WO2000045771A9

WO2000045771A9 - Suppression of transformation of cells by the transcription factor egr

Info

Publication number: WO2000045771A9
Application number: PCT/US2000/002799
Authority: WO
Inventors: Daniel Mercola; Eileen Adamson
Original assignee: Daniel Mercola; Eileen Adamson
Priority date: 1999-02-05
Filing date: 2000-02-04
Publication date: 2001-09-20
Also published as: WO2000045771A3; AU4448700A; WO2000045771A2

Abstract

The present invention provides a method for increasing expression of an anti-metastatic factor in a tumor cell or interfering with the metastasis of a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR. The present invention also includes a method for identifying compounds or compositions that increase the expression of EGR in a cell. The present invention further includes methods for identifying compounds or compositions that increase or decrease the expression of EGR in a cell, the expression of at least one anti-metastatic factor, or cell adhesion to a substrate, including pharmaceutical compositions, identified by these methods. The present invention also provides methods for interfering with the proliferation of a tumor cell by increasing the expression of a metastatic factor.

Description

SUPPRESSION OF TRANSFORMATION OF CELLS BY THE TRANSCRIPTION

FACTOR EGR

This invention was made partially with government support awarded by the National Institutes of Health under Grants CA63783, CA76173 and CA67888. The United States Government may have certain rights in the invention.

Technical Field

The present invention generally relates generally to the fields of molecular biology and cancer biology and treatment.

Background

EGR-1 is a nuclear phosphoprotein that comprises three zinc finger motifs in the C- terminal portion of the molecule that confers specific DNA-binding properties to the molecule. A growing body of circumstantial evidence indicates that EGR-1 may have functional effects in regulating cell growth, differentiation and development. An approach to understanding the underlying molecular basis for these EGR-1 functions has been to study signal transduction events associated with the expression of EGR-1 and the possible modulation of expression of TGF-βl.

TGF-βl belongs to the TGF superfamily of cytokines that have been implicated in the regulation of growth, differentiation, development, and apoptosis. TGF-βl may play a role in modulating the activity of PAI-1 and fibronectin (FN). PAI-1 is thought to participate in the stabilization of the extracellular matrix by either interacting with structures containing a urokinase plasminogen activator. PAI-1 is also thought to inhibit the serine protease activity of a urokinase plasminogen activator. FN plays a role in anchoring cells to the extracellular matrix. Inhibition of FN expression leads to a loss of FN from the cell surface and may be related to oncogenic transformation in vitro and may be related to tumorigenic and metastatic phenotypes in vivo.

Although these various cellular components are known, their complex interactions generally, and particularly how they relate to tumor cells, has not been appreciated. Thus, there exists a need to appreciate the role of these cellular components in pathological states, such as . tumors and cancers. This invention satisfies this need and provides related advantages as well by providing methods of regulating the growth of cancer cells by promoting or supplementing the expression of EGR, or fragments thereof, in cells.

Brief Description of the Figures

FIG. 1A depicts a Western blot analysis of EGR-1 expression in a series of EGR-1 stably transfected H4 cells. FIG. IB depicts the presence of radiolabled PAI-1 produced by a series of EGR-1 stably transfected H4 cells in the presence or absence of rhTGF-βl. FIG. 1C depicts densiometic analysis of PAI-1 expression in the absence (white bars) and presence (solid bars) of rhTGF-βl. The insert shows the correlation of EGR-1 expression and PAI-1 expression.

FIG. 2 A depicts the presence of radiolabled FN produced by a series of EGR-1 stably transfected H4 cells in the presence or absence of rhTGF-βl. FIG. IB depicts densiometic analysis of FN expression in the absence (white bars) and presence (solid bars) of rhTGF-βl. The insert shows the correlation of EGR-1 expression and FN expression.

FIG.3 A depicts the secretion of PAI-1 and FN in H4 cells in response to differing concentrations of rhTGF-βl. FIG. 3B depicts densiometric analysis of the relative expression levels of PAI-1 and FN in response to differing concentrations of rhTGF-βl.

FIG. 4 depicts the effect of anti-TGF-β₁₂₃ antibody on the secretion of PAI-1 and FN in a series of EGR-1 stably transfected H4 cells.

FIG. 5 depicts the effect of an application of antisense TGF-βl oligonucleotides on the secretion of PAI-1 and FN in a series of EGR-1 stably transfected H4 cells.

FIG. 6 A depicts a schematic representation of the human FN promoter with the location of two GCE sites and transcription start sites indicated as shaded boxes. FIG. 6B depicts gel- shift analysis of FLAG-tagged-EGR-1 (wild type) or mutant FLAG-tagged-EGR- shift analysis of FLAG-tagged-EGR-1 (wild type) or mutant FLAG-tagged-EGR- 1ΔS348A/A340S to detect binding with Probe A or Probe B in the presence or absence of anti- ERG-1 antibody or anti-Spl antibody. The protein-DNA complexes are indicated by the arrow. FIG. 6C depicts gel-shift analysis of GST-EGR-1 fusion protein to detect binding with Probe A or Probe B in the presence or absence of competing GCE sequences The protein-DNA complexes are indicated by the arrow. FIG. 6D depicts a gel-shift assay using nuclear protein extracts from a series of EGR-1 stably transfected H4 cells, Probe A or Probe B, in the presence or absence of anti-Spl antibody, anti-EGR-1 antibody or competing GCE sequences. DNA- protein complexes are indicated by the arrow.

FIG. 7A depicts the attachment of a series of EGR-1 stably transfected H4 cells to untreated polystyrene tissue culture dishes or Petri dishes over time. FIG. 7B depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well polystyrene ELISA plates pretreated with FN or PAI-1 over time.

FIG. 8 depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well polystyrene ELISA plates pretreated with PAI-1 in the presence or absence of anti-API-1 antibody or the peptides GRGDSP or GRGESP.

FIG. 9 A depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well ELISA plates pretreated with 5 μg/ml of FN in the presence or absence of anti- PAI-1 antibody or the peptides GRGDSP or GRGESP. FIG. 9B depicts the attachment of a series of EGR-1 stably transfected H4 cells to standard 96-well ELISA plates pretreated with 0.25 μg/ml of FN in the presence or absence of anti-PAI-1 antibody.

FIG. 10 depicts a proposed model for the mechanism of the suppression of transformation of cells by EGR-1. In this proposed model, which the inventors expressly do not wish to be bound to, but provide for clarification, the expression of FN receptor is regulated by FN and TGF-βl. EGR-1 transactivates both TGF-βl and FN genes leading to increased steady state synthesis and secretion of TGF-βl and FN. Secreted TGF-βl becomes activated and induces increased steady state PAI-1 expression via TGF-βl receptor-mediated signal transduction. Both FN and PAI-1 augment ECM formation and density-dependent growth control.

FIG. 11 presents a schematic diagram of the truncated forms of Egr-1 and their calculated molecular weights. Fragments were constructed as described in U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998, using standard recombinant DNA techniques. The truncated forms contain the thirteen initial amino acids from the first methionine to proline, in order to initiate translation. The fragments were cloned into pGEM-1 (Promega) to effect their synthesis in vitro, and into eukaryotic vectors driven by RSN for expression in eukaryotic cells such as ΝIH3T3 cells. The four principal domains of Egr-1 are designated A, B, C and D. The DNA-binding zinc-finger domain is indicated (domain C).

Summary

The present invention recognizes that the expression of EGR can decrease metastasis of tumor cells.

The methods of the present invention involve transfecting a cell with a nucleic acid molecule encoding a mammalian EGR or a fragment thereof comprising the zinc finger domain of an EGR-1 or a nucleic acid sequence that hybridizes to any of the appropriate EGR-1 nucleic acid sequences described herein under standard hybridization conditions and that encodes a polypeptide that increases the expression of an anti-metastatic factor in a cell or interferes with the metastasis of a tumor cell. The present invention also includes methods for identifying compounds or compositions that increase the expression of EGR-1 in a cell and compounds or compositions, including pharmaceutical compositions, that are identified by these methods.

A first aspect of the present invention is a method for increasing expression of an anti- metastatic factor in a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR. A second aspect of the present invention is a method for interfering with the metastasis of a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR.

A third aspect of the present invention is a method for identifying compounds or compositions that increase the expression of EGR in a cell.

. A fourth aspect of the present invention is a method for identifying compounds or compositions that increase or decrease the expression of EGR in a cell by contacting a cell with a test compound and measuring the expression of EGR in said cell and compositions, including pharmaceutical compositions, identified by this method.

A fifth aspect of the present invention is a method for interfering with proliferation of a tumor cell in a mammal by administering to a mammal a vector comprising a nucleic acid sequence encoding an EGR or a nucleic acid sequence that encodes an active portion of an EGR, wherein said tumor cell exhibits increased expression of at least one anti-metastatic factor upon said administering.

Detailed Description of the Invention

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, chemistry, microbiology, molecular biology, cell science and cell culture described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Where a term is provided in the singular, the inventors also contemplate the plural of that term. The nomenclature used herein and the laboratory procedures described below are those well known and commonly employed in the art. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

"Isolated polynucleotide" refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide (1) is not associated with the cell in which the isolated polynucleotide is found in nature, or (2) is operably linked to a polynucleotide that it is not linked to in nature. The isolated polynucleotide can optionally be linked to promoters, enhancers, or other regulatory sequences.

"Isolated protein" refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which by virtue of its origin the isolated protein (1) is not associated with proteins normally found within nature, or (2) is isolated from the cell in which it normally occurs, or (3) is isolated free of other proteins from the same cellular source, for example, free of cellular proteins), or (4) is expressed by a cell from a different species, or (5) does not occur in nature.

"Polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence.

"Active fragment" refers to a fragment of a parent molecule, such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combinations thereof, that retains at least one activity of the parent molecule.

"Naturally occurring" refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Control sequences" refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal biding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

"Polynucleotide" refers to a polymeric form of nucleotides of a least ten bases in length, either ribonucleotides or deoxynucleotides or a modified from of either type of nucleotide. The term includes single and double stranded forms of DNA or RNA.

"Directly" in the context of a biological process or processes, refers to direct causation of a process that does not require intermediate steps, usually caused by one molecule contacting or binding to another molecule (the same type or different type of molecule). For example, molecule A contacts molecule B, which causes molecule B to exert effect X that is part of a biological process.

"Indirectly" in the context of a biological process or precesses, refers to indirect causation that requires intermediate steps, usually caused by two or more direct steps. For example, molecule A contacts molecule B to exert effect X which in turn causes effect Y.

"Sequence homology" refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, for example 50%, the percentage denotes the proportion of matches of the length of sequences from a desired sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonuleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%).

"Selectively hybridize" refers to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to target nucleic acid strands, under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments thereof and a nucleic acid sequence of interest will be at least 30%, and more typically and preferably of at least 40%, 50%, 60%, 70%, 80% or 90%.

Hybridization and washing conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, a full length polynucleotide sequence can be labeled and used as a hybridization probe to isolate genomic clones from an appropriate target library as they are known in the art. Typical hybridization conditions and methods for screening plaque lifts and other purposes are known in the art (Benton and Davis, Science 196:180 (1978); Sambrook et al., supra, (1989)).

Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at least 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff, in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, volume 5, pp. 101-110 (1972) and Supplement 2, pp. 1-10). The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 30% identical when optimally aligned using the ALIGN program.

"Corresponds to" refers to a polynucleotide sequence is homologous (for example is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to all or a portion of a reference polypeptide sequence. In contradistinction, the term "complementary to" is u sed herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence TATAC corresponds to a reference sequence TATAC and is complementary to a reference sequence GTATA.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence can be a subset of a larger sequence, for example, as a segment of a full length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides can each (1) comprise a sequence (for example a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A comparison widow, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window can comprise additions and deletions (for example, gaps) of 20 percent or less as compared to the reference sequence (which would not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window can be conducted by the local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482 (1981)), by the homology alignment algorithm (Needleman and Wunsch, J. Mol. Bio., 48:443 (1970)), by the search for similarity method (Pearson and Lipman, Proc. Natl. Acid. Sci. U.S.A. 85:2444 (1988)), by the computerized implementations of these algorithms such as GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Page Release 7.0, Genetics Computer Group, Madison, WI), or by inspection. Preferably, the best alignment (for example, the result having the highest percentage of homology over the comparison window) generated by the various methods is selected.

"Sequence identity" means that two polynucleotide sequences are identical (for example, on a nucleotide-by-nucleotide basis) over the window of comparison.

"Percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

"Substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25 to 50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or addition which total 20 percent or less of the reference sequence over the window of comparison. "Substantial identity" as applied to polypeptides herein means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, and more preferably at least 50 percent sequence identity, and most preferably at lest 60 percent sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions.

"Conservative amino acid substitutions" refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chan is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine- isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine-glutamine.

"Modulation" refers to the capacity to either enhance or inhibit a functional property of a biological activity or process, for example, enzyme activity or receptor binding. Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.

"Modulator" refers to a chemical (naturally occurring or non-naturally occurring), such as a biological macromolecule (for example, nucleic acid, protein, non-peptide or organic molecule) or an extract made from biological materials, such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples or portions thereof. Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (for example, agonist, partial antagonist, partial agonist, antagonist, antineoplastic, cytotoxic, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like) by inclusion in assays described herein. The activity of a modulator may be known, unknown or partially known.

"Test compound" refers to a chemical, compound or extract to be tested by at least one method of the present invention to be a putative modulator. A test compound can be of any chemical nature, including but not limited to small molecules such as drugs, proteins or antibodies or active fragments thereof, nucleic acids such as DNA or RNA that can be provided in a vector such as a viral vector, antisense nucleic acid molecules and ribozymes. A test compound is usually not known to bind to the target of interest, but that not need be the case. "Control test compound" refers to a compound known to bind to the target (for example, a known agonist, antagonist, partial agonist or inverse agonist). Test compound does not typically include a chemical added to a mixture as a control condition that alters the function of the target to determine signal specificity in an assay. Such control chemicals or conditions include chemicals that (1) non-specifically or substantially disrupt protein structure (for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta- mercaptoethanol), (2) generally inhibit cell metabolism (for example mitochondrial uncouples) and (3) non-specifically disrupt electrostatic or hydrophobic interactions of a protein (for example, high salt concentrations or detergents at concentrations sufficient to non-specifically disrupt hydrophobic or electrostatic interactions). The term test compound also does not typically include chemicals known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject. Usually, various predetermined concentrations of test compound are used for determining their activity. If the molecular weight of a test compound is known, the following ranges of concentrations can be used: between about 0.001 micromolar and about 10 millimolar, preferably between about 0.01 micromolar and about 1 millimolar, more preferably between about 0.1 micromolar and about 100 micromolar. When extracts are uses a test compounds, the concentration of test compound used can be expressed on a weight to volume basis. Under these circumstances, the following ranges of concentrations can be used: between about 0.001 micrograms/ml and about 1 milligram/ml, preferably between about 0.01 micrograms/ml and about 100 micrograms/ml, and more preferably between about 0.1 micrograms/ml and about 10 micrograms/ml.

"Target" refers to a biochemical entity involved in a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic chemical typically binds to a target to alter or modulate its function. As used herein, targets can include, but not be limited to, cell surface receptors, G-proteins, G-protein coupled receptors, kinases, phosphatases, ion channels, Upases, phosholipases, nuclear receptors, intracellular structures, tubules, tubulin, and the like.

"Label' or "labeled" refers to incorporation of a detectable marker, for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin. Various methods of labeling polypeptide, nucleic acids, carbohydrates, and other biological or organic molecules are known in the art. Such labels can have a variety of readouts, such as radioactivity, fluorescence, color, chemiluminescence or other readouts known in the art or later developed. The readouts can be based on enzymatic activity, such as beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase, luciferase; radioisotopes such as ³H, ¹⁴C, ³⁵S, ¹²⁵I or ¹³¹I); fluorescent proteins, such as green fluorescent proteins; or other fluorescent labels, such as FITC, rhodamine, and lanthanides. Where appropriate, these labels can be the product of the expression of reporter genes, as that term is understood in the art. Examples of reporter genes are beta-lactamase (U.S. Patent No. 5,741,657 to Tsien et al., issued April 21, 1998) and green fluorescent protein (U.S. Patent No. 5,777,079 to Tsien et al.., issued July 7, 1998; U.S. Patent No. 5,804,387 to Cormack et al., issued September 8, 1998).

"Substantially pure" refers to an object species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified fraction is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present. Generally , as substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%, 90%, 95% and 99%. Most preferably, the object species or activity is purified to essential homogeneity, wherein contaminant species or activities cannot be detected by conventional detection methods) wherein the composition consists essentially of a single macromolecular species or activity. The inventors recognize that an activity may be caused, directly or indirectly, by a single species or a plurality of species within a composition, particularly with extracts.

"Pharmaceutical agent or drug" refers to a chemical, composition or activity capable of inducing a desired therapeutic effect when property administered by an appropriate dose, regime, route of administration, time and delivery modality.

"Pharmaceutically effective amount" refers to an appropriate dose, regime, route of administration, time and delivery modality associated with the delivery of an amount of a c'ompound or composition to cause a desired effect. Such pharmaceutically effective amount can be determined using methods described herein or by the United States Food and Drug Administration (USFDA).

As used herein a nucleic acid molecule "encodes" a polypeptide if transcription of the nucleic acid molecule and translation of the n RNA produce the polypeptide. Thus, nucleic acid molecules of the present invention include those whose nucleotide sequence encodes a polypeptide directly, such as cDNA, or whose nucleotide sequence includes introns that are spliced out upon transcription into mRNA, such as genomic DNA. It also includes nucleic acid molecules having sequences which are degenerate versions of any of the aforementioned nucleotide sequences. Other technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries, such as the McGraw-Hill Dictionary of Chemical Terms and the Stedman's Medical Dictionary.

Introduction

The present invention recognizes that the expression of EGR can decrease metastasis of tumor cells and increase the expression of anti-metastatic factors. As a non-limiting introduction to the breath of the present invention, the present invention includes several general and useful aspects, including:

1) a method for increasing expression of an anti-metastatic factor in a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR;

2) a method for interfering with the metastasis of a tumor cell including administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR;

3) a method for identifying compounds or compositions that increase the expression ofEGR in a cell;

4) compositions, including pharmaceutical compositions, identified by 3); and 5) a method for interfering with proliferation of a tumor cell in a mammal. „

These aspects of the invention, as well as others described herein, can be achieved by using the methods, articles of manufacture and compositions of matter described herein. To gain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention can be combined to make desirable embodiments of the invention.

I. A method for increasing expression of an anti-metastatic factor in a tumor cell, interfering with the metastasis of a tumor cell, and inhibiting the growth of a tumor cell.

According to one embodiment of the present invention, this method is used to increase the expression of at least one anti-metastatic factor in a tumor cell or interferes with the metastasis of a tumor cell. This method can also be used to inhibit the growth of a tumor cell. Tumors whose growth can be inhibited by the expression of mammalian EGR include cancer cells derived from mesoderm, ectoderm or endoderm, such as fϊbrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, acute myelogenous leukemia melanoma and prostate carcinoma. According to another embodiment of the invention, the cell is a transformed culture cell such as from fibrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, acute myelogenous leukemia, melanoma and prostate carcinoma.

"Mammalian EGR" or "EGR" refers to a polypeptide of the EGR gene family having activity as a transcription factor. This includes the polypeptides encoded by the mouse Egr-1 gene, the human EGR-1 gene, the human EGR2 gene, the human EGR3 gene, the human EGR4 gene and other mammalian EGR genes identifiable as follows. A mammalian EGR is characterized by having 30% overall amino acid sequence identity with at least one of the foregoing mammalian EGRs. It has a zinc finger domain with at least 80% amino acid identity with the zinc finger domain of at least one of the foregoing mammalian EGRs. A mammalian EGR is also characterized by its ability to bind with at least one GCE site.

The DNA and amino acid sequence of mouse Egr-1 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998. The DNA and deduced amino acid sequences of human EGRl is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998. The DNA and deduced amino acid sequence of human EGR2 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998. The DNA and deduced amino acid sequence of human EGR3 is given in U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998. A partial DNA and amino acid sequence of human EGR4 is given in Patwardhan et al., Oncogene. 6:917 (1991).

Mammalian EGR (early growth response) is a transcription factor that stimulates the activity of a number of mammalian genes and inhibits other genes. The mammalian EGRs contain three zinc fingers of the Cys₂His₂ class that binds to the GCE site in 5' enhancer region, which has the sequence GCGGGGGCG (SEQ ID NO:7), GCGTGGGCG (SEQ ID NO:8) or GCGAGGGCG (SEQ ID NO:9). However, the functions of EGR are obscure.

Egr-1, a mouse EGR gene, is associated with growth stimulation since it is rapidly induced by many growth factors and other stimuli. It is also known as Drox24, zif268 and TIS8. Typically, stimulation of quiescent cells leads to a transient peak of expression that returns to base values which are low but detectable. This is typical of most of the immediate early growth response genes such as c-fos and c-jun. However, Egr-1 is constitutively expressed in differentiated embryonal carcinoma (EC) cells where evidence supports its role in maintaining the differentiated state. When EC cells are induced to differentiate, the expression of Egr-1 increases markedly.

Egr-1 stimulates the activity of the GCE site in several promoters in transient expression assays. Genes whose transcription has been shown to be inhibited by Egr-1 include the PDGF-A gene (as tested in NIH-3T3 cells), adenosine deaminase (ADA) gene 1 (as tested in murine Cl- 1D cells), and the midkine (MK) gene (as tested in P19 cells). Moreover, it appears the Egr-1 can compete with Spl in binding to an overlapping consensus binding motif in the promoter region of murine ADA therefore abolishing the function of Spl . However, in several cell types Egr-1 stimulates the activity of the GC-rich DNA-binding element (GCE) while WT1 inhibits. Madden et al., Science 253:1550 (1991).

It is recognized that minor modifications can be made to EGR while retaining the growth inhibitory activity of the molecule. Minor modifications include simple substitutions, additions or deletions. Simple substitutions include the substitution of an amino acid for another having a side chain extending from the alpha carbon of the same class, i.e., non-polar (hydrophobic), neutral, positively charged or negatively charged. These modifications may be introduced deliberately through site-directed mutagenesis, or may be accidental, such as through mutation in hosts having DNA encoding these polypeptides. Any such modified protein can be easily tested for activity in the assays described herein.

Fragments of EGR that contain the zinc finger domain useful in the present invention also include the zinc finger domain that extends from amino acids 320 to 431 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) (nucleotides 1215 to 1551 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998)) of mouse Egr-1; amino acids 322 to 433 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) (nucleotides 1234 to 1567 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998)) of human EGRl; amino acids 214 to 380 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) (nucleotides 1023 to 1350 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) of human EGR2; amino acids 258 to 368 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998) (nucleotides 1132 to 1467 (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998)) of human EGR3; and the following sequence of EGR4 (Patwardham et al., supra): RGGKCSTRC FCPRPHAKAFA CPNESCVRS FARSDELΝRH LRIHTGHKPF QCRICLRΝFS RSDHLTTHVR THTGEKPFAC DNCGRRFARS DEKKRHSKNH LRQKARAEER (SEQ ID NO: 10). The zinc finger domain of other mammalian EGRs can be determined by inspecting their amino acid sequences and comparing them with the domains just described.

Recent studies (Gashler et al., Mole. Cell. Biol., 13:4556 (1993)) identified a small region on the 5' side of the zinc finger-encoding sequence of Egr-1 corresponding to amino acids 281 to 314 (U.S. Patent No. 5,837,692 to Mercola et al, issued November 11, 1998), called the "repression domain." The "repression domain" imparts a negative influence on certain biochemical activities of Egr-1, viz. transactivation of reporter constructs. However, it has been found that the zinc finger domain of mammalian EGR (fragment C of Egr-1, (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998)) inhibits the mitogenic activity of PDGF without these negative effects. It has also been found that a fragment of Egr-1 containing the zinc finger domain and the rest of the carboxy-terminal end of the molecule (fragment CD, (U.S. Patent No. 5,837,692 to Mercola et al., issued November 11, 1998)) has greater inhibitory effect than the zinc finger alone.

Accordingly, in preferred embodiments of the present invention the polypeptide fragments and the nucleic acid sequences encoding them have the zinc finger domain of a mammalian EGR, but exclude the "repression domain." In another preferred embodiment of the present invention, the polypeptide fragments and the nucleic acid sequences encoding them consist essentially of the zinc finger domain and the remainder of the carboxy terminal end of the molecule.

The methods of the present invention use a nucleic acid encoding a mammalian EGR, a nucleic acid sequence encoding a fragment of a mammalian EGR comprising the zinc finger domain or a nucleic acid sequence that hybridizes to any of the foregoing nucleic acid sequence under standard hybridization conditions and that encodes a polypeptide having the activity of increasing the expression of at least one anti-metastatic factor in a tumor cell or interfering with the metastasis of a tumor cell. Such nucleic acid molecules can be present in a vector, such as a viral vector or a liposome, or be present as naked DNA, such as linear DNA or plasmid DNA or other delivery vehicle know in the art. The nucleic acid encoding a mammalian EGR or a portion thereof can be linked to sequences that can directed homologous recombination into the genome of a host cell at desired locations, such as to be regulated by endogenous expression control sequences, such as promoters, using methods known in the art (WO 94/24301 to Smith et al., published October 27, 1994). Such nucleic acids can also be randomly integrated or targeted into the genome of a host cell (see, WO 98/13353 to Whitney, published April 2, 1998, and Skarnes et al., Genes and Development 6:903-918 (1992)). Alternatively, these nucleic acid molecules can be operatively linked to expression control sequences that are operable in a target cell. According to one embodiment of the present invention, the nucleic acid sequence encodes essentially the zinc finger domain of a mammalian EGR.

As used herein a nucleic acid molecule "encodes" a polypeptide if transcription of the nucleic acid molecule and translation of the mRNA produce the polypeptide. Thus, nucleic acid molecules of the present invention include those whose nucleotide sequence encodes a polypeptide directly, such as cDNA, or whose nucleotide sequence includes introns that are removed upon transcription into mRNA, such as genomic DNA. It also includes nucleic acid molecules having sequences which are degenerate versions of any of the aforementioned nucleotide sequences.

As used herein, the term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5 X SSC and 65° C for both hybridizations and washes. The activity of any such DNA sequence can be tested by the assays described in the Examples.

The nucleic acid molecules of the present invention can be produced by organic synthesis on a commercial nucleic acid synthesizer or through PCR of a nucleic acid encoding a polypeptide useful in the present invention. Nucleic acid sequences encoding mammalian EGR can be identified by probing cDNA libraries under standard hybridization conditions with probes derived from mouse Egr-1, human EGR-1, human EGR2, humanEGR3 or human EGR4 and by analyzing cDNA expression libraries with antibodies against a mammalian EGR. Alternatively, EGR from these mammals can be isolated and partially sequenced, and the sequence can be used to make sets of degenerate nucleic acid probes for probing gene libraries. Other methods for identifying and isolating genes are also known.

The expression vectors of this invention have an expression control sequence operatively linked to a nucleic acid molecule of the present invention. An expression control sequence is operatively linked to a nucleic acid molecule when it directs the transcription and translation of that molecule in an appropriate host cell. This includes provision of appropriate start and stop codons.

According to one embodiment of the invention, expression of EGR is constitutive. Expression vectors in which the expression control sequence comprises a RSN or CMN promoter will express EGR constitutively. Both promoters are commonly used in the art for the expression or recombinant nucleic acid molecules (Gorman et al., Molec. and Cell Biol., 2:1044 (1982) and Boshant et al., Cell 41:421 (1985)).

The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, Ν,.Y. (1989) provides many protocols in the art of molecular genetics.

Methods of transfecting genes into mammalian cells with expression vectors and obtaining their expression are well known to the art (Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, CA (D.N. Goeddel ed (1990) and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, Ν.Y. (1990)). Vectors useful in this invention include those capable of transferring genes into mammalian cells. These include viral vectors such as, but not limited to, retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, liposomes and other delivery vehicles as they are known in the art.

Retroviral vectors are one example of a vector useful for increasing the expression of at least one anti-metastatic factor in a tumor cell or interfering with the metastasis of a tumor cell by gene therapy. In one embodiment of this invention, a retroviral packaging cell line such as PA317 (ATCC NO: CRL 9078) is used to create infective amphotropic retroviral vectors. The retroviral plamid pLNCX (Miller and Rosman, Biotechniques 7:980 (1989)) can contain the expression control sequence operatively linked to the nucleic acid sequence to be expressed. That plasmid contains a Maloney murine leukemia virus LTR promoter/enhancer (L); neomycin resistance gene encoding neomycin phosphotransferase (N); a human cytomegalic virus LTR/enhancer (C) and the coding gene to be expressed (X). The gene is inserted by standard techniques at a pre-existing cloning site by replacement of the phosphotransferase gene for neomycin resistance for one encoding a phosphotrasferase of hygromycin resistance. Retroviruses such as pLHCC (pLHCX in which X is the C domain of Egr-1) or pLHCCD (pLHCX in which X is the CD domain of Egr-1) can be used directly to treat tumors. Alternatively, tumors can be treated with irradiated packaging cells that express these retroviruses (see U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998).

Vectors can be administered to a tumor cell in culture by simply adding the vector to the culture or by other means such as ballistics, electroporation or lipofection methods known in the art. Vectors can be administered to a tumor cell in a whole organism by a variety of methods, such as systemic administration, direct injection into a tumor or injection into a locus adjacent to the tumor, or electroporation directly into a tumor or adjacent thereto, or ballistic delivery to the tumor or adjacent thereto. Vectors can be administered to whole organisms that have naturally occurring tumors or tumors that arise from tumor cells injected or otherwise transplanted into the organism using known methods (see, U.S. Patent No. 5,837,692 to Mercola et al., issued November 17, 1998). The route of administration, dose and regiment of administration for such vectors can be determined as set forth herein for other pharmaceutical compositions.

The methods of the present invention operate to increase the expression of at least one anti-metastatic factor in a tumor cell, including but not limited to plasminogen activator inhibitor- 1, transforming growth factor beta-1, and fibronectin. Increased expression means that the expression of mRNA, protein, or modified protein (such as, for example, by glycosylation) is above the natural or normal expression. The increased expression of such anti-metastatic factors can be determined using the methods set forth in the Examples. In addition, the methods of the present invention can interfere with the metastasis of a tumor cell, which can be measured using the methods set forth in the Examples.

The methods of the present invention also operate to inhibit the growth of a tumor cell. To inhibit the growth of a tumor cell means to decrease the rate at which such a tumor cells replicates, or to decrease the number of living tumor cells in a sample. A sample can be at least one cell in culture or within an animal, such as a mammal, including test animals, such as mice, or humans. The inhibition of such growth as compared to appropriate control conditions and cells can be measured using methods known in the art. In culture, the growth and replication of cells can be monitored by microscopic examination, instrumentation counting or viability staining using suitable stains or dyes. In test animals or humans, the inhibition of such growth can be measured by the reduced increase in tumor mass or volume, or reduced tumor mass or volume. Such tumor mass or volume can be measured by established methods, such as visual examination, excising and weighing or measuring the volume of the tumor, or by imaging methods, such as x-rays or MRI instrumentation.

Tumor cells that are administered a vector of the present invention preferably do not express a detectable level of EGR. Preferably, the tumor cells express between about 0 and about 10% of the level of EGR normally expressed by these cells, and more preferably between about 2% and about 8%.

II. A method for identifying compounds or compositions that increase or decrease the expression of EGR or anti-metastatic factors in a cell, modulate metastasis or cell mobility or inhibit the growth of a tumor or tumor cell.

The present invention also includes a method for identifying compounds or compositions that increase or decrease the expression of an EGR, increase or decrease the expression of at least one anti-metastatic factors in a cell, or increase or decrease cell attachment to substrate. In operation, this method includes providing a cell that expresses or does not express EGR or at least one anti-metastatic factors. Such cells can be in culture or in an orgamsm, such as a mouse or other test animal, as a result natural generation of such cells, induced generation of such cells, transgenic manipulation of the organism or by injecting such cells into the organism using methods known in the art. The cell can either express EGR or at least one anti-metastatic factors endogenously, or express EGR or at least one anti-metastatic factor in response to at least one transfected nucleic acid molecule of the present invention that encode an EGR or a portion thereof.

The cells or organism are contacted with a test compound. The resulting increase or decrease of expression of EGR or increased or decreased expression of at least one anti- metastatic factors in the cell is then measured using appropriate methods, such as those described in the Examples, such as detecting EGR mRNA in the cell or EGR in the cell using established methods, such as PCR or immunological methods. Reporter genes can also be used to monitor the expression of a desired gene (see, Whitney et al.,WO 98/13353 to Whitney et al., published April 2, 1998; WO 94/24301 to Smith et al., published October 3, 1994; and Skarnes et al., Genes and Development, 6:903-918 (1992). The increase or decrease of at least one anti- metastatic factor, including but not limited to plasminogen activator inhibitor- 1, transforming growth factor beta-1 and fibronectin, can also be measured using such methods. Furthermore, the attachment of such cells to substrates, such as untreated plastics, or surfaces treated with plasminogen activator inhibitor- 1 or fibronectin, can be monitored using the methods of the present invention to evaluate the ability of a test compound to modulate cell metastasis or cell mobility.

Test compounds that increase the expression of EGR or at least one anti-metastatic factor are presumptive therapeutic compounds that can treat a tumor or cancer or interfere with the metastasis of a tumor cell. Test compounds that decrease the expression of EGR or at least one anti-metastatic factor are compounds that presumptive therapeutic compounds that can increase cell mobility. Test compounds that increase cell attachment to a substrate are presumptive therapeutic compounds that can treat a tumor or interfere with metastasis of a tumor. Test compounds that decrease cell attachment to a substrate are presumptive therapeutic compounds that increase cell mobility. These test compounds can be further evaluated for therapeutic activity and toxicity using the methods of the present invention.

III. Pharmaceutical Compositions

Pharmacology and toxicity of test compounds

The structure of a test compound can be determined or confirmed by methods known in the art, such as mass spectroscopy. For test compounds stored for extended periods of time under a variety of conditions, the structure, activity and potency thereof can be confirmed.

Identified test compounds can be evaluated for a particular activity using recognized methods and those disclosed herein. For example, if an identified test compound is found to have anticancer cell activity in vitro, then the bioactive compound or bioactivity would have presumptive pharmacological properties as a chemotherapeutic to treat cancer. Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, and toxicology, and be selected and performed. The methods described herein can also be used to assess pharmacological selectivity and specificity, and toxicity.

Identified test compounds can be evaluated for toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Patent Nos; 5,196,313 to Culbreth (issued March 23, 1993) and 5,567,952 to Benet (issued October 22, 1996)). For example, toxicology of a test compound can be established by determining in vitro toxicity toward a cell line, such as a mammalian, for example human, cell line. Test compounds can be treated with, for example, tissue extracts, such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the test compound after being metabolized by a whole organism. The results of these types of studies are predictive of toxicological properties of chemical s in animals, such as mammals, including humans. Alternatively, or in addition to these in vitro studies, the toxicological properties of a test compound in an animal model, such as mice, rats, rabbits, dogs or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)). Depending on the toxicity, target organ, tissue, locus and presumptive mechanism of the test compound, the skilled artisan would not be burdened to determine appropriate doses, LD₅₀ values, routes of administration and regimes that would be appropriate to determine the toxicological properties of the test compound. In addition to animal models, human clinical trials can be performed following established procedures, such as those set forth by the United States Food and Drug Administration (USFDA) or equivalents of other governments. These toxicity studies provide the basis for determining the efficacy of a test compound in vivo. Efficacy ofbioactive compounds and bioactivities

Efficacy of a test compound can be established using several art recognized methods, such as in vitro methods, animal models or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a test compound to extend the life-span of HIV-infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIN infection or AIDS (see, Daluge et al., Antimicro. Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of cyclosporin A (CsA) to prevent proliferation of T-cells in vitro has been established as an acceptable model to identify chemicals expected to be efficacious as immunosuppressants (see, Suthanthiran et al., supra (1996)). A battery of acceptable in vitro and in vivo models exist for anti-cancer compounds as are set forth in the literature and the National Institutes of Health, particularly the National Cancer Institute. For nearly every class of therapeutic, disease or condition, an acceptable in vitro or animal model is available. In addition, these in vitro methods can use tissue extracts, such as preparations of liver, such as microsomal preparations, to provide a reliable indication of the effects of metabolism on a test compound. Similarly, acceptable animal models can be used to establish efficacy of test compounds to treat various diseases or conditions. For example, the rabbit knee is an accepted model for testing agents for efficacy in treating arthritis (see, Shaw and Lacy, J. Bone Joint Surg. (Br.) 55:197-205 (1973)). Hydrocortisone, which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an appropriate model to determine efficacy of a test compound, the skilled artisan can be guided by the state of the art to choose an appropriate model, doses and route of administration, regime and endpoint and as such would not be unduly burdened.

In addition to animal models, human clinical trials can be used to determine the efficacy of bioactive compounds and bioactivities. The USFDA, or equivalent governmental agencies, have established procedures for such studies. Selectivity of bioactive compounds and bioactivities

The in vitro and in vivo methods described above also establish the selectivity of a candidate modulator. It is recognized that chemicals can modulate a wide variety of biological processes or be selective. Panels of cells as they are known in the art can be used to determine the specificity of the a bioactive compound or bioactivity (WO 98/13353 to Whitney et al., published April 2, 1998). Selectivity is evident, for example, in the field of chemotherapy, where the selectivity of a chemical to be toxic towards cancerous cells, but not toward non-cancerous cells, is obviously desirable. Selective modulators are preferable because they have fewer side effects in the clinical setting. The selectivity of a test compound can be established in vitro by testing the toxicity and effect of a test compound can be established in vitro by testing the toxicity and effect of a bioactive compound or bioactivity on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities. The data obtained form these in vitro toxicity studies can be extended to animal model studies, including human clinical trials, to determine toxicity, efficacy and selectivity of a test compound bioactive compound or bioactivity.

The selectivity, specificity and toxicology, as well as the general pharmacology, of a test compound can be often improved by generating additional test chemicals based on the structure/property relationship of a test compound originally identified as having activity. Test compounds can be modified to improve various properties, such as affinity, life-time in blood, toxicology, specificity and membrane permeability. Such refined test compounds can be subjected to additional assays as they are known in the art or described herein. Methods for generating and analyzing such compounds or compositions are known in the art, such as U.S. Patent No. 5,574,656 to Agrafiotis et al. Pharmaceutical compositions

The present invention also encompasses a test compound in a pharmaceutical composition comprising a pharmaceutically acceptable carrier prepared for storage and preferably subsequent administration, which have a pharmaceutically effective amount of the test compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro edit. (1985)). Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used.

The test compounds of the present invention can be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions or injectable administration; and the like. Injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like. In addition, if desired, the injectable pharmaceutical compositions can contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents and the like. If desired, absorption enhancing preparation, such as liposomes, can be used.

The pharmaceutically effective amount of a test compound required as a dose will depend on the route of administration, the type of animal or patient being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. In practicing the methods of the present invention, the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, preferably in a mammalian patient, preferably in a human, or in vitro. In employing them in vivo, the pharmaceutical compositions can be administered to the patient in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperiotoneally, employing a variety of dosage forms. Such methods can also be used in testing the activity of test compounds in vivo.

As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and type of patient being treated, the particular pharmaceutical composition employed, and the specific use for which the pharmaceutical composition is employed. The determination of effective dosage levels, that is the dose levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods as discussed above. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compound.

In non-human animal studies, applications of the pharmaceutical compositions are commenced at higher dose levels, with the dosage being decreased until the desired effect is no longer achieved or adverse side effects are reduced of disappear. The dosage for the test compound of the present invention can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the test compound. Typically, dosages can be between about 1 ng/kg and about 10 ng/kg, preferably between about 10 ng/kg and about 1 mgkg, more preferably between about 100 ng/kg and about 100 micrograms/kg, and most preferably between about 1 microgram/kg and about 10 micrograms/kg.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction or other adverse effects. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate. The magnitude of an administrated does in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight and response of the individual patient, including those for veterinary applications.

Depending on the specific conditions being treated, such pharmaceutical compositions can be formulated and administered systemically or locally. Techniques for formation and administration can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990). Suitable routes of administration can include oral, rectal, transdermal, otic, ocular, vaginal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For such transmucosal administration, penetrans appropriate to the barrier to be permeated are used in the formulation. Such penetrans are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the pharmaceutical compositions herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulation as solutions, can be administered parenterally, such as by intravenous injection. The pharmaceutical compositions can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administrations. Such carriers enable the bioactive compounds and bioactivities of the invention to be formulated as tables, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Substantially all molecules present in an aqueous solution at the time of liposome formation are incorporated into or within the liposomes thus formed. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse will cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amount of a pharmaceutical composition is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active chemicals into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tables, dragees, capsules or solutions. The pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, for example by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical formulations for parenteral administration include aqueous solutions of active chemicals in water-soluble form.

Additionally, suspensions of the active chemicals may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances what increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the chemicals to allow for the preparation of highly concentrated solutions.

Pharmaceutical compositions for oral use can be obtained by combining the active chemicals with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tables or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone. If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Dragee cores can be provided with suitable coatings. Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active doses.

The test compounds of the present invention, and pharmaceutical compositions that include such test compounds are useful for treating a variety of ailments in a patient, including a human. As set forth herein, the test compounds of the present invention have anti-metastatic effects, anti-tumor effects and anti-cancer effects. A patient in need of such treatment can be provided a test compound of the present invention, preferably in a pharmacological composition in an effective amount to reduce metastasis, reduce the growth rate of tumors, reduce to tumor mass of tumors or treat cancer or carcinomas. The amount, dosage, route of administration, Examples

Example 1: General Materials and Methods

The following general materials and methods where used in the performance of the various experiments described in this section. Cells and Cell Culture

Fibrosarcoma HT1080 sublone H4 cells (ATCC NO: ), ERG- 1 -expressing transfectants (H4E2 (ATCC NO: ), H4E3 (ATCC NO: ) and H4E9

(ATCC NO: )), neomycin-resistant control cells (H4N (ATCC NO: )), and EGR-1-null cells (H4E4 (ATCC NO: ) and H4E6 (ATCC NO: )) were prepared by transfection of H4 cells with expression vectors for mouse wild-type erg-1 (pCMV-erg-1) as described by Huang et al., Cancer Res. 55:5054-5062 (1995). These cells were maintained in DMEM supplemented with 5% fetal bovine serum and cultured in the presence of penicillin, streptomycin and 200 μg/ml of G-418. Cell numbers in culture were determined by direct cell counting following the general methods of Huang et al., Cancer Res. 55:5054-5062 (1995). Protein Preparation and Western Blot

Cells were initially cultured at a density of about 4 x 10⁴ cells/cm², incubated overnight, washed twice with ice-cold phosphate-buffered saline (PBS), and lysed by scraping using RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl floride, 1 mM aprotinin and 1 mM sodium orthovanadate). The lysates were passed though a 21 -gauge needle to shear DNA in the lysates, incubated for one hour on ice and centrifuged at 12,000 x g for twenty minutes and the supematants collected. Protein concentration in the supematants were determined using Bio-Rad protein assay reagent. Samples of the supematants containing 100 μg of protein were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7% gel. Protein in the gel was electrophoretically transferred onto polyvinylidene difluoride membrane (Millipore Corporation, Bedford, Mass) and incubated with polyclonal rabbit anti-Egr-1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). A secondary antibody, anti-rabbit IgG antibody with an appropriate label. Immunoreactive bands on the membrane were visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) and the intensity of chemiluminescence of bands determined using image analysis (Kodak Digital Science™ ID image analysis system, Eastman Kodak Co.). Cell Labeling, Extracellular Matrix Preparation and Immunoprecipitation

For the plasminogen activator inhibitor (PAI-1) assay, 2 x 10⁵ cells were initially placed in standard 6- well tissue culture plates in DMEM supplemented with 5% fetal bovine serum and incubated overnight. These cells were exposed to cysteine/methionine-free DMEM in the presence or absence of various concentrations (between about 0.001 ng/ml and about 100 ng/ml) of recombinant human TGF-βl (rhTGF-βl) (R&D Systems Inc., Minneapolis, MN) for about 30 μg/ml of mouse monoclonal anti-TGF-β_{! 23} (Genzyme Corp., Cambridge, MA) for two hours. The cells were then exposed to ³⁵S-cysteine/methionine at 50 μCi/ml of (1180 Ci/mmol; Trans- ³⁵S-label, ICN Biochemicals, Inc., Costa Mesa, CA) for two hours. Extracellular matrix from the cell cultures were obtained using established methods (Der et al., J. Cell Sci. 52:151-166 (1981). Briefly, cell monolayers in tissue culture wells were rinsed with PBS and the cytosolic and nuclear proteins were extracted by subsequence washes with hypotonic buffer and sodium deoxycholate. The remaining extracellular matrix proteins were recovered by the addition of electrophoresis buffer to the wells and scraping. The resulting samples were subjected to 10% SDS-PAGE. The resulting gels were treated with Fluoro-Hancer™ autoradiography enhancer (Research Products International Corp., Mt. Prospect, IL) for thirty minutes followed by drying and autoradiography.

For the fibronectin (FN) assay, 2 x 10⁵ cells were initially cultured in standard 6-well tissue culture plates and treated overnight with or without 10 ng/ml of TGF-βl or 30 μg/ml of mouse monoclonal anti-TGF-βj ₂₃ cysteine/methionine-free media. The media was collected and subjected to absorption of fibronectin using gelatin-Sepharose beads (Amersham Pharmacia Biotech) in the presence of 0.5% Triton X-100 as previously described (Carcamo et al., Mol. Cell. Biol. 14:3810-3821 (1994)). Samples were resolved using 7% SDS-PAGE and the gels were treated with Fluoro-Hancer™ followed by drying and autoradiography. Antisense TGF-βl Oligodeoxynucleotides and Cell Transfection

Anti-sense 14-base phosphorothioate oligodeoxynucleotides (5'-CGATAGTCTTGCAG- 3' (SEQ ID NO:l)) corresponding to the human TGF-βl mRNA (hTGF-βl antisense oligonucleotide) and a scrambled sequence control sequence (5'-GTCCCTATACGAAC-3^! (SEQ ID NO:2)) were synthesized by established methods. The hTGF-βl antisense oligonucleotide has been shown to specifically reduce TGF-βl expression (Paulus et al., J. Neuropathol. Exp. Neurol. 54:236-244 (1995)). Cationic liposome-mediated transfection was used to introduce oligonucleotides into fibrosarcoma HT1080 subclone H4 cells, or the erg-J transfected clones using established methods (Suzuki et al., Mol. Cell. Bio. 18:3010-3020 (1988)). Briefly, oligonuleotides were dissolved in one volume of antibiotic-free medium and mixed with Lipofectin™ reagent (Life Technologies, Inc.) dissolved in the same volume of antibiotic-free medium and incubated for fifteen minutes at room temperature. The oligonucleotide-liposome complexes thus formed were diluted with four volumes of antibiotic-free medium and added to cells that had be cultured to about 60% confluence and previously washed twice with antibiotic- free medium. The concentration of oligonuleotides and Lipofectin™ in the transfection was 1 mM and 1%, respectively. After four hours, fresh normal growth medium containing 5% fetal bovine serum as added to the cells. Forty-eight hours later the cells were analyzed for expression of plasminogen activator inhibitor-1 (PAI-1) or fibronectin (FN). Oligonucleotides and Electrophoretic Mobility Shift Assay

Nuclear extracts of clone H4H9 (ERG-1 expressing), clone H4 (non-ERG-1 expressing) and clone H4N (non-ERG-1 expressing) were prepared using established methods (Brown et al., Eur. J. Immunol. 22:2419-2420 (1992)). Protein concentrations in these nuclear extracts were determined using protein assay reagents (BioRad). Synthetic double-stranded oligonuleotides bearing sequences corresponding to either -75 to -52 base pairs of human FN promoter (Site A) or -4 to +18 base pairs of human FN promoter (Site B) (Brown et al., Eur. J. Immunol. 22:2419- 2420 (1992)). The DNA sequence for Site A is 5'-

GATCTCTCTCCTCCCCCGCGCCCCGGGG-3- (SEQ ID NO:3) with a prototypical EGR-1 binding site of 5'-CTCCCCCGC-3' (SEQ ID NO:4). The DNA sequence for Site B is 5'- GATCTCCGACGCCCGCGCCGGCTGTG-3' (SEQ ID NO:5) with a prototypical ERG-1 binding site of 5'-CGCCCGCGC-3' (SEQ ID NO:6). The full-length oligonucleotides for Site A and Site B were end-labeled with [γ-³²P] ATP using T4 polynucleotide kinase using established methods. These labeled oligonuleotides were uses as "Probe A" and "Probe B."

Gel shift assays were performed by incubating 20 μg of nuclear extract with Probe A or Probe B (1 x 10⁵ cpm) for twenty minutes at 4°C in a 20 μL reaction volume containing 25 mM HEPES buffer, pH 7.9, 60 mM KC1, 2 mM MgCl₂, 0.1 mM EDTA, 0.5 mM dithiothreitol, 100 μg/ml spermidine, 10% glycerol and 100 μg/ml bovine serum albumin. Protein-Probe complexes were separated from free Probe by electrophoresis through a 6% nondenaturing acrylamide gel in 0.5X Tris borate/EDTA buffer. The gels were dried and autoradiographed. Competition experiments were performed by adding excess unlabeled Probe A, Probe B, or unlabeled oligonucleotides comprising the prototypical EGR-1 binding sites (GCE), or mutated EGR-1 binding sequences (mGCE), either alone or in combinations, to the reaction mixture described in this section for fifteen minutes at 4°C prior to the addition of labeled Probe A, Probe B, or both.

Antibody supershift experiments were performed adding antibody specific for Spl (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal Egr-1 antiserum (Huang et al., Oncogene 9:1367-1377 (1994)) to the reaction mixture before the appropriate radiolabeled Probe A, Probe B, or both were added. For these experiments, recombinant GST-ERG- 1 fusion protein or wild-type FLAG-tagged-S348A/S350A-EGR-l and FLAG-tagged-ERG-lΔS348A/S350A mutant fusion proteins were used as controls. The FLAG-tagged proteins are fusion proteins produced analogues to GST-EGR proteins. Mutants were introduced into the DNA binding sites of Erg- 1 using the Quick Change method using polymerase chain reaction primers comprising the S348A/S350A mutations (Stratagene, La Jolla, CA). Cell Adhesion Assay

Cell adhesion assays were performed using ELISA plates (Sarstedt Inc., Newton, NC) or 35mm polystyrene Petri dishes (Falcon, Becton Dickinson, Bedford, MA). Cell adhesion assays performed in Petri dishes were performed by adding about 3 x 10⁴ cells/cm² of surface area. After incubation for between about four and about 7 hours, cells that were not attached to the surface were washed away using PBS at 37°C. Adherent cells were harvested from the surface using trypsin and counted using a Coulter counter.

In some cases, ELISA plates were pretreated by coated with 5 μg/ml or 0.25 μg/ml of human plasma fibronectin (Boehringer Mannheim) or 5 μg/ml recombinant active human PAI-1 (American Diagnostica, Inc. Greenwich, CT) for one hour at 37°C. Non-specific sites on the pretreated plates were blocked using 0.1% bovine serum albumin in PBS. Cells were dispensed into the wells at a density of about 4 x 10⁴ cells/well. After incubation of a variety of times, any non-adherent cells were washed away with gentle washing with warm PBS. Adherent cells were stained with 1% crystal violet in 20% methanol for fifteen minutes, washed with distilled water, and solubilized with 2% SDS. The absorbance of the solution in the wells at 590 nm was determined using a microtiter plate reader. For PAI-1 neutralization, 10 μg/ml of monoclonal antibody anti-human PAI-1 (American Diagnostics) were added to the wells at with cells and the effect on cell adhesion was observed. For FN inhibition, GRGDSP and GRGESP peptides were added to the wells at about 10 μg/ml and about 50 μg/ml with the cells and the effect on cell adhesion was observed. General Methods and Mechanisms

In these examples, established methods in molecular biology, nucleic acid chemistry, protein chemistry, chemistry and cell biology were used. Such methods are readily available from a number of sources, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Any reference to mechanisms in these examples is postulated based on the finding of these Examples. The inventors expressly do not wish to be bound by such postulated mechanism, which are provided for illustrative purposes.

Example 2: Effect of EGR-1 on the Expression of PAI-1 in Fibrosarcoma Cells

For this example, fibrosarcoma HT 1080 subclone H4 cells stably transfected with an expression vector for wild-type EGR-1 were used (Huang et al., Cancer Res. 55:5054-5062 (1995)). A series of transfectants of HT 1080 subclone H4 that express varying amounts of EGR-1 were used to determine whether PAI-1 or FN was synthesized and secreted in proportion to the amount of EGR-1 expressed by these cells. The relative expression of EGR-1 by these cells as confirmed by Western blot analysis, as is shown in FIG. 1 A. These results confirmed that clone H4E9 expressed the largest amount of EGR-1, which was normalized to 100% expression. The remaining clones expressed graded amounts of EGR-1, with H4E2 expressing 21% of EGR-1 expressed by H4E9 and H4E3 expressing about 9% of EGR-1 expressed by H4E9. Clones H4E4, H4E6, parental H4 and empty vector control cells H4N did not express detectable levels of EGR-1.

The expression of PAI-1 in these cells was examined. In metabolically labeled EGR-1 expressing clones, clone H4E expressed the greatest amount of PAI-1 and clone H4E2 expressed more PAI-1 than parental cell line H4 or negative control clones (see, FIG. IB). Quantitative analysis of the average of five independent experiments showed a greater than five fold elevation of PAI-1 expression in clone H4E9 as compared to control cells (see, FIG. 1C). In contrast, the pCMV empty vector-transfected clones or EGR-1 negative clones expressed low levels of PAI-1 at levels similar to parental H4 cells (see, FIG. IB and FIG. 1C). The correlation of PAI-1 expression and EGR-1 expression in these cells was highly correlated (R_PEARso_N ⁼ 0-971, p < 0.0003) (see, FIG. 1C, insert).

The expression and secretion of TGF-βl correlates to the levels of expression of EGR-1 in these cells (R_PEARso_N ~ 0.96) and is believed to function in an autocrine loop to regulate the growth of these cells (Lui et al., Proc. Natl. Acad. Sci. U.S.A. 93:11831-11836 (1996)). Also, TGF-βl can stimulate the expression of PAI-1 (Laiho et al., Cancer Res.49:2533-2553 (1989) and Laiho et al., Mol. Cell. Biol. 11 :972-978 (1991)). To test whether TGF-βl stimulate the expression of PAI-1 in these clones, the clones were labeled with [³⁵S]cysteine/methionine in the presence or absence of 10 ng/ml of recombinant human TGF-βl (rhTGF-βl) for four hours. The resultant expression of PAI-1, if any, was monitored by electrophoresis and autoradiography (see, FIG. IB). All of the clones tested exhibited a significant (p < 0.05) increase in the secretion of PAI-1 following treatment with rhTGF-βl (see, FIG. 1C). In addition, the total PAI-1 measured for each clone correlated with basal PAI-1 expression in these cells observed in the absence of stimulation with rhTGF-βl (see, FIG. 1C).

The results of this example establish that rhTGF-βl increases the expression of PAI-1 in fibrosarcoma cells and that the correlation between PAI-1 expression and EGR-1 expression is related to stimulation with TGF-βl .

Example 3: Induction of Fibronectin Expression in Fibrosarcoma cells by EGR-1.

The levels of FN secreted by the EGR-1 expressing and EGR-1 non-expressing cells were determined by metabolically labeling the cells for two hours. The labeled FN produced by the cells and presented in the culture medium was absorbed from the culture medium with gelatin- Sepharose beads known to bind FN (Scott et al., J. Immunol. Methods 43:29-33 (1981)). The absorbed FN was recovered and analyzed by SDS-PAGE. The characteristic band of FN at 220 KDa was observed at maximum intensity in the medium for H4E9 cells and band was noted in samples from H4E2 cells and H4E3 cells, with a band intensity between H4E9 cells, parental cells, and cells that do not express EGR-1 (see, FIG.2 A). Quantitative analysis of the average of three independent experiments showed a 3.5 fold average increase in FN in clone H4E2 and a 38 fold average increase of FN in clone H4E9 as compared to appropriate negative controls (see, FIG.2B). Similar to the analysis for PAI-1, we determined the correlation between EGR-1 expression and FN expression and found a high correlation (Rp_EARso_N ⁼ 0.985, p < 0.00005) (see, FIG. 2B, insert). These results establish that EGR-1 is a factor important for FN secretion by fibrosarcoma cells.

Example 4: Regulation of TGF-βl Expression, but not FN Expression, in EGR-1 Expressing Fibrosarchoma Cells

The expression of FN can be stimulated by TGF-βl in prostatic carcinoma cells (Frazen et al., Exp. Cell Res. 207:1-7 (1993)), colon cancer cells (Huang et al., Int. J. Cancer 261:4337- 4345 (1986)) and lung mink MvlLu cells (Laiho et al. Mol. Cell. Bio. 11 :972-978 (1991)). In order to determine whether EGR-1 -induced TGF-βl regulates the expression of FN in fibrosarchoma cells, the H4 clones of the present application were contacted with rhTGF-βl at the same concentration that stimulates a high level of expression of PAI-1. We observed a weak induction of FN by rhTGF-βl (see, FIG. 2A and FIG.2B) and a strong induction of PAI-1 by rhTGF-βl (data not shown), indicating that the regulation of FN expression is distinct from the regulation of PAI-1 expression.

The secretion of FN and PAI-1 in parental cell line H4 in response to exposure to rhTGF- βl (between about 0.001 ng/ml and about 100 ng/ml) was investigated further. The induction of PAI-1 expression induced by rhTGF-βl was found to be dose-dependent in H4 cells. When the concentration of rhTGF-βl was increased, the expression of PAI-1 increased up to about 9-fold compared with basal levels of PAI-1 in these cells. The half-maximal stimulation of PAI-1 by rhTGF-βl was found to be about 0.05 ng/ml rhTGF-βl (EC₅₀ of about 2 x 10^"12M) with the near- maximal effect being observed at about 100 ng/ml rhTGF-βl (4 x 10^"9 M) (see, FIG. 3 A and FIG.3B). These results indicate that H4 cells are relatively sensitive to rhTGF-βl.

In contract, the induction of FN by rhTGF-βl in H4 cells was not detectable at about 0.05 ng/ml of rhTGF-βl (see, FIG.3A). A weak response by was noted at a much higher concentration of rhTGF-βl, (4 x 10^"10 M) (see, FIG.3B). These data indicate that PAI-1, but not FN, is preferentially regulated by TGF-βl. The high correlation between EGR-1 expression and FN expression without the role of TGF-βl suggests, as one possible mechanism that the inventors expressly do not wish to be bound to, that EGR-1 directly regulates the expression of the FN gene.

Example 5: The Requirement of TGF-βl for the Secretion of PAI-1 and FN in egr-1 Regulated H4 Cells - Immunological Methods.

In order to determine the requirement of TGF-βl for the secretion of PAI-1 and FN in egr-1 regulated H4 cells, neutralizing anti-TGF-βl antibody preparations were added cells to block the effect of TGF-βl in cell cultures (see, FIG. 4). In control cells H4 and H4N, the basal level of PAI-1 was reduced 1.4 fold in response to anti-TGF-βl antibodies in the culture medium. In the EGR-1 expressing cells H4E2 and H4E9, there was a 2.7 fold reduction in the basal level of PAI-1 in response to anti-TGF-βl antibodies in the assay medium. These results indicate that the increased secretion of PAI-1 in response to EGR-1 may require the expression of TGF-βl . In contrast, the expression of FN in these cells was slightly altered by the addition of anti-TGF-βl antibodies to the culture medium (less than 1.4 fold change in expression of FN) in the EGR-1 expressing cell lines H4E2 and H4E9 (see, FIG. 4). These results are consistent with the previous Examples and suggest that the EGR-1 regulates the expression of PAI-1, but not FN.

Example 6: The Requirement of TGF-βl for the Secretion of PAI-1 and FN in egr-1 Regulated H4 Cells - Antisense Methods.

The results obtained in Example 5 were confirmed using antisense technologies. In these experiments, antisense TGF-βl molecules comprising SEQ ID NO:l, or comprising the control scrambled SEQ ID NO:2, were used as set forth in Example 1. Nucleic acid molecules comprising SEQ ID NO:l have been used to reduce the transcription levels and production of TGF-βl (LeRoy et al, J. Bio. Chem.271:11027-11033 (1996) and Paulus et al., J. Neuropathol. Exp. Neurol. 54:236-244 (1995)). Thus, we examined the expression of PAI-1 after lipofection with SEQ ID NO:l or SEQ ID NO:2 (see, FIG. 5). The level of PAI-1 in EGR-1 expressing H4E9 cells transfected with SEQ ID NO:l was reduced by over 75% in EGR-1 producing cell lines to levels of PAI-1 in control cells H4 and H4N in H4E9. In contrast, the level of PAI-1 in EGR-1 expressing cells was not influenced by transfection with the scrambled SEQ ID NO:2 or by lipofection reagents alone (see, FIG. 5). Furthermore, the expression of FN was not inhibited in cells expressing EGR-1 by transfection of either SEQ ID NO:l or SEQ ID NO:2 (see, FIG. 5). The results of these studies supports the results obtained in Example 5 and establish that TGF- βl is required for expression of PAI-1 but not FN by egr-1 transfected cells. Also, the lack of effect of antisense TGF-βl on FN section supports the observations on the dose-response studies presented in FIG.3. that TGF-βl is not involved in mediating expression of FN in the egr-1 transfected cells.

Example 7: Binding of Nuclear and Recombinant EGR-1 to the Proximal Region of the Human Fibronectin Promoter.

The correlation of FN secretion with EGR-1 expression independent of TGF-βl suggests that EGR-1 directly regulates the FN gene. To investigate this hypothesis, we examined the promoter region of the human FN gene (exon 1) consisting of 742 base pairs in the 5'-flanking region (Dean et al., Proc. Natl. Acad. Sci., U.S.A., 84:1876-1880 (1987)). We observed two potential EGR-1 binding sites in this locus by computer analysis using Transcription Elements Search Software. Two sequences containing potential EGR-1 consensus sites, termed A and B, were identified and double stranded probes were synthesized (Probe A and Probe B of Example 1) (see, FIG. 6A). Electrophoretic mobility assays were used to determine if EGR-1 binds to either Probe A or Probe B. A recombinant wild-type FLAG-tagged EGR-1 fusion protein and control mutant FLAG-tagged-EGR-lΔS348A/A350A fusion protein were sued for these experiments. The FLAG-tagged-EGR-lΔS348A/A350A fusion protein is a serine-alanine mutant at position 348 and 350 in the EGR-1 zinc finger domain, thereby reducing DNA binding activity (Wilson et al., J. Biol. Chem. 267:3718-3724 (1992) and Pavletich et al., Science 10:809- 817 (1991)). As shown in FIG. 6B, a specific DNA-protein complex occurred when wild-type protein was combined with either Probe A or Probe B and that the complexes were absent when the EGR-1 mutant protein was used in the reaction in place of the wild-type protein (compare lane 1 and lane 2, and lane 6 and lane 7, at the arrow). When anti-EGR-1 antibody was added to the reaction mixture, the DNA-protein complex was no longer detectable (see, FIG. 6B, lane 5 and lane 8). These results are consistent with previous results establishing that anti-EGR-1 did not promote a supershift, but caused a dissociation of the DNA-protein complex (Huang et al., DNA Cell Bio. 12:265-273 (1993 and Huang et al., J. Cell. Biochem. 66:489-499 (1997)). In contrast, the addition of an anti-Spl antibody, which binds with Sp-1, a GC-rich DNA binding protein which appears in nuclear extracts and mimic EGR-1, did not dissociate the DNA-protein complex or produce a supershift. The use of anti-Spl antibodies with FLAG-tagged-EGR-1 fusion protein preparations was associated with a slight but reproducible increase in band intensity (see, FIG. 6B, compare lane 1 and lane 3, and lane 2 and lane 4).

Experiments conducted with FLAG-tagged-EGR-1 fusion protein preparation and Probe B often lead to less intense bands than experiments performed with Probe A. In order to clarify these observations, similar experiments were performed using different EGR-1 preparation comprising GST-EGR-1 fusion protein, GST is glutathione-S-transferase (see, FIG. 6C). As before (FIG. 6B), specific DNA-protein complexes occurred when GST-EGR-1 protein was combined with either Probe A or Probe B (see, FIG. 6C, lane 2 and lane 6). Furthermore, for both Probe A and Probe B the addition of unlabeled oligonucleotides comprising the consensus sequence of Probe A or Probe B competed with Probe A or Probe B binding to protein (see, FIG. 6C, lane 3 and lane 7). Also, the addition of unlabeled olignocleotides with a mutated EGR-1- binding sequence (mGCE) (Y5'-GGATCCAGCTAGGGCGAGCTAGGGCGA-3' (SEQ ID NO:l 1) wherein the underlined sequences indicate repeated consensus binding sequences with the exception of the bolded letters which indicated changes from the consensus sequences) had no effect of probe-protein binding (see, FIG. 6C, lane 4 and lane 8). These results confirm the results of experiments presented earlier in this example using recombinant FLAG-tagged-EGR-1 fusion proteins. Also, these experiments show that the complexes formed with the GST-EGR-1 preparation under the same conditions as with the FLAG-tagged-EGR-1 fusion protein are of similar intensity, suggesting similar binding affinities between the Probe and the fusion protein. As shown in FIG. 6C (lanes 10 to 13 and lanes 15 to 18) directed titration experiments with unlabeled probes demonstrated similar biding for each site.

Similar results were obtained when nuclear extracts from the EGR-1 -expressing clone H4E9 and EGR-1 -lacking cells (H4 and H4N) were used in place of the recombinant fusion proteins used earlier in this Example. A prominent complex was observed when nuclear extracts from H4E9 cells were incubated with oligonucleotide Probe A or Probe B (see, FIG. 6D, line 3 and lane 13, at arrow). This complex was not detected for the nuclear extracts from control cells H4 and H4N (see, FIG. 6D, lane 1 and lane 2, and lane 11 and lane 12). Specificity of binding was determined by titration experiments using unlabeled oligonucleotides. The complex formation by the H4E9 nuclear extracts were inhibited in a dose-dependent manner by the addition of the unlabeled oligonuleotides Probe A or Probe B (see, FIG. 6D, lines 6 to 8, and lane 16 and lane 17). Also, competition with oligonuleotides containing two consensus EGR-1 binding sequences (mGCE) resulted in dissociation of complexes (see, FIG. 6D, lane 4 and lane 5; and lane 14 and lane 15). The addition of EGR-1 antibody dissociated the complex that was formed with H4E9 nuclear extracts (FIG. 6D, lane 10 and lane 19), but addition of the SP1 antibody had no effect (FIG. 6D, lane 9 and lane 18). These results argue against significant binding of any Spl that may be in these extracts to either sites (Suzuki et al., Mol. Cell. Biol. 18:3010-3020 (1998)). Thus, factors in nuclear extracts of EGR-1 -expressing cells but not control cells recognized both Probe A and Probe B sites of human FN promoter in a sequence- specific manner. The results of this example establish that EGR-1 binds specifically with the promoter region of human FN gene consistent with a direct role in the regulation of FN expression. Example 8: Enhanced Expression of Fibronectin and PAI-1 in EGR-1-Expressing Cells Increases Adherence to a Substratum.

To determine whether endogenous PAI-1 or FN influence the adhesion of HT1080 cells, we compared the attachment efficiency of the various cells described in Example 1 to untreated polystyrene Petri dishes. Polystyrene plastic tissue culture plates have high hydrophobic nature, inhibit cell adhesion and are commonly used for growing cells in suspension culture. Many types adherent cells fail to attach to this substratum (Ramsey et al., In Vitro, Rockville, 20:802- 808 (1984)), but EGR-1 expressing cell clone H4E9 can attach to this substratum significantly better than the parental cell line H4 or empty vector cells H4N (see, FIG. 7). We observed that 22% of H4E9 cells had attached to polystyrene Petri dishes after four hours compared with about 8% for the control cell lines (9% of H4 cells and 7% of H4N cells, p < 0.0006). After seven hours, 88% of H4E9 cells attached to the polystyrene Petri dishes, whereas only 58% of the control cells H4 or H4N attached to Petri dishes (p < 0.0001) (see, FIG. 7).

To test whether authentic FN or PAI-1 facilitates attachment, we carried out adhesion assays in the presence and absence of rhPAI-1 and human plasma FN (see, FIG. 7B). When the various cells were exposed to 96-well polystyrene "ELISA" plates precoated with 5 μg/ml of rhPAI-1 for 4 hours, they exhibited adhesion efficiencies similar to untreated plates (see, FIG. 7A and FIG. 7B). By seven hours, about 70% of the cells attached to the PAI-1-treated plates although no consistent differences were observed between EGR-1 -expressing cells and EGR-1- lacking control cells (see, FIG. 7B). Addition of neutralizing anti-PAI-1 antibodies had little effect on the adhesion of control cells, but partially inhibited the attachment of the FN-secreting H4E9 cells by about 20% (see, FIG. 8). This inhibition is reproducible and significant at p<0.03.

To test whether authentic FN the attachment of the various cells described in Example 1 to polystyrene surfaces, we tested the adhesion of cells on 96-well polystyrene plates coated with 5 μg/ml of FN. After four hours, 88% of H4E9 cells attached to the plates and about 70% of H4 and H4N control cells attached to the plates (see, FIG. 7B). Thus, FN promoted about 20 times greater adhesion than for uncoated or PAI-1 -coated plates. Virtually all cells attached to FN- coated plates after seven hours (see, FIG. 7B). To detemiine whether the increased adhesion depend on specific FN interactions, we added Arg-Gly-Asp (RGD) -containing peptides that block FN binding to its receptors, such as α^ or _vβ₃ integrins (Ruoslahti et al., Science 238:491-497 (1987) and Hynes, Cell 69:11-25 (1992)). The addition of moderate amounts of GRGDSP peptides (10 μg/ml) reduced the adhesion of H4 or H4N control cells to less than 20%, but had no effect on the adhesion of H4E9 cells (see, FIG. 9A). When the GRGDSP concentration was increased to 50 μg/ml, the adhesion of H4E9 cells was not detectable (see, FIG. 9A). The control peptide GRGESP had negligible effects on cell adhesion, even at concentrations of 50 μg/ml (see, FIG. 9A). These results indicate that H4 cells and related cells interact with FN in an RGD-dependent manner.

We noted that the attachment of FN-secreting H4E9 cells was partially inhibited by the addition of anti-PAI-1 antibodies (see, FIG.9A) and tested whether anti-PAI-1 antibodies inhibit attachment to FN-coated polystyrene plats. The addition of anti-PAI-1 antibodies to cells exposes to FN-coated plates had the opposite results compared with the effect of these antibodies on cell adhesion to PAI-1-coated plates (see, FIG. 9A, FIG. 8 and FIG. 9A). The anti-PAI-1 antibody blocked the attachment of the control cells by about 55% but had no effect on the attachment of EGR-1 -expressing cells to FN-coated plates (see, FIG.9A). Increased amounts of anti-PAI-1 had a weak inhibitory effect on the adhesion of H4E9 cells (data not shown). Addition of anti-PAI-1 antibodies to H4E9 cells when attached to plates coated with twenty times less FN substantially and significantly reduced adhesion of these cells to levels of adhesion of control cells (see, FIG. 9B). Also, the combination of small amounts of GRGDSP peptide (10 μg/ml) together with anti-PAI-1 antibody reduced the attachment of all tested cell types by approximately 50% compared to low doses of GRGDSP peptide alone (see, FIG.9A). These results indicate that PAI-1 facilitates attachment to surfaces in the context of FN-coated surfaces or FN-secreting cells. All publications, including patent documents and scientific articles, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

We claim:

4. A method for increasing expression of an anti-metastatic factor in a tumor cell, comprising: administering to a tumor cell a vector comprising a nucleic acid sequence encoding an EGR or a nucleic acid sequence that encodes an active portion of an EGR, wherein said tumor cell exhibits increased expression of at least one anti- metastatic factor upon said administering.

5. The method of claim 1 , wherein said at least one anti-metastatic factor is selected from the group consisting of plasminogen activator inhibitor- 1, ttansforming growth factor beta-1 and fibronectin.

6. The method of claim 1, wherein said vector is a viral vector.

7. The method of claim 1 , wherein said vector further comprises an expression control sequence operably linked to said nucleic acid sequence encoding an EGR or said nucleic acid sequence that encodes an active portion of EGR.

8. The method of claim 1 , wherein said tumor cell exhibits expression of between about 0% and about 10% of the normal level of EGR expression by those cells.

9. The method of claim 1 , wherein said tumor cell is selected from the group consisting of fibrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, acute myelogenous leukemia, melanoma and prostate carcinoma.

10. The method of claim 1 , wherein said EGR is a mammalian EGR.

11. The method of claim 7, wherein said mammalian EGR is human EGR or mouse EGR.

12. The method of claim 1 , wherein said active portion of EGR comprises a zinc finger domain and encodes a polypeptide that increases the expression of at least one of plasminogen activator inhibitor- 1, transforming growth factor beta-1 and fibronectin.

13. A method for interfering with the metastasis of a tumor cell, comprising: administering to a tumor cell a vector comprising a nucleic acid sequence encoding EGR or a nucleic acid sequence that encodes an active portion of EGR, wherein the metastasis of said tumor cell is interfered with upon said administering.

14. A method for identifying compounds or compositions that increase or decrease the expression of EGR in a cell, comprising: 1) contacting a cell with a test compound; and

2) measuring the expression of EGR in said cell.

15. The method of claim 11 , wherein said cell is a tumor cell.

16. The method of claim 11 , wherein said test compound comprises a small molecule.

17. The method of claim 11 , wherein said test compound comprises an antibody or active fragment thereof.

18. The method of claim 11 , wherein said test compound is a nucleic acid molecule.

19. A compound or composition identified by the method of claim 11.

20. A pharmaceutical composition comprising the compound of claim 11.

21. A method for identifying compounds or compositions that increase or decrease the expression of at least one anti-metastatic factor in a cell, comprising:

1) contacting a cell with a test compound; and

2) measuring the expression of at least one anti-metastatic factor in said cell.

22. A method for identifying compounds or compositions that increase or decrease the attachment of a cell to a substrate, comprising:

1) contacting a cell with a test compound; and 2) measuring the attachment of said cell to a substrate.

23. The method of claim 19, wherein said substrate comprises fibronectin.

24. A method for interfering with proliferation of a tumor cell in a mammal, comprising: administering to a mammal a vector comprising a nucleic acid sequence encoding an EGR or a nucleic acid sequence that encodes an active portion of an EGR, wherein said tumor cell exhibits increased expression of at least one anti-metastatic factor upon said administering.

25. The method of claim 21 , wherein said vector is a viral vector.

26. The method of claim 21 , wherein said vector further comprises an expression control sequence operably linked to said nucleic acid sequence encoding an EGR or said nucleic acid sequence that encodes an active portion of EGR.

27. The method of claim 21 , wherein said tumor cell exhibits expression of between about 0% and about 10% of the normal level of EGR expression by those cells.

28. The method of claim 21 , wherein said tumor cell is selected from the group consisting of fibrosarcoma, breast carcinoma, glioblastoma, osteogenic sarcoma, glioblastoma, myelodysplastic syndrome, small cell lung carcinoma, non-small cell lung carcinoma, leukemia, acute myelogenous leukemia, melanoma and prostate carcinoma.

29. The method of claim 21, wherein said EGR is a mammalian EGR.

30. The method of claim 26, wherein said mammalian EGR is human EGR or mouse EGR.

31. The method of claim 21 , wherein said active portion of EGR comprises a zinc finger domain and encodes a polypeptide that increases the expression of at least one of plasminogen activator inhibitor- 1, transforming growth factor beta-1 and fibronectin.