CA2377172A1 - Nfat transcriptional factors in tumor progression - Google Patents

Abstract

The present invention provides methods for monitoring or determining the progression of a cancer or metastatic potential of a cancer. The invention also applies to determining the prognosis for a treatment of cancer. Further features of the invention are methods for identifying target genes involved in cancer which requires NFAT biological activity.

Description

PATENT
ATTORNEY DOCKET NO.: 01948/086CA1 NFAT TRANSCRIPTION FACTORS IN TUMOR PROGRESSION
Statement as to Federally Sponsored Research The present research was supported by a grant from the National Institutes of Health/National Cancer Institute (Number CA8269S). The U.S. government has certain rights to this invention.
Field of the Invention The invention relates to cancer treatments and diagnostics for the monitoring of cancer progression and treatments.
Background of the invention Cancer progression is seen as the spreading or growing of the disease with or without treatment. The progression of cancer is marked by a series of events, which culminates in alterations with the molecular control of cell growth and survival. Cancer cells have overcome the barriers imposed in normal cells, which have a finite lifespan, to grow indefinitely. As the growth of cancer cells continue, genetic alterations may persist until the cancerous cell has manifested itself to pursue a more aggressive growth phenotype. If left untreated, metastasis, the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream, ensues.
Upon clinical diagnosis of a cancer, treatment of this disease is in part determined by the stage of the cancer. Depending on the severity of the cancer, a stage number is assigned, I, II, III, and IV. Stage I cancers are the least advanced and have a better prognosis. Higher stage cancers are more advanced with stage III and IV cancers associated with invasion to the lymph nodes and finally spread to other organs.

The transition of a benign; non-invasive tumor to an invasive and metastatic one involves alterations in intracellular signaling pathways, resulting in deviations in gene expression relative to normal cells. In epithelial cells, the process of cellular transformation can initiate a transition to a mesenchymal phenotype, resulting in the acquisition of motile and invasive characteristics. Some of these characteristics are manifested in the alteration of a cells complement of surface antigens. One such molecule that is associated with cellular migration and invasiveness is the a6[34 integrin.
Integrins, receptors for extracellular matrix ligands, are critical regulators of the invasive phenotype. Specifically, the a6~34 integrin has been linked with epithelial cell motility, cellular survival and carcinoma invasion, hallmarks of metastatic tumors. Engagement of the a6~4 integrin in cells derived from various human carcinomas mediates invasion through amplification of several intracellular signaling pathways such as the phosphoinositide 3-OH kinase (PI3-K) and the Met/HGF receptor tyrosine kinase pathways.
Typically a6(34 integrins are expressed primarily on the basal surface of most epithelial cells and function as receptors to laminins. In this capacity, a6(34 integrins function to maintain the integrity of the epithelia via the formation of rigid adhesive structures on the basal surface of the cell, adhering it to the basal membrane. Further functions of a6(34 integrins have been identified, whereby this integrin plays a role in cell migration and motility. In processes that facilitate migration, a6~i4 integrins are no longer confined to the basal surface of a cell and can be found localized in membrane protrusions such as filopodia, lamellipodia, and retraction fibers.
Summar~of the Invention The present invention features diagnostic methods to monitor the progression and metastatic potential of a cancer by monitoring the expression of NFAT polypeptides. We have found that increased NFAT levels are associated with cancer cell invasion and migration. We have also discovered that inhibition of NFAT biological activity results in decreased cancer cell invasion and migration. Thus, the invention includes inhibition of NEAT biological activity in cancer cells for the treatment of cancer.
Accordingly, in a first aspect, the invention features a method for monitoring the' progression of a cancer by measuring the amount of NFAT mRNA
or polypeptide expression in a sample from a subject, a change inNFAT mRNA
or polypeptide expression in the sample, relative to a control sample, indicating a change in the,progression of a cancer or a propensity for the progression of a cancer.
In a second aspect, the invention features a method of determining the prognosis for the treatment of cancer in a subject, comprising measuring the amount of NFAT mRNA or polypeptide expression in a sample where an increase or decrease in NFAT mRNA or polypeptide expression in the sample, relative to a control sample; indicates a worsened or improved prognosis for treatment of the cancer in the patient respectively.
In a third aspect, the invention features a method for determining the metastatic potential of a cancer in a subject, comprising measuring the amount of NFAT mRNA or polypeptide expression in a sample, where an increase or decrease in NFAT mRNA or polypeptide expression, relative to a control sample, indicates an increase or decrease, respectively, in the metastatic potential of the cancer in the subject.
In a desirable embodiment of the first three aspects, the NEAT polypeptides are selected from a group consisting of NFAT-l, NFAT-4 and NFAT-5:
Desirably, at least two of these NFAT mRNAs or polypeptides are measured in a given embodiment.
In other desirable embodiments to the first three aspects, an increase in NEAT mRNA or polypeptide expression indicates the progression of cancer, or poor prognosis cancer treatment, or an increase in the metastatic potential of the cancer.
In still other desirable embodiments to the first three aspects, measuring an increase of NFAT-1 mRNA or polypeptide expression indicates indicates an increase in cancer cell invasion or migration. Measuring an increase in NFAT-5 mRNA or polypeptide expression indicates an increase in cancer cell migration.
In yet still another desirable embodiment to the first three aspects, the method further comprises measuring the amount of the (34 subunit of integrin. More desirably the integrin is a6~34.
In a fourth aspect, the invention features a method for identifying a candidate compound that modulates cancer progression, tumor invasion, or tumor migration, this method comprising the steps of: i) contacting NFAT polypeptide to a candidate compound; and ii) measuring binding of the compound to the NFAT
polypeptide, wherein binding identifies the candidate compound as a compound that is useful for modulating cancer progression, tumor invasion or tumor migration.
In a fifth aspect, the invention features a method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, this method comprising the steps of i) contacting a cell or in vitro sample having NFAT biological activity; and ii) measuring NFAT biological activity, wherein a change in NFAT biological activity indicates a candidate compound that modulates cancer progression, tumor invasion, or tumor migration.
In a desirable embodiment to the fourth and fifth aspect, NFAT polypeptide or NFAT biological activity refers to NFAT-l, NEAT-4, or NFAT-S polypeptides or NEAT-1, NEAT-4, or NEAT-5 biological activities.
In other desirable embodiments to the fifth aspect, the NFAT biological activity is NFAT-1 biological activity; where a change in NFAT-1 biological activity results in a change in tumor invasion or tumor migration. Desirably, the change in NFAT-1 biological activity results in a decrease in tumor cell invasion or tumor cell migration.
In yet another desirable embodiment to the fifth aspect, the NFAT
biological activity is NFAT-5 biological activity, where a change in NFAT-5 biological activity results in a change in tumor migration. Desirably, the change in NFAT-5 biological activity results in a decrease in tumor cell migration.
In a sixth aspect, the invention features a method for identifying, a candidate compound that modulates cancer progression, tumor invasion, or tumor migration, the method comprises the steps of i) contacting a cell or ih vitro sample having NFAT biological activity, and an NEAT target gene, the NEAT target gene is regulated by NFAT biological activity, with a candidate compound; and ii) measuring the expression of the NFAT target gene, wherein the candidate compound is determined to modulate cancer progression, tumor invasion or tumor migration, if the candidate compound causes a change in expression of he NFAT
target gene.
In a desirable embodiment to the sixth aspect, the target gene is a reporter gene, and this reporter gene is operably linked to one, or a plurality of consensus NFAT DNA binding sequences. More desirably, the consensus NFAT DNA
binding sequence is selected from a group consisting of SEQ ID NO. 1, SEQ ID
NO. 2, and SEQ TD NO. 3.
In another desirable embodiments to the sixth aspect, NFAT biological activity refers to NEAT-l, NFAT-4, or NFAT-5 biological activities. Desirably, the NFAT biological activity is NFAT-1 or NFAT-5 biological activity; and the change in expression of the NFAT target gene or reporter gene is a decrease in target gene or reporter gene expression.
In a seventh aspect, the invention features a method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, said method comprising the steps of: i) contacting a candidate polypeptide with detectably labeled NEAT to for an NFAT-polypeptide complex;

ii) exposing the NFAT-polypeptide complex to a candidate compound; and iii) measuring binding of the NFAT-polypeptide complex, wherein a change in binding identifies a candidate compound as a compound that is useful for modulating cancer progression, tumor invasion or tumor migration.
In a desirable embodiment to the seventh aspect, the detestably labeled NFAT is an NEAT-1 or NEAT-5 polypeptide.
In another desirable embodiment to the seventh aspect, the candidate polypeptide is selected from a group consisting of, bZIP, AP-1, GATA; NF-KB, and NFAT-5 protein.
In yet another desirable embodiment to the seventh aspect, the candidate polypeptide is NEAT-5, and the NFAT is NEAT-5, where the NFAT-polypeptide complex is an NFAT-5 homodimer.
In still yet another desirable embodiment to the seventh aspect, the change in binding of the NFAT-polypeptide complex is a reduction in the binding of the NFAT-polypeptide complex.
In an eighth aspect, the invention features a method for identifying target genes involved in cancer which requires NEAT biological activity, the method comprising the steps of i) providing a first cell that has low NFAT biological activity or has been modified to have no NEAT biological activity; ii) providing a second cell that has high NFAT biological activity or has been modified to have high NFAT biological activity; and iii) measuring the differentially expressed genes, where differentially expressed genes are target genes of NFAT
transcriptional regulation.
In a desirable embodiment to the eighth aspect, the NEAT biological activity is NFAT-1 or NFAT-5 biological activity.
In another desirable embodiment to the eighth aspect, the transcribed genes are identified by a microarray of nucleic acids, ChIP, SAGE; or RNase protection assays.

In a ninth aspect, the invention features a method for treating the progression of cancer, this method comprising introducing a transgene encoding an NEAT polypeptide, where the transgene encodes a dominant negative NFAT
polypeptide, to a cell, the transgene being operably linked to expression control sequences, and the transgene being positioned for expression in said cell.
In a desirable embodiment to the ninth aspect, the NFAT transgene is an NFAT-1 or NEAT-S transgene.
In another desirable embodiment to the ninth aspect, the dominant negative NFAT transgene is provided by administration of a viral vector containing the transgene, and the transgene is positioned for expression in said cell ofa subject.
In yet another desirable embodiment to the ninth aspect, the dominant negative NFAT is selected from a group consisting of natural or artificial mutations affecting NEAT nuclear localization; dimerization, transactivation, and DNA-binding.
In yet still another desirable embodiment to the ninth aspect, expression of the transgene encoding an NFAT polypeptide results in a decrease in NEAT
biological activity.
In a tenth aspect, the invention features a method for treating the progression of cancer, this method comprising introducing an NEAT antisense nucleic acid that inhibits NFAT biological activity, regardless of length of the antisense nucleic acid.
In a desirable embodiment to the tenth aspect, the NFAT antisense nucleic acid is an NFAT-1 or NFAT-5 antisense nucleic acid.
In an eleventh aspect, the invention features a method for treating the progression of cancer, this method comprising introducing an NFAT consensus DNA-binding nucleic acid sequence, the nucleic acid sequence is double stranded, and the sequence inhibits NFAT biological activity, regardless of length of the nucleic acid sequence.

In a desirable embodiment to the eleventh aspect, the consensus NFAT
DNA binding sequence is selected from a group consisting of SEQ ID NO. l, SEQ
ID NO. 2, and SEQ ID NO. 3.
In a twelfth aspect, the invention features a method for treating the progression of cancer, comprising introducing an NFAT double-stranded interference ribonucleic acid or RNAi, that inhibits NFAT biological activity, regardless of length of the RNAi nucleic acid As used herein, by an "NFAT polypeptide" or an "NFAT nucleic acid" is meant an amino acid sequence or a nucleic acid sequence that has NFAT
biological activity and is substantially identical to any one of NFAT-1, NFAT-4, or NFAT-5 polypeptides. For example, an NFAT-1 polypeptide and nucleotide sequence may be substantially identical to GenBank Accession Number NM 012340, U43342, or U43341; anNFAT-4 polypeptide and nucleotide sequence may be substantially identical to GenBank Accession Number MVI_004555; and an NFAT-5 polypeptide and nucleotide sequence may be substantially identical to GenBank Accession Number AF163836; AJ243299, AJ243298, NM 006599, or to AF134870.
Known biological activities of NFAT include but are not limited to nuclear translocation by a nuclear localization signal, transactivation of target genes; the ability to interact with specific transcription factor binding partners such as bZIP
class proteins, Rel family members; and GATA; DNA binding activity, with specificities to nucleic acid sequences containing TGGAAANNYNY (where N is any nucleotide and Y represents any pyrimidine) bZIP class of transcription factors include but are not limited to Fos-Jun family members, Maf, Mef, CREB, and ATF. Rel family members include Rel and NF-KB.
NFAT polypeptides may homodimerize, in the case of NFAT-5, or heterodimerize, in the case of NFAT-1 through -4 with various transcription factors as previously mentioned. Dimerization of NFAT polypeptides and subsequent DNA binding results in an increase in target gene transcription, a group which includes, but are not limited to, cytokines such as GM-CSF, IFN-y, interleukins -2-4, -5, and -13, as well as lymphocyte markers, CD40L and CTLA-4. NEAT polypeptides also are able to recognize and transactivate NF- K B-like consensus sequences as seen in the promoters of the TNF- a, IL-8, E-selectin, GM-CSF and IL-2. Although NFAT polypeptides are able to bind DNA on their own, transactivation is increased when consensus NEAT DNA binding sequences are adjacent to:DNA binding sequences of the above-mentioned transcription factors.
The prototypical NEAT polypeptide comprises (from the amino-terminus):
an amino terminal acidic/hydrophobic transactivation domain; a calcineurin binding domain; also referred as the NEAT homology region; a nuclear localization sequence; a DNA binding domain; and a carboxy-terminal transactivation domain. Interspersed from the calcineurin-binding domain and DNA binding domain are serine-rich and serine/proline-rich regions, which are capable of being phosphorylated.
NFAT-1 to -4 mRNA and polypeptides are generally expressed in hematopoietic tissues and cells, however, expression can also be found in brain, muscle kidney, placental, and endothelial cells. NFAT-S is ubiquitously distributed. Elevated expression of NFAT has been found in cancer cells and is correlated with umor cell invasion and migration. Specifically, NEAT-1 overexpression is associated with tumor cell invasion and migration, whereas NFAT-5 overexpression is associated with tumor cell migration: It is implied that elevated expression of NFAT in cells result in increased biological activities of NEAT.
By "antisense," as used herein in reference to nucleic acids, is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of an NFAT gene. Preferably the antisense nucleic acid is capable of decreasing the expression of NFAT in a cell. Preferably the decrease is relative to a control, 90%, more preferably 75%, and most preferably 50% or more. Preferably an NFAT antisense nucleic acid includes from about 8 to 30 nucleotides. An NFAT antisense nucleic acid may also contain at least 40, 60;
85, 120, or more consecutive nucleotides that are complementary to a NFAT mRNA
or DNA, and may be as long as a full-length NEAT gene or mRNA. The antisense nucleic acid may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
By "assaying" is meant analyzing the effect of a treatment, be it chemical or physical, administered to whole animals or cells derived there from. The material being analyzed may be an animal, a cell, a lysate or extract derived from a cell, or a molecule derived from a cell: The analysis may be, for example, for the purpose of detecting altered gene expression, altered RNA stability;
altered protein stability; altered protein levels, or altered protein biological activity. The means for analyzing may include; for example, enzymatic assays, immunoprecipitation, phosphorylation assays, and methods known to those skilled in the art for detecting nucleic acids and polypeptides.
By "cancer" or "neoplasia" is meant a cell or tissue multiplying or growing in an abnormal manner. Cancer growth is uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
By "candidate compound" is meant a chemical, be it naturally-occurring or artificially-derived, that is assayed for its ability to modulate an alteration in reporter gene activity or protein levels, by employing one of the assay methods described herein. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By "detestably-labeled" is meant any means for marking and identifying the presence of a molecule, e.g., a polypeptide or fragment thereof, or a nucleic acid molecule. Methods for detestably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P or 35S) and non-radioactive labeling (e.g.; chemiluminescent labeling or fluorescein labeling) By "differentially expressed" refers to a difference in the expression level of a nucleic acid. This difference may be either an increase or a decrease in expression, when compared to control conditions.
By "dimer" is meant a protein-protein interaction composed an NEAT
monomer with a transcriptional binding partner. An NEAT monomer such as NFAT-5 is capable of binding to another NEAT-5, referred herein as a homodimer. Alternatively, NFAT-1 and NFAT-4 can interact with AF-l, NF-KB, GATA, and MAF to form heterodimers. Further guidance for assaying protein interactions or function may be found in, for example, Ausubel et al. (Current Protocols in Molecular Biology; John Wiley & Sons, New York, 2000) By "dominant negative" is meant a mutant cDNA allele of an NFAT, which when expressed as a protein in any given cell type, acts to selectively block or inhibit the activity or function of the endogenous NFAT proteins with subsequent inhibition of a given biological activity.
By "expression control sequences" or a "promoter" is meant a nucleic acid sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the native gene: Desirable promoters of the invention direct transcription of a protein in an epithelial cell and preferably to a tumor marker; such promoters include, without limitation, promoters from the following genes: keratin-14, keratin-19, Her2/lVeulerbB2, kallikrein-10; Gro-alpha, and MDM 2. Yet another desirable promoter of the invention directs transcription of a protein in an embryonal cell.

By a "greater than naturally-occurring amount of NEAT protein or NFAT
activity" is meant an amount of NFAT protein, or NFAT activity, that is at least 20%, 50%, 75%, 100%, 200%; 500%, or 1 U00% greater than the amount of NEAT
protein or NFAT activity that is naturally present within a particular cell or sample. A greater than naturally-occurring amount of NFAT protein may be generated by expressing a heterologous NFAT protein in a cell or sample.
Alternatively, a NEAT protein may be expressed in a cell or sample using a heterologous promoter, by using multiple copies of the endogenous gene, or by exploiting a mutation in the NFAT gene or elsewhere in a cell chromosome that results in increased NEAT expression or activity.
By a "heterologous nucleic acid" is meant a nucleic acid, for example, a DNA or RNA molecule, that is not normally present in a cell or sample obtained from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.
By a "heterologous promoter" is meant a promoter that regulates the expression of a nucleic acid with which it is not normally associated. A
"heterologous promoter" may be, for example, a viral promoter (e.g., a cytomegalovirus (CMV) promoter, a herpes simplex virus thymidine kinase promoter, or an adenovirus E l B promoter), a bacterial promoter, or a mammalian promoter, such as the ø-globin promoter.
By "integrin" is meant a receptor polypeptide composed of two subunits, a and [3. Several a and (3 subunits have been identified. An a subunit may combine with several (3 subunits. Preferably the a subunit is an a6 subunit with a polypeptide and nucleic acid sequence substantially identical to GenBank Accession No. XM 002335 and NM 000210. Preferably, the (3 subunit is the X34 subunit with a polypeptide and nucleic acid sequence substantially identical to GenBank Accession No. XM-036063 and NM 000.213. A X34 subunit can only bind to an a,6 subunit.

By "metastatic potential" is meant the ability of a cancer cell to progress to metastasis.
By "metastasis" is meant the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream. Metastasis involves cancer cell invasion and migration. Markers that may be associated with metastatic tumor growth include, but are not limited to, carcinoembryonic antigen (CEA), CA 19-9, p53 mutations, CA 15-3 (a mucin-like membrane glycoprotein); estrogen and progesterone receptors, c-erbB-2; and cathepsin-D.
A "microarray," as referred to herein, is a collection of nucleic acids from one or more cell types. For example, these nucleic acids may be arranged in a grid where the location of each nucleic acid remains fixed to aid in identification of the individual nucleic acids. A microarray may include; for example, DNA
representing all, or a subset, of the open reading frames of an organism.
Preferably, the DNA is derived from a region of the genome that shares limited homology to other regions of organism's genome.
By "modulating" is meant conferring a change, either by decrease or increase, in NFAT protein, mRNA or NEAT biological activity that is naturally:
present within a particular cell or sample. Preferably, the change in response is at least 5%, more preferably, the change in response is 20% and most preferably, the change in response level is a change of more than 50% relative to the levels observed in naturally occurring NFAT biological activity.
By "mutation" is meant an alteration in a naturally-occurnng or reference nucleic acid sequence, such ~as an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation. Preferably, the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from a naturally-occurring sequence. Examples of recombinant DNA
techniques for altering the genomic sequence of a cell, embryo, fetus, or mammal include inserting a DNA sequence from another organism (e.g., a human) into the genome, deleting one or more DNA sequences; and introducing one or more base mutations (e.g, site-directed or random mutations) into a target DNA sequence.
By "operably linked" is meant that a nucleic acid molecule and one or more regulatory sequences (e.g., a promoter) are connected in such a way as to permit expression andlor secretion ofthe product (i.e., a polypeptide) of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in "Remington: The Science and Practice of Pharmacy"
(20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins) By "positioned for expression" is meant that the DNA molecule is positioned adjacent to a DNA sequence, which directs transcription and translation of the sequence (i:e., facilitates the production of, e.g., CAR receptor polypeptide) By "progression" or "cancer progression" is meant as the spreading or growing of cancer with or without treatment. The progression of cancer is marked by a series of events, which culminates in alterations with the molecular control of cell growth and survival. As the growth of cancer cells continue, genetic alterations may persist until the cancerous cell has manifested itself to pursue a more aggressive growth phenotype. If left untreated, metastasis, the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream, ensues.
Upon clinical diagnosis of a cancer, treatment of this disease is in part determined by the stage of the cancer. Depending on the severity of the.
cancer, a stage number is assigned, I, II, III, and IV. Stage I cancers are the least advanced and have a better prognosis. Higher stage cancers are more advanced with stage III and IV cancers associated with invasion to the lymph nodes and finally spread to other organs.
By "protein" or "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation) By "reporter gene" is meant a gene whose expression may be assayed; such genes include, without limitation, those encoding glucuronidase (GUS), luciferase, chloramphenicol acetyl transferase (CAT); green fluorescent protein (GFP), alkaline phosphatase, and (3-galactosidase.
By "RNA;" or "RNA interference,"as used herein in reference to nucleic acids, is meant a ribonucleic acid sequence, regardless of length, that is double-stranded to the coding strand of the mRNA of an NFAT gene. Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates. Preferably; the RNA; is capable of decreasing the expression of NFAT in a cell. Preferably the decrease is relative to a control, 90%, more preferably 75%, and most preferably 50% or more. Preferably an NFAT RNAi includes from about 20 to 30 nucleotides. An NFAT RNA; may also contain at least 40, 60; 85, 120, or more consecutive nucleotides that are complementary to a NFAT mRNA or DNA, and may be as long as a full-length NFAT gene or mRNA. The RNA; nucleic acid may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
lVlethods of use of RNAi are are known to those skilled in the art and can found, for example, in, Zamore et al., 2000, Cell 101:25-33 or Tuschl et al., 1999, Genes Dev 13:3191-By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 75%, but preferably 85%; more preferably 90%, most preferably 95%, or even 99% identity to a reference amino acid or nucleic acid sequence.
For polypeptides, the length of comparison sequences will generally be at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 50 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and ether modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine;
valine, isoleucine~ leucine; aspartic acid, glutamic acid, asparagine, glutamine;
serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By "substantially pure polypeptide" is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure: A
substantially pure polypeptide maybe obtained; for example, by extraction from a natural source (e.g., a fibroblast) by expression of a recombinant nucleic acid encoding the polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants; which accompany it in its natural state.
'Thus, a protein, which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates, will be substantially frEe from its naturally associated components. Accordingly; substantially pure polypeptides not only include those derived from eukaryotic organisms but also those synthesised in E. coli or other prokaryotes.
By a "therapeutic amount" is meant an amount sufficient to result in the inhibition of cancer progression. It will be appreciated that there will be many ways known in the art to determine the therapeutic amount for a given application.
For example, the pharmacological methods for dosage determination may be used in the therapeutic context.
By "transcriptional activation" is meant the activation of a transcription factor, for example, NEAT polypeptides; as a consequence of increased NEAT
polypeptide expression; activation of upstream regulators of NFAT, leading to nuclear translocation, DNA binding, and subsequent promotion of events leading to target gene expression.
By "transgene" is meant any piece of nucleic acid that is inserted by artifice into a cell; or an ancestor thereof, and becomes part of the genome of the animal, which develops from that cell: Such a transgene rnay include a gene, which is partly or entireiy heterologous (i.e., foreign) to the transgenic animal, or may represent a gene homologous to an endogenous gene of the animal.
Other features and advantages of the invention will be apparent from the following description of the desirable embodiments thereof, and from the claims.

Brief Description of Drawings Figure 1. NEAT-1/4 are expressed and active in carcinoma cells. Figure 1 a. Nuclear extracts from MDA-MB-43 5 cells stably transfected with either vector alone (6D2, 6D7), ~i4.oCYT (3C12) and wild-type ~i4 (3A7, 5B3), or parental cells were incubated with a 32P-labeled NFAT probe (top panel) or a 32P-labeled AP-1 probe (bottompanel), in duplicate, and separated on 6% polyacrylamide gel.
Shown are the positions of the specific NEAT- and AP-1 probe complexes and a non-specific band (NS, right) Figure 1b specific anti-NFAT-1 antibody, (clone 67.1 ), or control non-immune serum (N.I. serum) were pre-incubated with nuclear extracts of the SB3 clone prior to the EMSA with NEAT probe. The position of the supershifted NFAT/antibody/probe complex is shown. Figure lc, demonstrates transcriptional activity of NFAT was measured by transiently transfecting a luciferase-NFAT reporter construct (NFAT-luc) into the MDA-MB-435 clones and parental cells. luciferase activity is shown as relative luciferase units.
Figure 1d shows total cell lysates from MDA-MB-435 (parental; vector alone (6D2), (34.dCYT and wild-type ~i4 (3A7)) were immunoblotted with the NEAT-1-specific antibody (clone 67.1) (top left panel), or as a control, with an antibody against the p85 subunit of PI 3-K (a-p85, bottom left panel) Nuclear extracts from he same cells were also prepared and immurioblotted with the same NFAT-1-specific antibody (right panel) Figure 1 a demonstrates total lysates from 3A7 and 5B3 clones were resolved by SDS PAGE and immunoblotted with anti-NEAT-1 (a-NEAT-l, clone 67.1) Figure if shows total lysates from MDA-MB-231 and clone A cells were resolved by SDS PAGE and immunoblotted with anti-NEAT-1 (a-NFAT-l, clone 67.1, left panel), anti-NFAT-4 (a-NFAT-4, right panel) or anti-p85 (a-p85, bottom panels) The positions of multiple phosphorylated NEAT
species (arrowheads, right) and molecular mass markers (kDa, left) is shown.
The data are representative of 3 independent experiments, and expressed as a mean ~
SEM, performed in triplicate (figure 1 c) Figure 2. NEAT activity is both necessary and sufficient to drive carcinoma invasion. Figure 2a MDA-MB-435 [34+-expressing cells (3A7) and Figure 2b represents MDA-MB-23lcells which were transiently transfected with DN-NFAT and the VIVIT peptide (GFP-VIVIT), as well as the constitutively active NFAT mutant (NFAT-mSPx3) and constitutively active calcineurin alleles OCnA and CnB, either alone or co-transfected with VIVIT. Following the transfection protocol, cells were assayed for their ability to invade Matrigel.
Relative invasion was determined by comparing the amount of invasion obtained from NFAT transfectants to that obtained from cells transfected with vector alone;
which was assigned a value of 100%. The data shown are mean values (t SEM) of 2 experiments performed in triplicate.
Figure 3. Role of NFAT-5 in mediating carcinoma invasion. Figure 3a shows MDA-MB-435 cells expressing vector alone, ~i~.oCYT (3C12) or wild-type [34+ (3A7 clone) were transiently transfected with an NFAT-5-luciferase reporter construct (NFAT-5-luc) Figure 3b shows lysates of Clone A, MDA-MB-435 (3A7, ~34.OCYT , 6D2 and parental) and MDA-MB-231 cells were resolved by SDS PAGE and immunoblotted with anti-NFAT-5 (a-NEAT-5, top panel) or anti-p85 (a-p85, bottom panel) The position of the 175 kDa and 90 kDa markers are shown (left) (Figure 3c) Dominant negative NFAT-5 constructs (NDS, DBDS), or control inactive D$DS (DIM1) were transiently transfected into MDA-MB-435 (34+-expressing cells (3A7 clone), MDA-MB-231 cells and clone A cells, as indicated. The ability of the transfected cells to invade Matrigel was then assayed and the data are shown as the relative invasion of NFAT-5-transfected cells compared to vector alone-transfected cells (100%) Figure 4. NFAT-5 transcriptional activity is induced in an a6(34-dependent manner. a, Parental, ~i4 (3A7) and ~i4.oCYT (3C12) MDA-MB-435 transfectants were transiently transfected with NEAT-5-luc, grown in complete media for 24 h, trypsinized, incubated with anti-a6 (clone 135-13C) for 30 min; then plated onto secondary antibody-coated plates, either in the presence (+) or absence (-) of NIH
3T3 conditioned medium (CM), for24 h. As a positive control, cells were also stimulated with PMA and ionomycin ( 1 OOnM each) for 24 h. Following stimulation, cells were lysecl and NEAT-5 transcriptional activity assayed. The data are presented as fold induction in NEAT-5 activity compared to control, unstimulated cells (-) Figure 4b show Clone A cells were transiently transfected with the NEAT-5-luc reporter and incubated as in 4A in the presence {+) or absence (-) of CMor with control PMA/ionomycin alone. NFAT-5 activity was measured as described above.
In Figure 4c, MDA-MB-435 cells expressing [34+ (3A7 clone) were transiently transfected with GFP-NDS and of NFAT-S-luc, and treated as described in SA. In Figure 4d, MDA-MB-435 [34+ transfectants (3A7) were transiently transfected with NEAT-5-luc, grown in complete media for 24 h, trypsinized and plated onto laminin-1-coated plates (lanes 3 and 4) or uncoated plates (lanes 1, 2 and 5) in the presence (+) or absence (-) of CM for 24 h: NFAT-5 activity was measured as described above. In all cases, the data are representative of 2 independent experiments performed.
Figure 5: Overexpression of wildtype NFAT-1 and NFAT-5 in MDA-MB-435 cells. Parental MDA-MB-435 (34-negative cells were transiently transfected with wild-type NFAT-1 (Figure Sa) tagged with the HA epitope (NEAT-1) or wild-type NFAT-5 (Figure Sb) tagged with the myc epitope (NFAT-5) or control vector alone {vector) Following the transfection protocol, cells were assayed for their ability to migrate or to invade Matrigel. Relative migration/invasion were determined by comparing the amount of migration/invasion obtained from NEAT
transfectants to that obtained from cells transfected with vector alone, which was assigned a value of 100%. An aliquot of the cells was lysed and nuclear extracts were resolved'by SDS PAGE and immunoblotted witch anti-HA or anti-myc (bottom panels) The data shown are mean values (~ SEM) of 3 experiments performed in triplicate.
Figure 6. NFAT-l, NFAT-5 and ~i4 integrin expression in human breast carcinoma. All sections were obtained from four different individuals (patients 1-4) with grade III invasive ductal breast carcinoma. Sections from two different individuals (patient 1, Figure 6a-c, patient 2, Figure 6d-f) were stained with anti-cytokeratin (Figure 6a, d) and anti-NEAT-1 (Figure 6b, e) The merged image is shown in panels (Figure 6c and f) Arrows indicate cells, which express NFAT-1 but not cytokeratin (Figure 6e, f) A separate carcinoma section and surrounding cells from patient 2 was stained with anti-NFAT-1 (Figure 6g) and anti-CD3 (Figure 6h) The overlay (Figure 6i) reveals infiltrating lymphocytes which express both NEAT-1 and CD3 (arrows) A section from patient 3 was stained with anti-cytokeratin (Figure 6j) and NFAT-5 (Figure 6k) and the overlay is shown in Figure 61. X34 integrin expression in invasive ductal carcinoma is shown in panels (Figure 6m-r) A section from patient 2 was stained with anti-(34 (Figure 6m) and anti-NFAT-1 (Figure 6n) A section from patient 4 was stained with anti-[34 (Figure 6p) and anti-NEAT-5 (Figure 6q) The merged images are shown in panels 6o and 6r respectively. Magnification is 20X for panels, (Figure 6a-lj, 60x for panels 6m-r.
Figure 7. Expression of NEAT does not alter, surface integrin expression.
24 hr after transfection of the indicated plasmids into Clone A, MDA-MB-231 and MDA-1VIB-435 cells; as indicated; cells were trypsinized, washed in PBS and labeled for 20 min with the anti-a6, anti-~i 1 and anti-(34 antibodies directly coupled to phycoerythrin. After labeling cells were washed twice in PBS and analyzed for a6, ~i l and ~i4 expression on a FACScan flow cytometer (Becton Dickinson) The data are plotted as relative cell number against log fluorescence intensity, for each of the indicated antibodies. Thus, expression of the various indicated NFAT mutants does not alter the expression of the surface a6~i4 and a6a1 integrins, such that the alteration in carcinoma cell migration is specifically linked to NFAT action, and not to alteration in integrin expression or function.
Figure 8. Expression of NFAT dominant negative alleles does not induce cell death: 24 h following transfection of the indicated plasmids into MDA-MB-435 ~i4+(3A7) cells, cells were trypsinizedwashed in PBS and labeled for 30 min with an anti-annexin-V antibody coupled to phycoerythrin, according to the manufacturer's instructions. After labeling; cells where washed twice in PBS
and annexin-V positive cells analyzed on a FACScan flow cytometer (Becton Dickinson) gated for transfected cells (GFP-positive) The data are presented as the percentage of annexin-V positive cells in the transfected population. The data demonstrate that expression of the indicated NFAT mutant proteins does not lead to increased cell death, and therefore increased apoptosis cannot account for the altered carcinoma migration and invasion observed when NFAT function is increased or decreased.
Figure 9. Integrin engagement does not increase conventional NFAT
transcriptional activity. MDA-MB-435 X34 transfectants (3A7) were transiently transfected with the NFAT-luc reporter, and after 24 hr cells were trypsinized, incubated with 'anti-a6 antibody for 20min, and plated onto secondary antibody-coated plates for 24 hr, either in the presence (+) or absence (-) of conditioned media. Control cells were also stimulated with PMA/ionomycin (100nM each) Cells were then lysed and assayed for NFAT-luciferase activity, plotted as relative luciferase units. Therefore, activation or engagement of the a6~i4 integrin does not specifically lead to an increase in the intrinsic transcriptional activity of in carcinoma cells, rather it is the increase in NEAT-1 expression (Fig. 1) which mediates the increased invasive phenotype in a6a4-expressing cells. In the same cells however, the control stimuli PMA+ ionomycin do increase NFAT-1 transcriptional activity indicating that a distinct signaling pathway is responsible _ _ __ __ _ - _ ._ .._ for a6~i4-dependent NFAT-1 activation.
Figure 10. FK506 blocks inducible NFAT activity. MDA-MB-435 X34 transfectants were transiently transfected with the NFAT-luc reporter construct, and treated with either FK506 (lOnM) or vehicle alone (methanol) for 1 hr, prior to stimulation with PMA+ionomycin (100nM each) for 24 hr. Cells were then lysed and NFAT-luciferase activity determined, plotted as relative luciferase units.
Therefore, in carcinoma cells, the Ca2+/calcineurin pathway leading to NFAT
activation does not appear to be necessary for induction of NFAT-1 activation, although this pathway is present because the control stimuli PMA/ionomycin do stimulate NFAT-1 activity and this can be blocked with FK506. This is reminiscent of previous studies which have also indicated that in T-cells, there exist two distinct mechanisms which are required for NFAT-1 activation, one which is FK506-sensitive (and thus Ca2+/calcineurin-dependent), and one which is not.
Detailed Description The results presented here provide evidence for a functional role of NEAT
transcription factors in promoting invasion of carcinoma cells and highlight a role for the a6~i4 integrin in the activation of conventional NFATs and NFAT-5.
This is the first demonstration that this family of transcription factors are expressed in human breast carcinomas and also that they are functionally relevant for mediating invasion, one of the hallmarks of tumor metastasis. A number of important concepts have emerged from these studies, the first being that NFAT-1 and NFAT-5 are expressed in tissues derived from invasive human breast carcinomas.
Secondly, increased expression and/or activation of conventional NEAT isoforms and NFAT-5 in breast and colon cancer cells is linked to the presence of a functional x6(34 integrin. In the case of NEAT-1; the functional consequence is increased constitutive transcriptional activity, which is mediated, at least in part, by an increase in protein expression. The net result is an increase in carcinoma invasion which can be blocked with dominant negative NEAT alleles, but surprisingly, not cyclosporin A or FK506. The most likely explanation for this observation is that in these cells, conventional NFATs are constitutively active and this basal activity is not sensitive to these antagonists, which target the ability of immunophilins to bind to and regulate calcineurin.
Moreover, our results also show that unlike NFAT-1, NEAT-5 transcriptional activity is increased by engagement of the a6[34 integrin.
Until now, only hyperosmotic stress, T cell activation through the T-cell receptor and PIViA/ionomycin treatment have been shown to activate NFAT-5. In kidney cells, the activation of NFAT-5 by hyperosmotic stress is necessary to enable the cell to re-equilibrate its osmolality: In carcinoma 'cells, full NEAT-5 activation is achieved by co-stimulation of the x6(34 integrin in the presence of chemoattractants present in conditioned medium derived from NIH 3T3 cells, and it is well established that it is also required for carcinoma invasion in the Matrigel assay. The identity of the ligands present in this medium which mediate this co-stimulation have yet to be identified, although potential candidates include chemokines such as CXCL12 and CCL21, which along with their receptors CXCR4 and CCR7 were recently shown to be highly expressed in breast cancer cells, malignant breast tumors and metastases.
These studies show for the first time the functional expression of NFAT in invasive human breast cancer and cells derived from breast carcinomas; and provide evidence for a previously uncharacterized signaling pathway leading to NFAT activation and carcinoma invasion.

Examine 1: The role of NEAT transcription factors in the regulation of integrin-mediated carcinoma invasion Signaling through the ~i4 integrin has been linked with epithelial cell motility. We sought to determine the relevance of NFAT expression in carcinoma cells which express a6~34. For this purpose, we used MDA-MB-435 breast carcinoma cells which endogenously express the a6~i 1 integrin, and which have been stably transfected with the (34 subunit leading to expression of functional a6~i4 (~i4+), or a mutated ~i4 subunit lacking the ~i4 cytoplasmic domain ((34.~CYT) which is unable to activate downstream signaling pathways. MDA-MB-231 breast carcinoma and Clone A colon carcinoma cells which endogenously express x6(34 were also used. We discovered MDA-MB-435 cells were found to constitutively express nuclear NEAT as judged by an electrophoretic mobility shift assay (EMSA) using a specific 32P-labeled'nucleotide probe for the NFAT DNA
recognition sequence (Fig. l a) Our results indicate that the NFAT complex is predominantly found in two separate clones of cells expressing a6~i4, whereas in parental, (34.OCYT or vector alone-transfected cells, this complex is present at lower levels. In many instances, NFATs co-operate with other transcription factors; most notably AP-1 (Fos/Jun) to mediate gene expression, although there are examples of AP-1-independent NEAT function. AP-1 was detected in all MDA-MB-435 clones and unlike NFAT, its DNA-binding activity was not increased in cells expressing a6(34 (Fig. 1 a) Therefore, we focused on the relevance of increased nuclear NFAT in these cells.
A supershift experiment was performed by pre-incubating nuclear extracts with an anti-NEAT-1 antibody; or non-immune serum (Fig. 1b) The results show that the NEAT-1 isoform is present in nuclear extracts of ~4+-expressing cells. The NFAT-1 detected in MDA-MB-435 /34-expressing cells was transcriptionally active with a high basal activity, measured using an NEAT luciferase reporter construct (Fig. 1 c) Expression of NEAT-1 in 1VIDA-MB-435 cells was further confirmed by immunoblotting total cell lysates with an NFAT-l specific antibody.
NFAT-1 resolved as multiple bands on 8DS-PAGE (Fig. 1d, left panel), indicative of differentially phosphorylated species and correlating with an increase in nuclear NEAT-1 in MDA-MB-435 (34-expressing cells (Fig 1d, right panel) Two separate clones which express the a6(34 integrin showed distinct NFAT transcriptional activities and interestingly; the clone with the highest NFAT transcriptional activity (5B3) also showed the highest expression of total cellular NFAT-1 (Fig.
1e) and nuclear NEAT (Fig. 1d, right panel) Moreover,. to ensure that the presence of NEAT-1 in X34 -expressing-cells was not restricted to clonally derived cell lines, expression of NEAT-1 was also evaluated in MDA-MB-231 breast cancer cells and Clone A colon cancer cells which endogenously express a6~34. Imrnunoblotting experiments revealed that NEAT-1 is also expressed at high levels in MDA-MB~231 cells, whereas clone A
cells predominantly express NFAT-4 (Fig. lfj As a control for equivalent protein loading, lysates were also probed with an antibody, which recognizes the p85 subunit of PI3-K. Equal amount of p85 were detected in all cell lines tested (Fig.
1 d, a and f) We demonstrate three distinct epithelial carcinoma cell lines express NFATs, and this correlates with expression of the (34 subunit.
To investigate the role of NFAT in carcinoma invasion, we used a molecular genetic approach with dominant negative alleles of NFAT as well as constitutively active mutants. Two: distinct NFAT dominant negative mutants were used, one deleted in the carboxyl terminus in the sequence necessary for DNA-binding and the VIVIT peptide, which block the ability of calcineurin to dephosphorylate and activate NEAT. Both mutants reproducibly inhibited carcinoma invasion when transfected into MDA-MB-435 ~i4+-transfectants, as measured in a standard Matrigel chemoinvasion assay (Fig. 2a) Conversely, transfection of an activated NFAT allele; mutated in three serines required for NFAT nuclear export, caused an increase in the invasive phenotype (Fig. 2a) Similarly, a constitutively active calcineurin allele, comprising a mutant catalytic A subunit and wild type regulatory B subunit, was also able to significantly increase carcinoma invasion, and this was also inhibited by the VIVIT peptide.
Collectively, these data implicate conventional NFATs in carcinoma migration and invasion. Expression of the NFAT mutants did not quantitatively alter the expression of the surface integrins, as judged by FRCS analysis, and did not modify cell viability compared to cells transfected with vector alone (Fig. 7-10, inclusive) Identical results were obtained in the Clone A colon cancer cell line, where the VIVIT peptide blocked invasion, and the activated NFAT and calcineurin mutants increased the basal level of carcinoma invasion (Fig. 2b) Importantly, the parental cell line (MDA-MB-435) which does not express a6~i4 but does express NFAT-1 is also invasive, but to a lesser extent that the ~i4+-.
expressing transfectants. In the parental cells, invasion is also blocked by the two dominant negative NEAT alleles, demonstrating that regardless of the expression of the ~i4 integrin, the invasive phenotype is NFAT-dependant. Therefore, as previously proposed, expression of this integrin mediates amplification of PI
3-K, Met and NFAT pathways leading to invasion. These results show that NFAT is both necessary and sufficient to induce carcinoma invasion and this is the first demonstration, to our knowledge; that NFAT plays a role in mediating this phenotype.
In a6~i4-expressing cells, integrin engagement did not enhance the transcriptional activity of conventional NFAT, suggesting that the high basal NEAT activity observed in these cells is sufficient to promote invasion (see Fig. 7-10, inclusive) In addition, neither eyclosporin A nor FK506 significantly inhibited x6(34-dependent carcinoma invasion in the Matrigel assay, although FK506 quantitatively blocked inducible NFAT activity when cells were stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin. However; these antagonists did not block the high basal NFAT transcriptional activity also observed in these cells (Fig. 7-10, inclusive); correlating with the lack of inhibition in the invasion assay.
The activity of the novel and ubiquitously-expressed NFAT-5 family member is not regulated by calcineurin because it lacks the amino-terminal NFAT
homology region, although it does contain an NFAT-related DNA-binding domain. As with conventional NFATs, the transcriptional activity of NFAT-5 was significantly increased in MDA-MB-435 cells expressing (34+; compared to parental cells or cells expressing ~4:~CYT (Fig. 3a) The presence of NFAT-5 was confirmed by evaluating the expression of NFAT-5 using an NFAT-5-specific antibody in immunoblotting experiments. Expression of NEAT-5 in (34+-expressing cells ((34+ (3A7, 5B3), MDA-MB-231 and clone A) was significantly higher than in non-~i4+-exprressing cells (Fig. 3b) Thus, as with conventional NFAT-l, increased NFAT-5 expression correlated with (34 expression. We therefore evaluated the role of NEAT-5 in mediating carcinoma invasion in a6~i4-expressing cells. Homodimerization of NEAT-5 is required to achieve full transcriptional activity. A dominant negative NFAT-5 mutant (ND5) which blocks dimerization of endogenous NFAT-5 significantly inhibited invasion when transiently transfected into MDA-MB-435 MDA-MB-231 and Clone A cells (Fig.
3c) Moreover, a distinct NFAT-5 dominant negative mutant (DBDS) also inhibited invasion in clone A cells, and as a control, an inactive DBDS allele (DIMI) which does not block dimerization did not lead to inhibition of invasion (Fig. 3c) As with NEAT-1, the dominant negative NFAT-5 alleles were able to inhibit invasion in. the parental MDA-MB-4f 5 cell line.
Unlike ca,lcineurin-sensitive NFAT, the transcriptional activity of NEAT-5 was inducible. The standard Matrigel chemoinvasion assay is carried out in the presence of conditioned media obtained from confluent NIH 3T3 fibroblasts, which contains the necessary chemoattractants, which carcinoma cells migrate towards in a chemotactic fashion. Thus, both integrin ligation and conditioned media are required to mediate the full invasive phenotype. We found that clustering x6(34 with a6 monoclonal antibodies, or addition of conditioned media alone to MDA-MB-435 cells resulted in a small, but significant increase im NFAT-transcriptional activity (Fig. 4a) However, when integrin clustering was performed in the presence of conditioned media, them was an additive; 3-fold increase in NFAT-5 transcriptional activity, which was comparable to levels achieved with control PMA and ionomycin. Importantly, this co-stimulation of NFAT-5 activity required the presence of an intact (34 cytoplasmic tail; as it was not observed in X34-negative or (34.OCYT-expressing cells. Co-stimulation of NEAT-5 transcriptional activity by a6(34 clustering and conditioned media was also observed in Clone A colon cancer cells (Fig: 4b) Finally, co-stimulation of NFAT-5 transcriptional activity by a6~i4 and conditioned media; as well as PMA/ionomycin treatment was inhibited when cells were co-transfected with the dominant negative NFAT-5 mutant (Fig. 4c) This inhibition correlates with the ability of this mutant to block carcinoma invasion in the Matrigel assay (Fig: 3c), and thus NFAT-5 is not only transcriptionally active in these cells, but also participates in integrin-mediated invasion. NFAT-S
activity was also induced by plating cells on the a6(34 ligand, laminin-1, and maximal transcriptional activity was also observed when cells were plated on laminin-1 in the presence of conditioned media (Fig. 4d) The analysis of NFAT in the carcinoma cell lines tested here showed elevated levels of NFAT-1 and NFAT-5 in (34+-expressing cells when compared to cells which do not express this integrin subunit (Fig. 1 d and 3b) Increased cell migration is intimately linked with carcinoma invasion. Therefore we tested if overexpression .of either wildtype NEAT-1 or NFAT-5 in the parental cell line MDA-MB-435 (which does not express the (34 integrin) is sufficient to promote cellular migration and/or invasion. Chemotactic migration assays were carried in vector alone-, NFAT-1- or NFAT-5-transiently transfected parental MDA-MB-435 cells, in a'transwell chamber in the absence of Matrigel and in presence of conditioned media.
The results presented in Fig. 5 demonstrate that both wild-type NFAT-1 and wild-type NEAT-5 are sufficient to increase chemotactic migration of MDA-MB-435 cells. Strikingly, whereas expression of NEAT-1 also induced carcinoma invasion (Fig. 5a), this was not observed with the wild-type NFAT-5 allele (Fig.
5b) Immunoblot analysis of the nuclear extracts of transfected cells revealed expression of the expressed NEAT 1 and NFAT-5 (Fig. 5) These results suggest that NEAT-1 and NEAT-5 may induce a distinct subset of genes important for migration (NFAT-5) and invasion (NFAT-1) Since expression of the wildtype NFAT-1 allele is sufficient to promote invasion, likely targets of NFAT-1 transcriptional activity are the promoters of matrix metalloproteases which are also critical for inducing the invasive phenotype.
The above results clearly point to an increase in migration as a phenotype induced by NFAT in carcinoma cells. It is equally important to note that endogenous NEAT may promote carcinoma invasion in the absence of a functional a6~i4 integrin; because wild-type NEAT alhles are able to promote invasion in parental MDA-MB-435 cells (Fig. 5), and dominant negative NFAT
mutants block invasion in the same cells (data not shown) This is reminiscent of PI 3-K-dependent 'invasion in these cells, which can occur in the absence of a6(34 signaling.
We have: discovered that NFA.Ts are :functionally expressed in cell lines derived from human carcinomas. NFATs are not restricted to cells of the immune system, and recent studies have revealed expression in other tissues including heart, muscle, and brain. However, there is currently no information available on NFAT expression in epithelial tissues or tumors. Therefore, we evaluated the presence of NEAT in tissue samples derived' from five breast cancer patients.
Sections of carcinoma were studied in all five patients who had grade III
invasive ductal carcinoma. Sections of adjacent histologically normal breast tissue were examined in two patients. Four of the five patients studied had lymph node metastases at the time of diagnosis.
To evaluate NFAT expression, sections were labeled with an anti-cytokeratin antibody to identify the carcinoma, and with an anti-NFAT-1 antibody.
The results in Fig. 6 (panels a-c) show two distinct breast carcinomas which scored positive for cytokeratin (panel a), and NEAT-1 expression (panel b) Only one of these carcinomas expressed NFAT-1 as judged by the overlay (panel c) Breast carcinoma from a distinct breast cancer patient (Fig. 6, panels d-f) was also positive for NEAT-1 expression (Fig: 6, panel e) Interestingly, in this section certain cells scored positive for NFAT-1 expression but not for cytokeratin (arrows in Fig. 6, panels a and f) We hypothesized that these cells might represent infiltrating lymphocytes, which are known to express NFAT-1. To confirm this, a tissue section from this patient was labeled with anti-NFAT-1 (Fig. 6, panel g) and an anti-CD3 antibody (Fig. 6, panel h) to identify lymphocytes. Both the breast carcinoma and the surrounding cells scored positive for NEAT-1 (Fig. 6, panel g), and cells present in the center of the section scored positive for both CD3 and NFAT-1 (Fig. 6, panels h and i) confirming their identity as infiltrating lymphocytes. In similar labeling experiments, we evaluated the presence of the recently identified NEAT-5 isoform. Like NFAT-l, tissue sections also derived from grade III invasive ductal breast carcinoma showed a strong reactivity with an anti-NFAT-5 specific antibody (panel k); and this was found to co-localize with the carcinoma as judged by the anti-cytokeratin overlay (Fig. 6, panel 1) The x6(34 integrin has been shown to be highly expressed in human breast carcinomas. Conversely, X34 expression in normal tissue is primarily restricted to the surrounding myoepithelial cells. We therefore evaluated the expression of the ~i4 integrin in these tissue sections. All sections scored positive for (34 expression (Fig. 6, panels m and p), and interestingly we detected co-localization with both NEAT-1 (Fig. 6, panel o) and NFAT-5 (Fig. 6, panel r) Therefore, human breast carcinomas, which express he ~4 integrin also express NFAT-1 and NFAT-5.
Experimental "Procedures Tissue samples and immunofluorescence. Tissue samples of primary tumors and adjacent breast'tissue from untreated patients (n=5) with grade III invasive ductal breast carcinomas were taken. Histopathological diagnosis was confirmed for each specimen: Institutional review board (IRB) approval for specimen collection was obtained. Tissues were collected fresh, thinly sliced; fixed in 4%
paraformaldehyde for 4 h at 4°C, left in 30% sucrose in PBS overnight at 4°C and embedded in OCT (optimal cutting temperature) compound. Immunostaining was performed on frozen sections. The anti-NFAT-1 antibody (clone 67.1, rabbit polyclonal) was used at 1:200, the anti-NFAT-5 (rabbit polyclonal, Affinity BioReagents) at 1:200, the anti-cytokeratin (mouse monoclonal, Sigma) at 1:100 and the anti-CD3 (mouse monoclonal, UCHT 1 clone, Pharmingen) at 1:200 and the anti-~i4 (mouse monoclonal, Ancell) at 1:200: Secondary fluorophore-coupled antibodies were an anti-rabbit coupled to Cy-2 and an anti-mouse to Cy-3, and used at 1:200 and 1:800 according to manufacturer's instructions (Jackson-ImmunoResearch Laboratories) Before incubating the sections with specific antibodies, slides were blocked with anon-specific goat non-immune serum (Life Technologies) Mig~ationllnvasion Assays. Invasion assays were performed as described3, using Transwell chambers coated with Matrigel. Briefly, cells were co-transfected with the indicated NEAT constructs, 1 pg EGFP-Nl and 1 ~g pCS2-(n)(3-Gal using the Lipofectamine procedure (Life Technologies), and after 24 h, resuspended in serum-free medium containing 0.1% BSA and added to each well. After 6 h, cells that had migrated to the lower surface of the filters were fixed in 4%
paraformaldehyde and stained with PBS containing lmg/ml bluo-gal. Cells, which stained positive for ~3-galactosidase expression were counted, and the mean of triplicate assays for each experimental condition was used as % relative invasion. The co-transfection of EGFP-Nl plasmid was used to analyze by FACS
the expression of the a6, ~i 1 and [34 integrins of transfected cells. For migration assays, the same procedure was performed except that Transwell chambers were not coated with Matrigel.
Elect~ophoretic Mobility Shift Assay. Growing cells were washed in PBS and resuspended in hypotonic buffer (lOmM Hepes pH 7.9, lOmM KCI, O.lmM
EDTA, O.lmM EGTA, 1mM DTT, 1mM PMSF) with 0.1% NP-40. After centrifugation, nuclear pellets were washed twice in hypotonic buffer and lysed in high salt buffer (20mM Hepes pH 7.9; 400mM NaCI, 1 mM EDTA, 1 mM EGTA, 1mM DTT, 1mM PMSF) The same quantity of protein from the resulting nuclear extracts for each clone was used in an EMSA using NEAT or AP-1 nucleotide probes. For supershift experiments; nuclear extracts were preincubated with non-immune serum or specific antibodies against NFAT-1 (anti-NFAT-1, clone 67-1) prior to addition of the labeled probe, and separated on a 6% polyacrylamide gel.
Cell T~ansfections. Cells were transiently co-transfected with an NEAT
luciferase reporter construct (NFAT-luc, 10~,g) and lp,g pCS2-(n)(3-gal using the Lipofectamine procedure (Life Technologies) 24 h after the transfection, luciferase and betagalactosidase assays were performed using standard assays on total cell lysates and measured on a lurninometer. For each clone the luciferase activity was normalized to the betagalactosidase activity.
Therapeutic Uses The present invention features methods for treating cancer or the progression of cancer by administering nucleic acid compounds. Compounds of the present invention may be administered by any appropriate route for treatment or prevention of a disease or condition associated with angiogenesis associated diseases. These may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be parenteral, intravenous, infra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration.
Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed.
A.R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e:g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, fox example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. The concentration of the compound in the formulation will vary depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

The compound may be optionally administered as a pharmaceutically acceptable salt; such as a non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, malefic, citric, malic, ascorbic, succinic; benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like:
Administration of compounds in controlled release formulations is useful where the compound of formula I has (i) a narrow therapeutic index (e.g:, the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defined as the ratio of median lethal dose (LDso) to median effective dose (EDSO)); (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half life, so that frequent dosing during a day is required in-order to sustain the plasma level at a therapeutic level.
Many strategies can be pursued to obtain contr~lled release in which the rate of release outweighs the rate of metabolism of the therapeutic compound.
For example, controlled release can be-obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions; oil solutions; suspensions; emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.
Formulations for oral use include tablets containing the active ingredients) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g:, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc) Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent; or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
Gene therapy. Gene therapy is another potential therapeutic approach in which nucleic acid encoding dominant negative or inactivating mutants of NFATs, are introduced into cells. The transgene must be delivered to those cells in a form in which it can be taken up and encode for sufficient protein to provide effective function.
Transducing retroviral, adenoviral, and human immunodeficiency viral (HIS vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression (see, for example, Cayouette and Gravel, Hum. Gene Ther., 8:423-430, 1997; Kido et al.
Curr. Eye Res., 15:833-844, 1996; Bloomer et al., J. Virol., 71:6641-6649, 1997;
Naldini et al., Science 272:263-267, 1996; Miyoshi et al., Proc. Natl. Acad.
Sci.
USA, 94:10319-10323, 1997) For example, NFAT nucleic acid, or portions thereof, can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest (such as epithelial carcinoma cells) Other viral vectors, which can be used, include adenovirus, adeno-associated virus, vaccinia virus, bovine papilloma virus; vesicular stomatitus virus, or a herpes virus such as Epstein-Barr Virus:
Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA into a cell. Although these methods are available, many of these are of lower efficiency.
Identification of compounds that modulate NFAT biological activity. Methods of observing changes to NFAT interactions and subsequent biological activity are exploited in high throughput assays for the purpose of identifying compounds that modulate this protein-protein or protein-nucleic acid interaction. Compounds that inhibit NFAT from binding to transcription factor partners or to NEAT
consensus DNA sequences or from functioning as a transcription~l activator or repressor, may be identified by such assays. Such identified compounds may have utility as therapeutic agents in the treating cancer.
There are many methods known in the art to screen for modulators of protein-protein interactions. One method is to immobilize one component (for example, the an NFAT-binding polypeptide) to a solid support matrix. The second component (for example, NFAT) is labeled (either through the use of radioactive isotopes such as 32P or 355, or through non-radioactive alternatives such as fluorophores) and allowed to form a complex with the immobilized polypeptide.
This complex is ready for screening with candidate compounds. A compound that displaces NFAT from the NEAT-binding polypeptide can be readily measured by release of radioactivity or fluorescence.
Alternatively, compounds can be contacted to immobilized NFAT-binding polypeptide, followed by addition of the labeled NFAT polypeptide. Compounds that modulate NFAT interactions could also be measured by release of either radioactivity or fluorescence.
Target gene identification. It is well established in the art, processes to ascertain the identification of target genes. Such methods include, but are not limited to, SAGE, differential display, DNA or RNA oligonucleotide microarray analysis, yeast 2-hybrid analysis (or variations thereof), chromatin immunoprecipitation (ChIP analysis), Rnase protection assays. Many of the above-mentioned techniques are readily found in the art (see for example, in "Expression Genetics:
Accelerated and high-throughput methods", Eds. McClelland, M. and Pardee, A.
1999, BioTechniques Press; Takahashi et al., 2000, Science's STKE, 31 October;
Carter et al., 1999, Proc. Natl. Acad. Sci. 96:13118-23; Velculescu et al., 1995, Science 270:484-487). Typically, microarray analysis is accomplished by hybridizing mRNAs (or cDNAs) extracted from the cells of interest onto fixed arrays of nucleotides. Control and NEAT over-expressing cells can be analysed, and differences in gene expression noted. Variations to this technique include a subtractive hybridization step. A further variation includes extracting mRNAs following polysome fractionation, whereby highly translated RNA species can be determined.
Test extracts ahd compounds. In general, compounds that affect NFAT or NFAT-dependent signaling are identified from large libraries of both natural products, synthetic (or semi-synthetic) extracts or chemical libraries, according to methods known in the art.
Those skilled in the art will understand that the precise source of test extracts or compounds is not critical to the screening procedures) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to; plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. Numerous methods are also available for generating random or directed synthesis (e:g:, semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from, for example, Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI) Alternatively, libraries of natural compounds in the form of bacterial;
fungal; plant, and animal extracts are commercially available from a number of sources, including; but not limited to; Biotics (Sussex, UK), Xenova (Slough, UK), Harbor:Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A.
(Cambridge, MA). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art (e.g., by combinatorial chemistry methods or standard extraction and fractionation methods).
Furthermore, if desired, any library or compound may be readily modified using standard chemical, physical, or biochemical methods.
Other Embodiments. From the foregoing description, it is apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Beth Isreal Deaconess Medical Center (ii) TITLE OF INVENT20N: NFAT Transcription Factors in Tumor (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: 650 WEST GEORGIA STREET, SUITE 2200 (C) CITY: VANCOUVER
(D) STATE: BRITISH COLUMBIA
(E) COUNTRY: CANADA
(F) ZIP: V6B 4N8 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: ROBINSON, J. CHRISTOPHER
(C) REFERENCE/DOCKET NUMBER: 81331-95 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (604) 682-7780 (B) TELEFAX: (604) 682-0274 (2) INFORMATION FOR SEQ ID NO.: 1 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence (D) OTHER INFORMATION: based on Homo Sapiens (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 2 (D) OTHER INFORMATION: N at position 2 is A or T
(A) NAME/KEY: misc_feature (B) LOCATION: 1, 10, 11, 12, 13 (D) OTHER INFORMATION: N = A,T,C or G

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 1 (2) INFORMATION FOR S~Q ID NO.: 2 {i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 11 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: ARTIFICIAL SEQUENCE
(D) OTHER INFORMATION: based on Homo Sapiens (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 9,11~
(D) OTHER INFORMATION: Y at position 9 and 11 is A or T.
{A) NAME/KEY: misc_feature (B) LOCATION: 7, 8, 10 (D) OTHER INFORMATION: N ~ P.,T,C or G
{XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 2 (2) INFORMATION FOR SEQ ID NO.: 3 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 13 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: ARTIFICIAL SEQUENCE
(D) OTHER INFORMATION: based on Homo Sapiens (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1, 13 (D) OTHER INFORMATION: N = A,T,C or G
(XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 3 (2) INFORMATION FOR SEQ ID NO.: 4 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2986 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant {D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (XI) SEQUENCE DESCRIPTTON: SEQ ID NO.: 4 (2) INFORMATION FOR SEQ ID NO.: 5 (I) SEQUENCE CHARACTERISTICS

(A) LENGTH: 921 (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 5 Met Asn Ala Pro Glu Arg Gln Pro Gln Pro Asp Gly Gly Asp Ala Pro Gly His Glu Pro Gly Gly Ser Pro Gln Asp Glu Leu Asp Phe Ser Ile Leu Phe Asp Tyr Glu Tyr Leu Asn Pro Asn Glu Glu Glu Pro Asn Ala His Lys Val Ala Ser Pro Fro Ser Gly Pro Ala Tyr Pro Asp Asp Val Met Asp Tyr Gly Leu Lys Pro Tyr Ser Pro Leu Ala Ser Leu Ser Gly Glu Pro Pro Gly Arg Phe Gly Glu Pro Asp Arg Val Gly Pro Gln Lys Phe Leu Ser Ala Ala Lys Pro Ala Gly Ala Ser Gly Leu Ser Pro Arg Ile Glu Ile Thr Pro Ser His Glu Leu Ile Gln Ala Val Gly Pro Leu Arg Met Arg Asp Ala Gly Leu Leu Val Glu Gln Pro Pro Leu Ala Gly Val Ala Ala Ser Pro Arg Phe Thr Leu Pro Val Pro Gly Phe Glu Gly Tyr Arg Glu Pro Leu Cys Leu Ser Pro Ala Ser Sex Gly Ser Sar Ala Ser Phe Ile Ser Asp Thr Phe Ser Pro Tyr Thr Ser Pro Cys Val Ser Pro Asn Asn Gly Gly Pro Asp Asp Leu Cys Pro Gln Phe Gln Asn Ile Pro Ala His Tyr Ser Pro Arg Thr Ser Pro Ile Met Ser Pro Arg Thr Ser Leu Ala Glu Asp Ser Cys Leu Gly Arg His Ser Pro Val Pro Arg Pro Ala Ser Arg Ser Sex Ser Pro G1y Ala Lys Arg Arg His Ser Cys Ala Glu Ala Leu Val Ala Leu Pro Pro Gly Ala Ser Pro G1n Arg Ser Arg Ser Pro Ser Pro Gln Pro Ser Ser His Va1 Ala Pro Gln Asp His Gly Ser Pro Ala Gly Tyr Pro Pro Val Ala Gly Ser Ala Val Ile Met Asp Ala Leu Asn Ser Leu Ala Thr Asp Ser Pro Cys Gly Ile Pro Pro Lys Met Trp Lys Thr Ser Pro Asp Pro Ser Pro Val Ser Ala Ala Pro Ser Lys A1a Gly Leu Pro Arg His Ile Tyr Pro Ala Val Glu Phe Leu Gly Pro Cys Glu Gln Gly Glu Arg Arg Asn Ser Ala Pro Glu Ser Ile Leu Leu Val Pro Pro Thr Trp Pro Lys Pro Leu Val Pro Ala Ile Pro Ile Cys Ser Ile Pro Val Thr Ala Ser Leu Pro Pro Leu Glu Trp Pro Leu Ser Ser Gln Ser Gly Ser Tyr Glu Leu Arg Ile Glu Val Gln Pro Lys Pro His His Arg Ala His Tyr Glu Thr Glu Gly Ser Arg Gly A1a Val Lys Ala Pro Thr Gly Gly His Pro Val Val Gln Leu His Gly Tyr Met Glu Asn Lys Pro Leu Gly Leu Gln Ile Phe Ile Gly Thr Ala Asp 450. 455 460 Glu Arg Ile Leu Lys Pro His Ala Phe Tyr,Gln Val His Arg Ile Thr Gly Lys Thr Val Thr Thr Thr Ser Tyr Glu Lys Tle Val Gly Asn Thr Lys Val Leu Glu Ile Pro Leu Glu Pro Lys Asn Asn Met Arg Ala Thr Tle Asp Cys Ala Gly Ile Leu Lys Leu Arg Asn Ala Asp Ile Glu Leu Arg Lys Gly G1u Thr Asp Ile Gly Arg Lys Asn Thr Arg Val Arg Leu Val Phe Arg Val His Ile Pro Glu Ser Ser Gly Arg I1e Val Ser Leu Gln Thr Ala Ser Asn Pro Ile Glu Cys Ser Gln Arg Ser Ala His Glu Leu Pro Met Val Glu Arg Gln Asp Thr Asp Ser Cys Leu Va1 Tyr Gly Gly G1n Gln Met Ile Leu Thr Gly Gln Asn Phe Thr Ser Glu Ser Lys Val Val Phe Thr Glu Lys Thr Thr Asp Gly Gln Gln Ile Trp Glu Met Glu Ala Thr Val Asp Lys Asp Lys Ser Gln Pro Asn Met Leu Phe Val Glu Ile Pro Glu Tyr Arg Asn Lys His Ile Arg Thr Pro Val Lys Val Asn Phe Tyr Val Ile Asn Gly Lys Arg Lys Arg Ser Gln Pro Gln His Phe Thr Tyr His Pro Val Pro Ala Ile Lys Thr Glu Pro Thr Asp Glu Tyr Asp Pro Thr Leu Ile Cys Ser Pro Thr His Gly Gly Leu Gly Ser Gln Pro Tyr Tyr Pro Gln His Pro Met Val Ala Glu Ser Pro Ser Cys Leu Val Ala Thr Met Ala Pro Cys Gln Gln Phe Arg Thr Gly Leu Ser Ser Pro Asp Ala Arg Tyr G1n Gln Gln Asn Pro Ala Ala Val Leu Tyr 740 ?45 750 Gln Arg Ser Lys Ser Leu Ser Pro Ser Leu Leu Gly Tyr Gln Gln Pro Ala Leu Met Ala Ala Pro Leu Ser Leu Ala Asp Ala His Arg Ser Val Leu Val His Ala Gly Ser Gln Gly Gln Ser Ser Ala Leu Leu His Pro Ser Pro Thr Asn Gln Gln Ala Ser Pro Val Ile His Tyr Ser Pro Thr Asn Gln Gln Leu Arg Cys Gly Ser His Gln Glu Phe Gln His Ile Met Tyr Cys Glu Asn Phe Ala Pro Gly Thr Thr Arg Pro Gly Pro Pro Pro Val Ser Gln Gly Gln Arg Leu Ser Pro G1y Ser Tyr Pro Thr Val Ile Gln Gln Gln Asn Ala Thr Ser Gln Arg Ala Ala Lys Asn Gly Pro Pro Val Ser Asp Gln Lys Glu Val Leu Pro Ala Gly val Thr Ile Lys Gln Glu Gln Asn Leu Asp Gln Thr Tyr Leu Asp Asp Glu Leu Ile Asp Thr His Leu Ser Trp Ile Gln Asn Ile Leu (2) INFORMATION FOR SEQ ID NO.: 6 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 3969 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens {XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 6 CTCAATGCT'T CTGTCCATGG TGGGTCAGGC CTTGGCCCTG CAGTTTTTCC ATTTCAGTAC 1200 GCAGTTACAG CTGCAGTGCA GGTGCACTTT TATCTTTGCA ATGGCAAGAG ~GC 2280 CAGCATAGGC TGTT'TGAGCT TTGGGGAAAT GAACTTTGCT TTTTATATTT AACTAGGATA 3780 (2) INFORMATION FOR SEQ ID NO.: 7 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1068 (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (VI) ORIGINAL SOURCE;
(A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 7 Met Thr Thr Ala Asn Cys Gly Ala His Asp Glu Leu Asp Phe Lys Leu Val Phe Gly Glu Asp Gly Ala Pro Ala Pro Pro Pro Pro Gly Ser Arg Pro Ala Asp Leu Glu Pro Asp Asp Cys Ala Ser Ile Tyr Ile Phe Asn Val Asp Pro Pro Pro Ser Thr Leu Thr Thr Pro Leu Cys Leu Pro His His Gly Leu Pro Ser His Sex Ser Val Leu Ser Pro Ser Phe Gln Leu Gln Ser His Lys Asn Tyr Glu Gly Thr Cys Glu Ile Pro Glu Ser Lys Tyr Ser Pro Leu Gly Gly Pro Lys Pro Phe Glu Cys Pro Ser Ile Gln Ile Thr Ser Ile Ser Pro Asn Cys His Gln Glu Leu Asp Ala His Glu Asp Asp Leu Gln Ile Asn Asp Pro Glu Arg Glu Phe Leu Glu Arg Pro Ser Arg Asp His Leu Tyr Leu Pro Leu Glu Pro Ser Tyr Arg Glu Ser Ser Leu Ser Pro Ser Pro A1a Ser Ser Ile Ser Ser Arg Ser Trp Fhe Ser Asp Ala Ser Ser Cys Glu Ser Leu Ser His Ile Tyr.Asp Asp Val Asp Ser Glu Leu Asn Glu Ala Ala Ala Arg Phe Thr Leu Gly Ser Pro Leu Thr Ser Pro Gly Gly Ser Pro Gly Gly Cys Pro Gly Glu Glu Thr Trp His Gln Gln Tyr Gly Leu Gly His Ser Leu Ser Pro Arg Gln Sex Pro Cys His Ser Pro Arg Ser Ser Val Thr Asp Glu Asn Trp Leu Ser Pro Arg Pro Ala Ser Gly Pro Ser Ser Arg Pro Thr Ser Pro Cys Gly Lys Arg Arg His Ser Ser Ala Glu Val Cys Tyr Ala Gly Ser Leu Ser Pro His His Ser Pro Val Pro Ser Pro Gly His Ser Pro Arg G1y Ser Val Thr Glu Asp Thr Trp Leu Asn Ala Ser Val His G1y Gly Ser Gly Leu Gly Pro Ala Val Phe Pro Phe Gln Tyr Cys Val Glu Thr'Asp Ile Pro Leu Lys Thr Arg Lys Thr Ser Glu Asp Gln Ala Ala Ile Leu Pro Gly Lys Leu Glu Leu Cys Ser Asp Asp Gln Gly Ser Leu Ser Pro Ala Arg Glu Thr Ser I1e Asp Asp Gly Leu Gly Ser Gln Tyr Pro Leu Lys Lys Asp Ser Cys Gly Asp Gln Phe Leu Ser Val Pro Ser Pro Phe Thr Trp Ser Lys Pro Lys Pro Gly His Thr Pro Ile Phe Arg Thr Ser Ser Leu Pro Pro Leu Asp Trp Pro Leu Pro Ala His Phe Gly Gln Cys Glu Leu Lys Ile Glu Val Gln Pro Lys Thr His His Arg Ala His Tyr Glu Thr Glu Gly Ser Arg Gly Ala Val Lys Ala Ser Thr Gly Gly His Pro Val Val Lys Leu Leu Gly Tyr Asn Glu Lys Pro Ile Asn Leu Gln Met Phe Ile Gly Thr Ala Asp Asp Arg Tyr Leu Arg Pro His Ala Phe Tyr Gln Val His Arg Ile Thr Gly Lys Thr Val Ala Thr Ala Ser Gln Glu Ile Ile Ile Ala Ser Thr Lys Val Leu Glu Ile Fro Leu Leu Pro Glu Asn Asn Met Ser Ala Ser Ile Asp Cys Ala Gly Ile Leu Lys Leu Arg Asn Ser Asp Ile Glu Leu Arg Lys Gly Glu Thr Asp Ile Gly Arg Lys Asn Thr Arg Val Arg Leu Val Phe Arg Val His Ile Pro Gln Pro Ser Gly Lys Val Leu Ser Leu Gln Ile Ala Ser Ile Pro Val Glu Cys Ser Gln Arg Ser Ala Gln Glu Leu Pro His Ile Glu Lys Tyr Ser Ile Asn Ser Cys Ser Val Asn Gly Gly His Glu Met Val Val Thr Gly Ser Asn Phe Leu Pro Glu Ser Lys Ile Ile Phe Leu Glu Lys Gly Gln Asp Gly Arg Pro Gln Trp Glu Val Glu Gly hys Ile Ile Arg Glu Lys Cys Gln Gly Ala His Ile Val Leu Glu Val Pro Pro Tyr His Asn Pro Ala Val Thr Ala Ala Val Gln Val His Phe Tyr Leu Cys Asn Gly Lys Arg Lys Lys Ser Gln Ser Gln Arg Phe Thr Tyr Thr Pro Val Leu Met Lys Gln Glu His Arg Glu Glu Ile Asp Leu Ser Ser Val Pro Ser Leu Pro Val Pro His Pro Ala Gln Thr Gln Arg Pro Ser Ser Asp Ser Gly Cys Ser His Asp Ser Val Leu Ser Gly Gln Arg Ser Leu Isle Cys Ser Ile Pro Gln Thr Tyr Ala Ser Met Val Thr Ser Ser His Leu Pro Gln Leu Gln Cys Arg Asp Glu Ser Val Ser Lys Glu Gln His Met Ile Pro Ser Pro Ile Val His Gln Pro Phe Gln Val Thr Pro Thr Pro Pro Val Gly Ser Ser Tyr Gln Pro Met Gln Thr Asn Val Val Tyr Asn Gly Pro Thr Cys Leu Pro Ile Asn Ala Ala Ser Ser Gln Glu Phe Asp Ser Va1 Leu Phe Gln Gln Asp Ala Thr Leu Ser Gly Leu Val Asn Leu Gly Cys Gln Pro Leu Ser Ser Ile Pro Phe His Ser Ser Asn Ser Gly Ser Thr Gly His Leu Leu Ala His Thr Pro His Ser Val His Thr Leu Pro His Leu G1n Ser Met Gly Tyr His Cys Ser Asn Thr Gly Gln Arg Ser Leu Ser Ser Pro Val Ala Asp Gln Ile Thr Gly Gln Pro Ser Ser Gln Leu Gln Pro Ile Thr Tyr Gly Pro Ser His Ser Gly Ser Ala Thr Thr Ala Ser Pro Ala Ala Ser His Pro Leu Ala Ser Ser Pro Leu Ser Gly Pro Pro Ser Pro Gln Leu Gln Pro Met Pro Tyr G1n Ser Pro Ser Ser Gly Thr Ala Ser Ser Pro Ser Pro Ala Thr Arg Met His Sex Gly Gln His Ser Thr Gln Ala Gln Ser Thr Gly Gln Gly Gly Leu Ser Ala Pro Ser Ser Leu Ile Cys His Ser Leu Cys Asp Pro Ala Ser Phe Pro Pro Asp Gly Ala Thr Val Ser Ile Lys Pro Glu Pro Glu Asp Arg Glu Pro Asn Phe Ala Thr Ile Gly Leu Gln Asp Ile Thr Leu Asp Asp Asp Gln Phe Ile Ser Asp Leu Glu His G1n Pro Ser Gly Ser Ala Glu Lys Trp Pro Asn His Ser Val Leu Ser Cys Pro Ala Pro Phe Trp Arg Ile (2) INFORMATION FOR SEQ ID NO.: 8 (I) SEQUENCE CHARACTERISTICS

{A) LENGTH: 4455 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
{A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 8 TCAAAAGCGG GAAATGGAAC ATTGGAAAAC CAAA.AAGGAA CTGGAGTAAA GAAGAGCCCT 660 (2) INFORMATION FOR SEQ ID NO.: 9 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1484 (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 9 Met Ser Gln Thr Ser Gly Gly Glu Ala Gly Ser Pro Pro Pro Ala Val Val A1a Ala Asp Ala Ser Ser Ala Pro Ser Ser Ser Ser Met Gly Gly Ala Cys Ser Ser Phe Thr Thr Ser Ser Ser Pro Thr Ile Tyr Ser Thr Ser Val Thr Asp Ser Lys Ala Met Gln Val Glu Ser Cys Ser Ser Ala Val Gly Val Ser Asn Arg Gly Val Ser Glu Lys Gln Leu Thr Ser Asn Thr Val Gln Gln His Pro Ser Thr Pro Lys Arg His Thr Val Leu Tyr Ile Ser Pro Pro Pro Glu Asp Leu Leu Asp Asn Ser Arg Met Ser Cys Gln Asp Glu Gly Cys Gly Leu Glu Ser Glu Gln Ser Cys Ser Met Trp Met Glu Asp Ser Pro Ser Asn Phe Ser Asn Met Ser Thr Ser Ser Tyr Asn Asp Asn Thr Glu Val Pro Arg Lys Ser Arg Lys Arg Asn Pro Lys Gln Arg Pro Gly Va1 Lys Arg Arg Asp Cys Glu Glu Ser Asn Met Asp Ile Phe Asp Ala Asp Ser Ala Lys Ala Pro His Tyr Val Leu Ser Gln Leu Thr Thr Asp Asn Lys Gly Asn Ser Lys Ala Gly Asn Gly Thr Leu Glu Asn Gln Lys Gly Thr Gly Val Lys Lys Ser Pro Met Leu Cys Gly Gln Tyr Pro Val Lys Ser Glu Gly Lys Glu Leu Lys I1e Val Val Gln Pro Glu Thr Gln His Arg Ala Arg Tyr Leu Thr Glu Gly Ser Arg Gly Ser Val Lys Asp Arg Thr Gln Gln Gly Phe Pro Thr Val Lys Leu Glu Gly His Asn Glu Pro Val Val Leu Gln Val Phe Val Gly Asn Asp Ser Gly Arg Val Lys Pro His Gly Phe Tyr Gln Ala Cys Arg Val Thr G1y Arg Asn Thr Thr Pro Cys Lys Glu Val Asp Ile Glu Gly Thr Thr Va1 Ile Glu Val G1y Leu Asp Pro Ser Asn Asn Met Thr Leu Ala Val Asp Cys Val Gly I1e Leu Lys Leu Arg Asn Ala Asp Val Glu Ala Arg Ile Gly Ile Ala G1y Ser Lys Lys Lys Ser Thr Arg Ala Arg Leu Val Phe Arg Val Asn Ile Met Arg Lys Asp Gly Ser Thr Leu Thr Leu Gln Thr Pro Ser Ser Pro Ile Leu Cys Thr Gln Pro Ala Gly Val Pro Glu Ile Leu Lys Lys Ser Leu His Ser Cys Ser Val Lys Gly Glu Glu Glu Val Phe Leu Ile G1y Lys Asn Phe Leu Lys Gly Thr Lys Val Ile Phe Gln Glu Asn Val Ser Asp Glu Asn Ser Trp Lys Ser Glu Ala Glu Ile Asp Met Glu Leu Phe His Gln Asn His Leu Ile Val Lys Val Pro Pro Tyr His Asp Gln His Ile Thr Leu Pro Val Ser Val Gly Ile Tyr Val Val Thr Asn Ala Gly Arg Ser His Asp Val Gln Pro Phe Thr Tyr Thr Pro Asp Pro Ala Ala Ala Gly Ala Leu Asn Val Asn Val Lys Lys Glu Ile Ser Ser Pro Ala Arg Pro Cys Ser Phe Glu Glu Ala Met Lys Ala Met Lys Thr Thr Gly Cys Asn Leu Asp Lys Val Asn Ile Ile Pro Asn Ala Leu Met Thr Pro Leu Ile Pro Ser Ser Met Ile Lys Ser Glu Asp Val Thr Pro Met G1u Va1 Thr Ala Glu Lys Arg Ser Ser Thr Ile Phe Lys Thr Thr Lys Ser Val Gly Ser Thr Gln Gln Thr Leu Glu Asn Ile Ser Asn Ile Ala Gly Asn Gly Ser Phe Ser Ser Pro Ser Ser Ser His Leu Pro Ser Glu Asn Glu Lys Gln Gln Gln Ile Gln Pro Lys Ala Tyr Asn Pro Glu Thr Leu Thr Thr Ile Gln Thr Gln Asp Ile Ser Gln Pro Gly Thr Phe Pro Ala Val Ser A1a Ser Ser Gln Leu Pro Asn Ser Asp Ala Leu Leu Gln Gln Ala Thr Gln Phe Gln Thr Arg Glu Thr Gln Ser Arg Glu Ile Leu Gln Ser Asp Gly Thr Val Val Asn Leu Ser Gln Leu Thr Glu Ala Ser Gln Gln Gln Gln Gln Ser Pro Leu Gln Glu Gln Ala Gln Thr Leu Gln Gln Gln Ile Ser Ser Asn Ile Phe Pro Ser Pro Asn Ser Val Ser Gln Leu Gln Asn Thr Ile Gln Gln Leu Gln Ala Gly Ser Phe Thr Gly Ser Thr Ala Ser Gly Ser Ser Gly Ser Val Asp Leu Val Gln Gln Val Leu Glu Ala Gln Gln Gln Leu Ser Ser Val Leu Phe Ser Ala Pro Asp Gly Asn Glu Asn Val Gln Glu.Gln Leu Ser Ala Asp Ile Phe Gln Gln Val Ser Gln Ile Gln Ser Gly Va1 Ser Pro G1y Met Phe Ser Ser Thr Glu Pro Thr Val His Thr Arg Pro Asp Asn Leu Leu Pro Gly Arg Ala Glu Ser Val His Pro Gln Ser Glu Asn Thr Leu Ser Asn Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val Met Glu Ser Ser Ala Ala Met Val Met Glu Met Gln Gln Ser Ile Cys Gln Ala Ala Ala Gln Ile Gln Ser Glu Leu Phe Pro Ser Thr Ala Ser Ala Asn Gly Asn Leu Gln Gln Ser Pro Val Tyr Gln Gln Thr Ser His Met Met Ser Ala Leu Ser Thr Asn Glu Asp Met Gln Met Gln Cys Glu Leu Phe Ser Ser Pro Pro Ala Val Ser Gly Asn Glu Thr Ser Thr Thr Thr Thr Gln Gln Val Ala Thr Pro Gly Thr Thr Met Phe Gln Thr Ser Ser Ser Gly Asp Gly Glu Glu Thr Gly Thr Gln Ala Lys Gln Ile Gln Asn Ser Val Phe Gln Thr Met Val Gln Met Gln His Ser Gly Asp Asn Gln Pro Gln Val Asn Leu Phe Ser Ser Thr Lys Ser Met Met Ser Val Gln Asn Ser Gly Thr Gln Gln Gln Gly Asn G1y Leu Phe Gln Gln Gly Asn Glu Met Met Ser Leu Gln Ser Gly Asn Phe Leu Gln Gln Ser Ser His Ser Gln Ala Gln Leu Phe His Pro Gln Asn Pro Ile Ala Asp Ala Gln Asn Leu Ser Gln Glu Thr Gln Gly Ser Leu Phe His Ser Pro Asn Pro Ile Val His Ser Gln Thr Ser Thr Thr Ser Ser Glu Gln Met Gln Pro Pro Met Phe His Ser Gln Ser Thr Ile Ala Val Leu Gln Gly Ser Ser Va1 Pro Gln Asp Gln Gln Ser Thr Asn Ile Phe Leu Ser Gln Ser Pro Met Asn Asn Leu Gl:n Thr Asn Thr Val Ala Gln Glu A1a Phe Phe Ala Ala Pro Asn Ser Ile Ser Pro Leu Gln Ser Thr Ser Asn Ser Glu Gln Gln Ala Ala Phe Gln Gln Gln Ala Pro Ile Ser His Ile Gln Thr Pro Met Leu Ser Gln Glu Gln Ala Gln Pro Pro Gln Gln Gly Leu Phe Gln Pro Gln Val Ala Leu Gly Ser Leu Pro Pro Asn Pro Met Pro Gln Ser Gln G1n Gly Thr Met Phe Gln Ser Gln His Ser Ile Val Ala Met Gln Ser Asn Ser Pro Ser Gln Glu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln G1n Gln Gln Gln Gln Gln Gln Ser Ile Leu Phe Ser Asn Gln Asn Thr Met Ala Thr Met Ala Ser Pro Lys Gln Pro Pro Pro Asn Met Ile Phe Asn Pro Asn Gln Asn Pro Met Ala Asn Gln Glu Gln Gln Asn Gln Ser Ile Phe His Gln Gln Ser Asn Met Ala Pro Met Asn Gln Glu Gln Gln-Pro Met Gln Phe Gln Ser Gln Ser Thr Val Ser Ser Leu Gln Asn Pro Gly Pro Thr Gln Ser Glu Ser Ser Gln Thr Pro Leu Phe His Ser Ser Pro Gln Ile Gln Leu Val Gln Gly Ser Pro Ser Ser Gln Glu Gln Gln Val Thr Leu Phe Leu Ser Pro Ala Ser Met Ser Ala Leu Gln Thr Ser Ile Asn Gln Gln Asp Met Gln Gln Ser Pro Leu Tyr Ser Pro Gln Asn Asn Met Pro Gly Ile Gln Gly Ala Thr Ser Ser Pro Gln Pro Gln Ala Thr Leu Phe His Asn Thr Ala Gly Gly Thr Met Asn Gln Leu Gln Asn Ser Pro Gly Ser Ser Gln Gln Thr Ser Gly Met Phe Leu Phe Gly Ile Gln Asn Asn Cys Ser Gln Leu Leu Thr Ser Gly Pro Ala Thr Leu Pro Asp Gln Leu Met Ala Ile Ser Gln Pro Gly Gln Pro Gln Asn Glu Gly Gln Pro Pro Val Thr Thr Leu Leu Ser Gln Gln Met Pro Glu Asn Sex Pro Leu Ala Ser Ser Ile Asn Thr,Asn Gln Asn Ile Glu Lys Ile Asp Leu Leu Val Ser Leu Gln Asn Gln Gly Asn Asn Leu Thr Gly Ser Phe (2) INFORMATION FOR SEQ ID NO.: 10 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 5575 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens Gln Ser Thr Ile Ala Val Leu Gln Gly Ser Ser V

(XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 10 GCTCACCGAT ATG~AAAAAAG GCAGCATGTT AATACGAAGC AGGAATCCCG AGACATCTTT 600 TGAAAAGTAT ATTGATAACC TTGAAAAf~AA ACAGTGGATC ACAAAGTGGA ACGAAAATGA 3540 (2) INFORMATION FOR SEQ ID NO.: 11 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH:. 1073 (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 11 Met Ala Ala Ala Gly Gln Leu Cys Leu Leu Tyr Leu Ser Ala Gly Leu Leu Ser Arg Leu Gly Ala Ala Phe Asn Leu Asp Thr Arg Glu Asp Asn Val Ile Arg Lys Tyr Gly Asp Pro Gly Ser Leu Phe Gly Phe Ser Leu Ala Met His Trp Gln Leu Gln Pro Glu Asp Lys Arg Leu Leu Leu Val Gly Ala Pro Arg Ala Glu Ala Leu Pro Leu Gln Arg Ala Asn Arg Thr Gly Gly Leu Tyr Ser Cys Asp Ile Thr Ala Arg Gly Pro Cys Thr Arg Ile Glu Phe Asp Asn Asp Ala Asp Pro Thr Ser Glu Ser Lys Glu Asp Gln Trp Met Gly Val Thr Val Gln Ser Gln Gly Pro Gly Gly Lys Val Val Thr Cys Ala His Arg Tyr Glu Lys Arg Gln His Val Asn Thr Lys Gln G1u Ser Arg Asp Ile Phe Gly Arg Cys Tyr Val Leu Ser Gln Asn Leu Arg Ile Glu Asp Asp Met Asp G1y Gly Asp Trp Ser Phe Cys Asp Gly Arg Leu Arg Gly His Glu Lys Phe Gly Ser Cys Gln Gln Gly Val Ala Ala Thr Phe Thr Lys Asp Phe His Tyr Ile Val Phe Gly Ala Pro Gly Thr Tyr Asn Trp Lys Gly Ile Val Arg Val Glu Gln Lys Asn Asn Thr Phe Phe Asp Met Asn Ile Phe Glu Asp Gly Pro Tyr Glu Val Gly Gly Glu Thr Glu His Asp Glu Ser Leu Val Pro Val Pro Ala Asn Ser Tyr Leu Gly Phe Ser Leu Asp Ser Gly Lys Gly Ile Val Ser Lys Asp Glu Ile Thr Phe Val Ser Gly Ala Pro Arg Ala Asn His Ser Gly Ala Val Val Leu Leu Lys Arg Asp Met Lys Ser AIa His Leu Leu Pro Glu His Ile Phe Asp Gly Glu Gly Leu Ala Ser Ser Phe Gly Tyr Asp Va1 Ala Val Val.Asp Leu Asn Lys ASp Gly Trp G1n Asp Ile Val Ile Gly Ala Pro Gln Tyr Phe Asp Arg Asp Gly Glu Val Gly Gly Ala Val Tyr Val Tyr Met Asn Gln Gln Gly Arg Trp Asn Asn Val Lys Pro Ile Arg Leu Asn Gly Thr Lys Asp Ser Met Phe Gly Ile Ala Val Lys Asn Ile Gly Asp Ile Asn Gln Asp Gly Tyr Pro Asp Ile Ala Val Gly Ala Pro Tyr Asp Asp Leu Gly Lys Val Phe Ile Tyr His Gly Ser Ala Asn Gly Ile Asn Thr Lys Pro Thr Gln Val Leu Lys Gly Ile Ser Pro Tyr Phe Gly Tyr Ser Ile Ala Gly Asn Met Asp Leu Asp Arg Asn Ser Tyr Pro Asp Val Ala Val Gly Ser Leu Ser Asp Ser Val Thr Ile Phe Arg Ser Arg Pro Val Ile Asn Ile Gln Lys Thr Ile Thr Val Thr Pro Asn Arg Ile'Asp Leu Arg Gln Lys Thr Ala Cys Gly Ala Pro Ser Gly Ile Cys Leu Gln Val Lys Ser Cys Phe Glu Tyr Thr Ala Asn Pro Ala Gly Tyr Asn Pro Ser Ile Ser Ile Val Gly Thr Leu Glu Ala Glu Lys Glu Arg Arg Lys Sex Gly Leu Ser Ser Arg Val Gln Phe Arg Asn Gln Gly Ser Glu Pro Lys Tyr Thr Gln Glu Leu Thr Leu Lys Arg Gln Lys Gln Lys Val Cys Met Glu Glu Thr Leu Trp Leu Gln Asp Asn Tle Arg Asp Lys Leu Arg Pro Ile Pro Ile Thr Ala Ser Val Glu Ile Gln G1u Pro Ser Ser Arg Arg Arg Val Asn Ser Leu Pro Glu Va1 Leu Pro I1e Leu Asn Ser Asp Glu Pro Lys Thr Ala His Ile Asp Val His Phe Leu Lys Glu Gly Cys Gly Asp Asp Asn Val Cys Asn Ser Asn Leu Lys Leu Glu Tyr Lys Phe Cys Thr Arg Glu Gly Asn Gln Asp Lys Phe Ser Tyr Leu Pro Ile Gln Lys Gly Val Pro Glu Leu Val Leu Lys Asp Gln Lys Asp Ile Ala Leu Glu Ile Thr Val Thr Asn Ser Pro Ser Asn Pro Arg Asn Pro Thr Lys Asp Gly Asp Asp Ala His Glu Ala Lys Leu Ile Ala Thr Phe Pro Asp Thr Leu Thr Tyr Ser Ala Tyr Arg Glu Leu Arg Ala Phe Pro Glu Lys Gln Leu Ser Cys Val Ala Asn Gln Asn Gly Ser Gln Ala Asp Cys Glu Leu Gly Asn Pro Phe Lys Arg Asn Ser Asn Val Thr Phe Tyr Leu Val Leu Ser Thr Thr Glu Val Thr Phe Asp Thr Pro Asp Leu Asp Ile Asn Leu Lys Leu Glu Thr Thr Ser Asn Gln Asp Asn Leu A1a Pro Ile Thr Ala Lys Ala Lys Val Val Ile Glu Leu Leu Leu Ser Val Ser Gly Val Ala Lys Pro Ser Gln Val Tyr Phe Gly Gly Thr Val Val Gly Glu Gln Ala Met Lys Ser Glu Asp Glu Val Gly Ser Leu Ile Glu Tyr Glu Phe Arg Val Ile Asn Leu Gly Lys Pro Leu Thr Asn Leu G1y Thr Ala Thr Leu Asn Ile Gln Trp Pro Lys Glu Ile Ser Asn Gly Lys Trp Leu Leu Tyr Leu Val Lys Val Glu Ser Lys Gly Leu Glu Lys Val Thr Cys Glu Pro Gln Lys G1u Ile Asn Ser Leu Asn Leu Thr Glu Ser His Asn Ser Arg Lys Lys Arg Glu Ile Thr Glu Lys Gln Ile Asp Asp Asn Arg Lys Phe Ser Leu Phe Ala Glu Rrg Lys Tyr Gln Thr Leu Asn Cys Ser Val Asn Val Asn Cys Val Asn Ile Arg Cys Pro Leu Arg Gly Leu Asp Ser Lys Ala Ser Leu Ile Leu Arg Ser Arg Leu Trp Asn Ser Thr Phe Leu Glu Glu Tyr Ser Lys Leu Asn Tyr Leu Asp Ile Leu Met Arg Ala Phe Ile Asp Val Thr Ala Ala Ala Glu Asn Ile Arg Leu Pro Asn Ala Gly Thr Gln Val Arg Val Thr Val Phe Pro Ser Lys Thr Val Ala Gln Tyr Ser Gly Val Pro Trp Trp Ile Ile Leu Val Ala Ile Leu A1a Gly Ile Leu Met Leu Ala Leu Leu Val Phe Ile Leu Trp Lys Cys G1y Phe Phe Lys Arg Asn Lys Lys Asp His Tyr Asp Ala Thr Tyr His Lys Ala Glu Ile His Ala Gln Pro Ser Asp Lys Glu Arg Leu Thr Ser Asp Ala (2) INFORMATION FOR SEQ ID NO.: 12 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 5642 (B) TYPE: nucleic acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: DNA
(VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 12 CACATCCGGC GGGGGGAAGT GGGTATATAC 'CAGGTGCAGC TGCGGGCCCT TGAGCACGTG 1380 GGCCGCTGCC ACTGCCACCA GCAGTCGCTC TACACGGACA CCATCTGCGA GATCAAC'TAC 1980 CAGCGGCCTG GCTTTGCCAC TCATGCCGCC AGCATCAACC CCACAGAGCT GGTGCCCTAC '2580 ACTACTCCAC CCTCACCTCC GTCTCCTCCC ACGCTCTCGC CTGACTGCTG GTGTGCCCGA 4'500 TGGGAGG.CAT GAAGGGGGCA AGGTCCGTCC TCTGTGGGCC CAAACCTATT TGTAACCAAA 5580 TG

(2) INFORMATION FOR SEQ ID NO.: 13 (I) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1158 (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (VI) ORIGINAL SOURCE:
(A) ORGANISM: Homo Sapiens (XI) SEQUENCE DESCRIPTION: SEQ ID NO.: 13 Met Ala Gly Pro Arg Pro Ser Pro Trp Ala Arg Leu Leu Leu Ala Ala Leu Ile Ser Val Ser Leu Ser Gly Thr Leu Ala Asn Arg Cys Lys Lys Ala Pro Val Lys Ser Cys Thr Glu Cys Val Arg Val Asp Lys Asp Cys Ala Tyr Cys Thr Asp Glu Met Phe Arg Asp Arg Arg Cys Asn Thr Gln Ala Glu Leu Leu Ala Ala Gly Cys Gln Arg Glu Ser Ile Val Val Met Glu Ser Ser Phe Gln Ile Thr Glu Glu Thr Gln Ile Asp Thr Thr Leu Arg Arg Ser Gln Met Ser Pro Gln Gly Leu Arg Val Arg Leu Arg Pro Gly Glu Glu Arg His Phe Glu Leu Glu Val Phe Glu Pro Leu Glu Ser Pro Val Asp Leu Tyr Ile Leu Met Asp Phe Ser Asn Sex Met Ser Asp Asp Leu Asp Asn Leu Lys Lys Met Gly Gln Asn Leu Ala Arg Val Leu Ser Gln Leu Thr Ser Asp Tyr Thr Ile Gly Phe Gly Lye Phe Val Asp Lys Val Ser Val Pro Gln Thr Asp Met Arg Pro Glu Lys Leu Lys Glu Pro Trp Pro Asn Ser Asp Pro Pro Phe Ser Phe Lys Asn Val Ile Ser Leu Thr Glu Asp Val Asp Glu Phe Arg Asn Lys Leu Gln Gly Glu Arg Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Leu Gln Thr Ala Val Cys Thr Arg Asp Ile Gly Trp Arg Pro Asp Ser Thr His Leu Leu Val Phe Ser Thr Glu Ser Ala Phe His Tyr Glu Ala Asp Gly Ala Asn Val Leu Ala Gly Ile Met Ser Arg Asn Asp Glu Arg Cys His Leu Asp Thr Thr Gly Thr Tyr Thr Gln Tyr Arg Thr Gln Asp Tyr Pro Ser Val Pro. Thr Leu Val Arg Leu Leu Ala Lys His Asn Ile Ile Pro Ile Phe Ala Val Thr Asn Tyr Ser Tyr Ser Tyr Tyr Glu Lys Leu His Thr Tyr Phe Pro Val Ser Ser Leu Gly Val Leu Gln Glu Asp Ser Ser Asn Ile Val Glu Leu Leu G1u Glu Ala Phe Asn Arg Ile Arg Ser Asn Leu Asp Ile Arg Ala Leu Asp Ser Pro Arg Gly Leu Arg Thr Glu Val Thr Ser Lys Met Phe Gln Lys Thr Arg Thr Gly Ser Phe His Ile Arg Arg Gly Glu Val Gly Ile Tyr Gln Val Gln Leu Arg Ala Leu Glu His Val Asp Gly Thr His Val Cys G1n Leu Pro Glu Asp Gln Lys Gly Asn Ile His Leu Lys Pro Ser Phe Ser Asp Gly Leu Lys Met Asp Ala Gly Ile Ile Cys Asp Val Cys Thr Cys Glu Leu Gln Lys Glu Val Arg Ser Ala Arg Cys Ser Phe Asn Gly Asp Phe Val Cys Gly Gln Cys Va1 Cys Ser Glu Gly Trp Ser Gly Gln Thr Cys Asn Cys Ser Thr Gly Ser Leu Ser Asp Ile Gln Pro Cys Leu Arg G1u Gly Glu Asp Lys Pro Cys Ser Gly Arg Gly Glu Cys Gln Cys Gly His Cys Val Cys Tyr Gly Glu Gly Arg Tyr Glu Gly Gln Phe Cys Glu Tyr Asp Asn Phe Gln Cys Pro Arg Thr Ser Gly Phe Leu Cys Asn Asp Arg Gly Arg Cys Ser Met Gly Gln Cys Val Cys Glu Pro Gly Trp Thr Gly Pro Ser Cys Asp Cys Pro Leu Ser Asn Ala Thr Cys Ile Asp Ser Asn Gly Gly Ile Cys Asn Gly Arg Gly His Cys Glu Cys Gly Arg Cys His Cys His Gln Gln Ser Leu Tyr Thr Asp Thr Ile Cys Glu Ile Asn Tyr Ser Ala Ile His Pro Gly Leu Cys Glu Asp Leu Arg Ser Cys Val Gln Cys Gln Ala Trp Gly Thr Gly G1u Lys Lys Gly Arg Thr Cys Glu Glu Cys Asn Phe Lys Val Lys 645 650 . 655.
Met Val Asp Glu Leu Lys Arg Ala G1u Glu Val Val Val Arg Cys Ser Phe Arg Asp Glu Asp Asp Asp Cys Thr Tyr Ser Tyr Thr Met Glu Gly Asp Gly Ala Pro Gly Pro Asn Ser Thr Val Leu Val His Lys Lys Lys Asp Cys Pro Pro Gly Ser Phe Trp Trp Leu Ile Pro Leu Leu Leu Leu Leu Leu Pro Leu Leu Ala Leu Leu Leu Leu Leu Cys Trp Lys Tyr Cys Ala Cys Cys Lys Ala Cys Leu Ala Leu Leu Pro Cys Cys Asn Arg Gly His Met Val Gly Phe Lys G1u Asp His Tyr Met Leu Arg Glu Asn Leu Met Ala Ser Asp His Leu Asp Thr Pro Met Leu Arg Ser Gly Asn Leu Lys Gly Arg Asp Val Val Arg Trp Lys Val Thr Asn Asn Met Gln Arg Pro Gly Phe Ala Thr His Ala Ala Ser Ile Asn Pro Thr Glu Leu Val 805 810 815 .
Pro Tyr Gly Leu Ser Leu Arg Leu Ala Arg Leu Cys Thr Glu Asn Leu Leu Lys Pro Asp Thr Arg Glu Cys Ala Gln Leu Arg Gln Glu Val Glu Glu Asn Leu Asn Glu Val Tyr Arg Gln Ile Ser Gly Val His Lys Leu Gln Gln Thr Lys Phe Arg Gln Gln Pro Asn Ala Gly Lys Lys Gln Asp His Thr Ile Val Asp Thr Val Leu Met Ala Pro Arg Ser Ala Lys Pro Ala Leu Leu Lys Leu Thr Glu Lys Gln Val Glu Gln Arg Ala Phe His Asp Leu Lys Val Ala Pro Gly Tyr Tyr Thr Leu Thr Ala Asp Gln Asp Ala Arg Gly Met Val Glu Phe Gln Glu Gly Val Glu Leu Val Asp Val Arg Val Pro Leu Phe Ile Arg Pro Glu Asp Asp Asp Glu Lys Gln Leu Leu Val Glu Ala Ile Asp Val Pro Ala Gly Thr Ala Thr Leu Gly Arg Arg Leu Val Asn Ile Thr Ile Ile Lys Glu Gln Ala Arg Asp Val Val Ser Phe Glu Gln Pro Glu Phe Ser Val Ser Arg Gly Asp Gln Val Ala Arg Ile Pro Val Ile Arg Arg Val Leu Asp Gly Gly Lys Ser Gln Val Ser Tyr Arg Thr Gln Asp Gly Thr Ala Gln Gly Asn Arg Asp Tyr Ile Pro Val Glu Gly Glu Leu Leu Phe Gln Pro Gly Glu Ala Trp Lys Glu Leu Gln Val Lys Leu Leu Glu Leu Gln Glu Val Asp Ser Leu Leu Arg Gly Arg Gln Val Arg Arg Phe His Val Gln Leu Ser Asn Pro Lys Phe Gly Ala His Leu Ala Ser Pro Thr Pro Pro Pro Ser Ser Ser Gly Thr Gln Met Asn Trp Thr Gly Ala Ser Arg Val Arg Cys Cys His His Ser His Pro Leu Thr Ala Thr Trp Ala Pro Arg Arg Thr Pro Met Leu Arg Pro Leu Gly Pro Gly Arg Ser Ile Ser Thr Gly Cys Pro Leu Leu Ala Ser Gln Trp Gly Thr Gly

Claims (35)

1. A method of monitoring the progression of a cancer, said method comprising, measuring the amount of NFAT mRNA or polypeptide expression in a sample from a subject, an increase or decrease in said NFAT mRNA or polypeptide expression in said sample, relative to a control sample, indicating a progression of a cancer or a propensity thereto in said subject.

2. A method of determining the prognosis for a treatment of cancer in a subject, said method comprising measuring the amount of NFAT mRNA or polypeptide expression in a sample from said subject, an increase or decrease in said NFAT mRNA or polypeptide expression in said sample, relative to a control sample, determining the prognosis of said cancer treatment in said subject.

3. A method for determining the metastatic potential of a cancer in a subject, said method comprising measuring the amount of NFAT mRNA or polypeptide expression in a sample from said subject, an increase or decrease in said NFAT mRNA or polypeptide expression in said sample, relative to a control sample, indicating metastatic potential of said cancer in said subject.

4. The method of claims 1, 2, or 3, wherein said NFAT is selected from a group consisting of NFAT-1, NFAT-4, and NFAT-5.

5. The method of claims 1, 2, or 3, wherein said measuring of NFAT indicates an increase in said NFAT mRNA or polypeptide expression in said sample, and said increase indicates progression of said cancer, poor prognosis of said cancer treatment, or increases metastatic potential of said cancer.

6. The method of claim 4, wherein said measuring of said NFAT can be any two of said group consisting of NFAT-l, NFAT-4, and NFAT-5.

7. The method of claim 4, wherein said measuring of said NFAT-1 mRNA or polypeptide expression indicates an increase in said NFAT-1 mRNA or polypeptide expression, and said increase indicates an increase in cancer cell invasion or migration.

8. The method of claim 4, wherein said measuring of said NFAT-5 mRNA or polypeptide expression indicates an increase in said NFAT-5 mRNA or polypeptide expression, and said increase indicates an increase in cancer cell migration.

9. The method of claim 4, further comprising measuring the amount of the .beta.4 subunit of integrin.

10. The method of claim 9, wherein said integrin is .alpha.6.beta.4.

11. A method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, said method comprising the steps of:
i) contacting NFAT polypeptide to a candidate compound; and ii) measuring binding of said compound to said NFAT polypeptide, wherein said binding identifies said candidate compound as a compound that is useful for modulating cancer progression, tumor invasion or tumor migration.

12. A method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, said method comprising the steps of:
i) contacting a cell or in vitro sample having NFAT biological activity;
and ii) measuring said NFAT biological activity, wherein a change in said NFAT biological activity indicates a candidate compound that modulates cancer progression, tumor invasion or tumor migration.

13. The method of claim 12, wherein said NFAT biological activity is NFAT-1 biological activity, and said NFAT-1 biological activity is tumor invasion or tumor migration.

14. The method of claim 12, wherein said NFAT biological activity is NFAT-5 biological activity and said NFAT-5 biological activity is tumor migration.

15. The method of claims 12-14, wherein said wherein said change in said NFAT biological activity is a decrease in NFAT biological activity.

16. A method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, said method comprising the steps of:
i) contacting a cell or in vitro sample having NFAT biological activity, and an NFAT target gene, said NFAT target gene is regulated by said NFAT biological activity, with a candidate compound; and ii) measuring expression of said NFAT target gene, wherein said candidate compound is determined to modulate cancer progression;
tumor invasion or tumor migration, if said candidate compound causes a change in expression of said NFAT target gene.

17. The method of claim 16, wherein said target gene is a reporter gene, said reporter gene is operably linked to one, or a plurality of consensus NFAT
DNA binding sequences.

18. The method of claim 17, wherein said consensus NFAT DNA binding sequence is selected from a group consisting of SEQ ID NO. 1, SEQ ID
NO. 2, and SEQ ID NO. 3.

19. A method for identifying a candidate compound that modulates cancer progression, tumor invasion or tumor migration, said method comprising the steps of:
i) contacting a candidate polypeptide with detectably labeled NFAT to for an NFAT-polypeptide complex;
ii) exposing said NFAT-polypeptide complex to a candidate compound;
and iii) measuring binding of said NFAT-polypeptide complex, wherein a change in said binding identifies said candidate compound as a compound that is useful for modulating cancer progression, tumor invasion or tumor migration.

20. The method of claim 19, wherein said candidate polypeptide is selected from a group consisting of, bZIP, AP-1, GATA, NF-.kappa.B, and NFAT-5.

21. The method of claim 19, wherein said candidate polypeptide is NFAT-5, said NFAT is NFAT-5, and said NFAT-polypeptide complex is an NFAT-5 homodimer.

22. The method of claim 19, wherein said change in said binding of said NFAT-polypeptide complex, is a reduction in said binding of said NFAT-polypeptide complex.

23. A method for identifying target genes involved in cancer which requires NFAT biological activity, said method comprising the steps of:
i) providing a first cell that has low NFAT biological activity or has been modified to have no NFAT biological activity;
ii) providing a second cell that has high NFAT biological activity or has been modified to have high NFAT biological activity; and iii) measuring the differentially expressed genes, wherein said differentially expressed genes are target genes of NFAT
transcriptional regulation.

24. The method of claim 23, wherein said transcribed genes are identified by a microarray of nucleic acids, ChIP, SAGE, or RNase protection assays.

25. A method for treating the progression of cancer, said method comprising introducing a transgene encoding an NFAT polypeptide, wherein said transgene encodes a dominant negative NFAT polypeptide, to a cell, said transgene is operably linked to expression control sequences, and said transgene being positioned for expression in said cell.

26. The method of claim 25, wherein said dominant negative NFAT transgene is provided by administration of a viral vector containing said transgene, said transgene positioned for expression in said cell of said subject.

27. The method of claim 25, wherein said dominant negative NFAT is selected from a group consisting of natural or artificial mutations affecting NFAT
nuclear localization, dimerization, transactivation, and DNA-binding.

28. The method of claim 25, wherein expression of said transgene encoding an NFAT polypeptide results in a decrease in NFAT biological activity.

29. A method for treating the progression of cancer, said method comprising introducing an NFAT antisense nucleic acid that inhibits NFAT biological activity; regardless of length of said antisense nucleic acid.

30. The method of claims 11, 12, 16, 19, 23, 25, or 29, wherein said NFAT or said NFAT transgene is NFAT-1 or NFAT-5.

31. A method for treating the progression of cancer, said method comprising.
introducing an NFAT consensus DNA-binding nucleic acid sequence, said nucleic acid sequence is double stranded, said sequence inhibits NFAT
biological activity, regardless of length of said nucleic acid sequence.

32. The method of claim 31, wherein said consensus NFAT DNA binding sequence is selected from a group consisting of SEQ ID NO. 1, SEQ ID
NO. 2, and SEQ ID NO. 3.

33. The method of claims 16 or 17, wherein said NFAT biological activity is NEAT-1 biological activity, said change in expression of said NFAT target gene or said reporter gene is a decrease in target gene or reporter gene expression.

34. The method of claims 16 or 17, wherein said NFAT biological activity is NEAT-5 biological activity, said change in expression of said NFAT target gene or said reporter gene is a decrease in target gene or reporter gene expression.

35. A method for treating the progression of cancer, said method comprising introducing an NFAT double-stranded interference ribonucleic acid or RNAi, that inhibits NFAT biological activity, regardless of length of said RNAi nucleic acid.