (RPMI1640 + FCS 10% + 1 mg / ml Gentamicin) is replaced twice a week with fresh medium.Colonies appear after 2 to 3 weeks.On a plate with well-growing colonies separated by pDEST-EPF5, they are scraped 24 using a pipettor with a tip of 100 μ? while simultaneously aspirating eagerly scraped cells towards the tip. The tip cells are then transferred to a 24-well plate and 0.5 ml of selective medium is added. After 5 to 10 days the taken cells have formed a confluent monolayer. The cells of such a confluent of 24 wells are trypsinized, divided into two "wells" of 24 wells and 1 of 6 wells. A few days later, the total RNA is isolated from one of the 24 wells of each clone and the mRNA level of E2-EPF5 is measured by RT-PCR as described in example 5. This RT-PCR measures the combined level of the recombinant E2-EPF5 mRNA from the integrated copy (or copies) of pDEST-EPF5 and the native endogenous E2-EPF5 mRNA. In NCI-H1299 cells successfully transformed by pDEST-EPF5 the combined E2-EPF5 mRNA is more than 2.5 x greater than in native H1299 cells or cells transformed with pDEST12.2. Three independent NCI-H1299 / pDEST-EPF5 clones are amplified (nrs 1, 5 and 15). Expression of recombinant E2-EPF5 by NCI-H 1299 / pDEST-EPF5 cells is confirmed in Western blots as described in Example 6 using anti-FLAG epitope antibodies (Sigma). As a control several Petri dishes with approximately 200 colonies of NCI-H1299 cells transformed by pDEST12.2 are trypsinized and propagated to be used as a negative control in experiments with NCI-H1299 / pDEST-EPF5 cells. 13. Increased secretion of VEGF in PDEST-EPF5 cell lines NCI-H1299 To determine the effect of forced overexpression of E2-EPF5-FLAG the cell lines described in example 12 are coated in plates from 24 wells to 20 wells., 000 cells per well. 20 h later, the medium is replaced by means of RPMI1640 + 10% FCS or RPMI1640 medium + 10% FCS also containing 150 μ? of DFO. 6 h later the medium is harvested and frozen for determination of the VEGF protein levels (example 7), while the total RNA is isolated from the cells and the level of a mRNA species group is measured by RT-PCR (example 5). The amount of VEGF protein secreted by NCI-H1299 cells transformed with pDEST-EPF5, which express a high level of E2-EPF5, is significantly higher (2 ± 0.3 times more) than the amount secreted by pDEST12.2 cells, which express normal E2-EPF5 levels (Table 7). In contrast, induction by DFO remains essentially unchanged (Table 7). However, an equivalent DFO induction in combination with high basal VEGF levels implies that the absolute levels of VEGF after induction with DFO is twice as high as in NCI-H1299 cells that do not express elevated E2-EPF5. Protein level STDEV Induction STDEV na VEGF% DFO% NCI-H1299 Parent line 100 4 419 33 NCI-H1299 / vector pool 87 3 366 4 NCI-H1299 / EPF5 # 1 191 31 396 13 NCI-H1299 / EPF5 # 5 236 21 309 7 NCI-H1299 / EPF5 # 15 185 7 355 14 NCI-H1299 / EPF53 lines 204 28 353 44 Table 7: VEGF protein levels in 6 h conditioned medium of pDEST12.2 and NCI-H1299 cells transformed with pDEST-EPF5 compared to the level in NCI-H1299 parent cells. 14. Levels of messenger RNA in cell lines pDEST-EPF5 NCI-H1299 Using the total RNA isolated from NCI-H1299 cells transformed with plasmid (Example 13), the levels of several species of mRNAs are determined by RT-PCR (example 5). ). The mRNA level of E2-EPF5 (recombinant + native E2-EPF5 mRNA) is in cells transformed with pDEST-EPF5 2.3 to 3 times more than in the parental NCI-H1299 cells (Table 8). This is accompanied by a significantly elevated level of the mRNA of mRNA of 3 genes regulated by known HIFs: VEGF mRNA (38 ± 10% increased), hexocinase-2 mRNA (HK2, 4.9 ± 2.5 times increased) and GLUT-1 mRNA ( 3.8 ± 0.2 times increased). In contrast, the basal levels of mRNAs from a number of genes not regulated by HIF are not significantly changed (β-actin, Bcl-xL, CYPA and HIF1a). The rate of induction of genes regulated by HIF by DFO was not altered in cells overexpressing E2-EPF5, but the combined effect of higher basal mRNA levels and induction by normal DFO results, however, in higher levels of these mRNAs than respond to HIF than those observed in NCI-H1299 cells stimulated with DFO that do not express extra E2-EPF5.

Regulated by HIF Reference genes EPF5 VEGF HK2 GLUT1 actin Bd-xL CYPA HIF1a H1299 parental line 108 89 275 170 114 120 98 131 H1299 / vector pool 100 100 100 100 100 100 100 100 H1299 / EPF5 # 1 245 143 334 382 145 102 80 124 H1299 / EPF5 # 5 304 143 362 347 90 51 86 121 H1299 / EPF5 # 15 235 127 771 415 118 154 98 136 H1299 / EPF5 all 3 261 138 489 381 118 102 88 127 H1299 parental line STDEV 19 12 120 65 8 18 20 14 H1299 / vector pool STDEV 9 4 31 38 10 30 14 22 H1299 / EPF5 # 1 STDEV 85 28 117 11 5 20 22 42 H1299 / EPF5 # 5 STDEV 79 6 98 11 18 2 22 12 H1299 / EPF5 # 15 STDEV 40 15 227 42 10 64 1 13 H1299 / EPF5 all 3 STDEV 37 10 251 18 27 55 16 22% Inductton bv DFO EPF5 VEGF HK2 GLUT1 actln Bcl-xL CYPA HIF1a? 12T9 parental line n.d. 419 479 254 59 53 74 49 H1299 vector pool No. 326 1395 198 58 60 74 31 H1299 / EPF5 # 1 No. 403 701 258 59 81 108 49 H1299 / EPF5 # 6 No. 256 725 292 65 76 78 87 H1299 / EPF5 # 15 nd 249 344 248 63 34 89 90 H1299 EPF5 all 3 nd 303 590 266 63 64 92 69 H1299 parental line STDEV n.d .. 78 278 45 4 7 11 6 H1299 / vector pool STDEV No. 33 345 4 12 14 8 8 H1299 / EPF5 # 1 STDEV No. 18 145 60 4 13 18 10 H1299 / EPF5 # 5 STDEV No. 19 143 78 15 6 12 6 H1299 / EPF5 # 15 STDEV nd. 7 58 83 5 6 6 5 H1299 / EPF6 all 3 STDEV n.d .. 87 169 23 3 18 16 14 Table 7: mRNA expression levels in cells lines pDEST1 2.2 and NC IH 1 299 transformed with pDEST-EP F5 (n. D. = Not determined) 1 5. Production of recombinant E2-EPF5-FLAG protein in E . coli The plasmid insert pBM2537 / N PL002981 (example 11) is recombined according to the manual of Gateway ™ systems with the vector pDEST17 (Invitrogen Corp.) to give the plasmid E2-12-flag-pDESTI 7 / NPL006782. The encoded amino acid sequence of E2-EPF5 recombinant protein is MSYYHHHHHHLESTSLYKKAGLIEGRMNSNVENLPPHIIRLVYKEVTTLT ADPPDGIKVFPNEEDLTDLQVTIEGPEGTPYAGGLFRMKLLLGKDFPASP PKGYFLTKIFHPNVGANGEICVNVLKRDWTAELGIRHVLLTIKCLLIHPNP ESALNEEAGRLLLENYEEYAARARLLTEIHGGAGGPSGRAEAGRALASG TEASSTDPGAPGGPGGAEGPMAKKHAGERDKKLAAKKKTDKKRALRAL RRLDYKDDDDK (SEQ ID NO: 49) This plasmid is used to express recombinant E2-EPF5 in E. coli. By means of the N-terminal 6-His tag it can be purified by standard metal chelate affinity chromatography and this results in a pure preparation of more than 95% labeled E2-EPF5. It is active in an in vitro auto-ubiquitination assay and can be detected using antibodies to the C-terminal FLAG-tag peptide sequence (DYKDDDK). 16. Rabbit antibody production for recombinant E2-EPF5-FLAG protein Rabbits are immunized with recombinant E2-EPF5 derived from E. coli (Example 15) by subcutaneous injection of 0.2 mg with complete Freund's adjuvants followed, at weekly intervals, by a booster with 0.1 mg of protein with incomplete Freund's aids. 20 to 30 ml of blood are harvested after 5, 6, 7, 8 and 9 weeks followed by a terminal bleeding of 100 to 120 ml of blood, after 10 weeks, from which serum is prepared.

From the terminal bleeding serum, anti-E2-EPF5 polyclonal antibodies are purified by affinity chromatography on immobilized recombinant E2-EPF5. Briefly: Recombinant E2-EPF5 derived from E. coli (labeled FLAG) (Example 15) was covalently coupled to anti-FLAG sepharose using dimethyl pimelimidate in borate buffer as described in: Harlow, E. and Lane, D .. Using Antibodies: A Laboratory Manual, (1999) Cold Spring Harbor Press, pp. 522-523. To isolate E2-EPF5-specific antibodies, 1.5 ml of rabbit anti-E2-EPF5 crude serum is applied to 150 microliter packed beads equilibrated with PBS. The unbound antibodies are removed with an excess of PBS. Anti-E2-EPF5 antibodies are eluted using 0.2 M glycine-HCl (pH 2.5), immediately neutralized by addition of 1/10 volume of 1 M Tris-HCl, pH 9 and stored at 4 ° C. E2-EPF5 in mouse tumor tissue The tumor used for staining is from B16BL6 melanomas grown in C57BL / 6 mice. C57BL / 6 mice, black females, weighing between 17 and 20 g, are obtained from Iffa Crédo animal breeding facilities (L'Arbresie, France). They are identified via tags in the tail and are kept in groups (6 to 7 animals per cage) under normal conditions and were observed daily. Six mice are used for treatment group. The melanin produced by the B16 / BL6 tumor cell line of murine melanomas, derived from a spontaneous tumor of C57BL / 6 mice, has been extensively characterized and obtained from Dr. Isaiah J. Fldler, Texas Medical Center, Houston, USA. The cultured tumor cells used in all experiments are free of mycoplasma. Cultured at 37 ° C and 5% C02 in MEM (MEM EBS, AMIMED, Allschwil) with stable glutamine supplemented with 5% fetal calf serum, 1% sodium pyruvate, 1% non-essential amino acids and 2% vitamins and left to grow until confluence. Subsequently, 0.25% -EDTA 0.02% (2 min at 37 ° C) are shed with trypsin, and then processed. Viability is assessed by trypan blue exclusion, and only suspensions with > 90% The tumor cells are resuspended in Hanks Buffer and a suspension of 5 x 104 cells / μ is prepared. for intradermal injection (i.d.) in the ears of immuno-competent female sympathetic C57BL / 6 mice. For the injection of tumor cells, anesthesia is induced by an inhalation of Isoflurane 3% (Forene, Abbott AG, Cham, Switzerland). The animals are placed in a heated operating field maintained at a temperature of 39 ° C and their ears spread smoothly on a steel cone conditioned with a double-sided adhesive. With the help of a microscope, a 30G hypodermic needle is then inserted into the periphery of the ear and a 4 to 5 mm tunnel is made in a subcutaneous plane to allow delivery of the tumor cells to a site distal to the entry point of the needle The site of the injection is always located on the back of the ear between the first and second neurovascular bundles. Using a microliter syringe (250 μ ?, Hamilton, Bonaduz, Switzerland), 1 μ is injected? of suspension (5 x 104 cells) of tumor cells in the subcutaneous plane of the mouse ear forming a subdermal blister of 2 x 2 mm. After a week, the primary tumor begins to develop and you can easily see a black dot in the middle part of the ear. The size of the primary tumor was monitored on days 7, 14 and 21. After three weeks (day 21) the animals were sacrificed by CO 2 inhalation, the cervical lymph nodes were weighed, fixed in 4.2% formaldehyde and embedded in paraffin. Paraffin sections of 5 μ are placed ?? (prepared with a Microtome, MICRON) on SuperFrost + glass slides (Menzel), dried overnight at 37 ° C and heated for 5 minutes at 59 ° C on a hot plate. The sections are stripped of 2x wax in xylene, rehydrated in decreasing alcohol solutions (100%, 95%, 90%, 70%), rinsed in water dd and subsequently subjected to a technique to unmask antigen at high temperature. The sections are subjected to microwaves (14 minutes of heat up to 98 ° C, 10 minutes maintained at 98 ° C; Milestone # T / TMEGA) in Na citrate 0.1 m, pH 6.0. The sections are cooled to room temperature, rinsed in double distilled water (ddH20) and immersed in PBS. To block the endogenous peroxidase activity, the sections are incubated for 30 minutes in H2020.3% in PBS and rinsed in PBS. To block the binding of nonspecific antibodies, the sections are incubated for 20 minutes with PBS containing 1.5% goat serum (Vector Laboratories) at room temperature in a humid chamber. The blocking serum is dried and the sections are incubated for 1 hour with the primary antibody (0.5-1.0 μ9 /? T? Of rabbit polyclonal anti-EPF5) diluted in PBS containing Tween 200.1%. Sections are rinsed 3 x 2 minutes in PBS and subsequently incubated with the secondary binding antibody (biotinylated goat anti-rabbit IgG, Vector Laboratories) diluted in PBS containing 1.5% blocking serum. The sections are rinsed 3 x 2 minutes in PBS and incubated for 30 minutes at room temperature with avidin radish H complex (VECTASTAIN Elite ABC PK 6101 package). Sections are washed 3 x 2 minutes in PBS and stained for 5 to 10 minutes with Vector NovaRed (peroxidase substrate pack, Vector Laboratories # SK-480) and rinsed 3 x 2 minutes in ddH20. The sections are then counter-stained for 30 seconds in Mayer's hematoxylin (Fluka # 51275), rinsed for 5 minutes in tap tap water, rinsed in ddH20 and air-dried. The sections are assembled with Eukitt (Fluka # 03989) and analyzed by bright field microscopy. The lymph node metastasis test of B16BL6 reveals an amazingly intense staining of the tumor cells immediately adjacent to the necrotic nucleus of the tumor. These are the live tumor cells farthest from the blood supply vessels, which is a presumably hypoxic area. A high level of E2-EPF5 protein in this area is, therefore, consistent with a role of E2-EPF5 in the hypoxia response of tumor cells. As the hypoxia response is very important for the survival of the tumor cells, it is expected that the inhibition of E2-EPF5 interferes with the growth of the tumors, an effect that can be exploited therapeutically. 18. Detection of E2-EPF5 in areas of neovascularization in a disease model in the mouse eye Ischemic retinopathy occurs in C57 / BL6J mice (Smith et al., Oxygen-induced retinopathy in the mouse Invest Ophthalmol Vis Sci 35: 101 -111). Briefly, seven-day-old mice and their mothers are placed in an air-tight incubator and exposed to an oxygen atmosphere 75 ± 3% for 5 days (hypoxia). The temperature of the incubator is maintained at 23 ± 2o C, and oxygen is measured every 8 hours with an oxygen analyzer. After 5 days, the mice are removed from the incubator, placed in room air and after 5 days at P17, the mice are sacrificed, the eyes are quickly removed and frozen in the embed compound at the optimum cut-off temperature (OCT). Miles Diagnostics, Elkhart, IN) or fixed in formaldehyde buffered with 4% phosphate and embedded in paraffin. C67BL6J mice of the same genus and age, which were not exposed to hyperoxia, served as controls. They were treated by gavage with vehicle and after 5 days, they were sacrificed and their eyes were processed for frozen or paraffin sections. Frozen sections of 5 μp? they are air dried, fixed for 10 minutes at 4 ° C in dry acetone and rinsed in PBS. Peroxidase activity in endogenous tissue is blocked by incubating sections 30 minutes in H202 0.3% in PBS. Sections are incubated and incubated for 20 minutes with PBS containing 1.5% goat serum. All subsequent immunostaining steps are performed as described in 1. 7. The immunostaining of EPF5 in paraffin sections is performed as described in 1. 7. Examination of the tissue slices reveals significantly higher levels of stained E2-E PF5. in the areas of pathological neovasculature in ROP eyes including the same cells of blood vessels, but not in the corresponding areas of the control eyes. The observation of high levels of E2-E protein P F5 in a tissue that reacts to hypoxia is consistent with a functional role of E2-EPF5 in the cellular hypoxia response. Conversely, due to the relative hypoxia experienced when mice are transferred from high to normal oxygen levels, an abnormal retinal rization occurs. This premature model retinopathy (RO P) is a widely used retinal angiosurgery model. It is possible that the inhibition of E2-EPF5 interferes with this hypoxia response, an effect considered beneficial in several disease states. SEO ID NO: 1 January ggcggaccga agaacgcagg aagggggccg gggggacccg cccccggccg gccgcagcca 61 tgaactccaa cgtggagaac ctacccccgc acatcatccg cctggtgtac aaggaggtga 121 cgcagaccca cgacactgac cccgatggqa tcaaggtctt tcccaacgag gaggacctca 181 ccgacctcca ggtcaccatc gagggccctg aggggaccce atatgctgga ggtctgttee 241 gcatgaaact cctgctgggg aaggacttcc ctgcctcccc.acccaagggc tacttcctga 301 ccaagatctt ccacccgaac gtgggcgcca atggcgagat ctgcgtcaac gtgctcaaga 361 gggactggac ggctgagctg ggcatccgac acgtactgct gaccatcaag tgcctgctga 421 tccaccctaa ccccgagtct gcactcaacg aggaggcggg ccgcctgctc ttggagaact 481 acgaggagta tgcggctcgg gcccgtctgc tcacagagat ccacgggggc gccggcgggc 541 ccagcggcag ggccgaagcc ggtcgggccc tggccagtgg cactgaagct tcctccaccg 601 accctggggc cccagggggc ccgggagggg ctgagggtcc catggccaag aagcatgctg 661 taagaagctg gcgagcgcga gcggccaaga aaaagacgga caagaagcgg gcgctgcggg 721 cgctgcggcg gctgtagtgg gctctcttcc tccttccacc gtgaccccaa cctctcctgt 781 cccctccctc caactctgtc tctaagttat ttaaattatg gctggggtcg gggagggtac 841 agggg gcact gggacctgga tttgtttttc taaataaagt tggaaaagca SEQ ID NO: 2 MNSNVENLPPHIIRLVYKEVTTLTADPPDGIKVFPNEEDLTDLQ VTIEGPEGTPYAGGLFR KLLLGKDFPASPPKGYFLTKIFHPNVGANGEICVNVLKRD WTAELGIRHVLLTIKCLLIHPNPESALNEEAGRLLLENYEEYAARARLLTEIHGGAGG PSGRAEAGRALASGTEASSTDPGAPGGPGGAEGPMAKKRAGERDKKLAAKKKTDKKRA LRALRRL

Claims (19)

1. CLAIMS 1. A method for the treatment of a disease related to aberrant neovascularization comprising administering an effective amount of an agent that inhibits the expression of the agent encoding E2-EPF5 above ubiquitin conjugation or that inhibits an activity of the product of the E2-EPF5 gene.
2. 2. A method according to claim 1, wherein the aberrant neovascularization is vascularization dependent on VEGF.
3. A method according to claim 1 or 2, wherein the VEGF-dependent vascularization is angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neovascularization as seen in diabetic retinopathy, skin angiogenesis in psoriasis, or induced angiogenesis by hypoxia in cirrhosis of the liver.
4. 4. A method according to claim 1, 2 or 3, wherein said agent is an inhibitory nucleic acid capable of specifically inhibiting the E2-EPF5 of ubiquitin conjugation enzyme.
5. 5. A method according to claim 4, wherein said inhibitory nucleic acid is an antisense oligonucleotide or a siRNA.
6. 6. A method according to claim 1, 2 or 3, wherein said agent is an antibody that specifically binds to E2-EPF5 of ubiquitin conjugation enzyme.
7. A pharmaceutical composition comprising an effective amount of an agent that inhibits the expression of the gene encoding E2-EPF5 of ubiquitin conjugation enzyme or that inhibits an activity of the E2-EPF5 gene product and a pharmaceutically acceptable carrier.
8. 8. A pharmaceutical composition according to claim 7, wherein the E2-EPF5 inhibitor is an antisense oligonucleotide or a siRNA.
9. 9. A pharmaceutical composition according to claim 8, wherein the E2-EPF5 inhibitor is an antibody that specifically binds E2-EPF5 on top of ubiquitin conjugation.
10. A method for identifying a compound useful for the inhibition of aberrant neovascularization, comprising: (a) contacting E2-EPF5 of ubiquitin conjugation enzyme with a test compound, (b) detecting the modulation of biological activity of E2-EPF5 of ubiquitin conjugation enzyme.
11. A method for identifying a compound useful for treating a disease related to aberrant neovascularization, comprising: i) contacting a test compound with an E2-EPF5 of ubiquitin conjugation enzyme under sample conditions permissive for biological activity of E2-EPF5; ii) determining the level of said at least one biological activity of E2-EPF5; iii) comparing said level with that of a control sample that lacks said test compound; and optionally iv) selecting a test compound that causes said level to change for further testing as a potential therapeutic for the prophylactic and / or therapeutic treatment of a disease with dysregulated serum glucose or a metabolic disorder.
12. 12. A method according to claim 10 or 11, wherein the biological activity of E2-EPF5 is reduced.
13. A method according to claim 12, wherein the aberrant vascularization is angiogenesis in tumors, synovial angiogenesis in rheumatoid arthritis, ocular neovascularization as seen in diabetic retinopathy, skin angiogenesis in psoriasis, or hypoxia-induced angiogenesis in cirrhosis of the liver
14. 14. A compound identified by a method according to claim 10 to 13.
15. 15. A method for the inhibition of an expression regulated by HIF-1 which comprises inhibiting the expression of E2-EPF5 of enzyme related to the conjugation of ubiquitin.
16. 16. A method according to claim 15, wherein said gene regulated by HIF-1 is selected from the group consisting of GLUT-1, GLUT-3, HK2, EPO, NOS2, VEGF, TGF-alpha, TGF-beta , VEGFR-2, C-Met, UPAR, CXCR4, carbonic anhydrase IX (CAIX).
17. 17. A method according to claim 15, wherein said gene regulated by HIF-1 is VEGF.
18. 18. A method for the inhibition of tumor angiogenesis comprising inhibiting E2-EPF5 expression of enzyme relative to the conjugation of ubiquitin.
19. 19. A method according to claim 15, wherein said inhibition is effected via a nucleic acid or inhibitory antibody.