BACKGROUND OF THE INVENTION Deregulation of beta-catenin signaling is a frequent and early event in the development of a variety of human tumors, including colon cancer, melanoma, ovarian cancer, and prostate cancer. Activation of beta-catenin signaling can occur in tumor cells by loss-of-function mutations in the tumor suppressor genes APC (adenomatus polyposis coli protein) or Axin, as well as by gain-of-function mutations in the oncogene beta- catenin itself. APC, Axin, and beta-catenin normally bind to each other, as well as to the serine/threonine kinase GSK3-beta. Assembly of this degradation complex allows GSK3- beta to phosphorylate beta-catenin, which leads to beta-catenin ubiquitination and degradation by the proteasome. In the absence of APC or Axin activity, beta-catenin protein becomes stabilized and accumulates in the nucleus where it acts as a transcriptional co-activator with TCF for the induction of target genes, including the cell cycle regulators cyclin D 1 and c-Myc.

The C. elegans gene pry-1 is the structural and functional ortholog of vertebrate Axin (Korswagen HC et al. (2002) Genes Dev. 16: 1291-302). PRY-1 is predicted to contain conserved RGS and DIX domains that, in Axin, bind APC and Dishevelled, respectively. Overexpression of the C. elegans pst-l gene in zebrafish can fully rescue the mutant phenotype of masterblil7d, the zebrafish Axinl mutationO p7w-l loss-of- function mutations produce several phenotypes that appear to result from increased beta- catenin signaling (Gleason JE et al. (2002) Genes Dev. 16: 1281-90; Korswagen et al., supra). We find that the temperature-sensitive, reduction-of-function pry-l mutant mu38 grown at 25°C produces a multivulva (Muv) phenotype in which approximately 30% of animals are induced to form ectopic vulvae. The pry-1 Muv mutant phenotype is suppressed by RNAi-mediated inactivation the beta-catenin ortholog bar-l and the TCF ortholog pop-1. The Muv phenotype can also be generated by gain-of-function mutations

in bar-1/beta-catenin that eliminate the consensus GSK3-beta phosphorylation sites and are predicted to prevent Axin-mediated degradation of BAR-1.

The C. elegans gene product APR-1 shows significant structural similarity to human APC and can bind to both BAR-1/beta-catenin and PRY-1/Axin (Rocheleau et al.

(1997), Cell, Vol. 90,707-716 ; Natarajan et al. (2001), Genetics, Vol. 159,159-172 ; Korswagen et al., supra).

The ability to manipulate the genomes of model organisms such as C. elegarzs provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms.

Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Dulubova I, et al, J Neurochem 2001 Apr; 77 (1) : 229-38; Cai T, et al., Diabetologia 2001 Jan; 44 (1) : 81-8; Pasquinelli AE, et al., Nature. 2000 Nov 2; 408 (6808): 37-8; Ivanov IP, et al. , EMBO J 2000 Apr 17; 19 (8): 1907-17; Vajo Z et al. , Mamm Genome 1999 Oct ; 10 (10): 1000-4). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e. g. knockout) or overexpression of a gene (referred to as genetic entry point") that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a "modifier"involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as APC and axin, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.

All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.

SUMMARY OF THE INVENTION We have discovered genes that modify the APC and axin pathways in C. elegant, and identified their human orthologs, hereinafter referred to as modifier of APC and Axin (MAPCAX). The invention provides methods for utilizing these APC and axin modifier

genes and polypeptides to identify MAPCAX-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired APC and axin function and/or MAPCAX function. Preferred MAPCAX- modulating agents specifically bind to MAPCAX polypeptides and restore APC and axin function. Other preferred MAPCAX-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress MAPCAX gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i. e. DNA or mRNA).

MAPCAX modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with a MAPCAX polypeptide or nucleic acid. In one embodiment, candidate MAPCAX modulating agents are tested with an assay system comprising a MAPCAX polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate APC and axin modulating agents. The assay system may be cell-based or cell-free. MAPCAX- modulating agents include MAPCAX related proteins (e. g. dominant negative mutants, and biotherapeutics); MAPCAX-specific antibodies; MAPCAX-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with MAPCAX or compete with MAPCAX binding partner (e. g. by binding to a MAPCAX binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.

In another embodiment, candidate APC and axin pathways modulating agents are further tested using a second assay system that detects changes in the APC and axin pathways, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the APC and axin pathways, such as an angiogenic, apoptotic, or cell proliferation disorder (e. g. cancer).

The invention further provides methods for modulating the MAPCAX function and/or the APC and axin pathways in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a MAPCAX polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be

administered to a mammalian animal predetermined to have a pathology associated with the APC and axin pathways.

DETAILED DESCRIPTION OF THE INVENTION Genetic screens were designed to identify modifiers of the axin and APC pathway in C. elegans. The function of apr-1 was depleted by RNAi in a pry-1 hypomorphic allele mu38. Various specific genes were then silenced by RNA inhibition (RNAi). Methods for using RNAi to silence genes in C. elegans are known in the art (Fire A, et al. , 1998 Nature 391: 806-811; Fire, A. Trends Genet. 15,358-363 (1999); W09932619). Genes causing altered phenotypes in the worms were identified as modifiers of the APC and axin pathways. Modifiers of particular interest, were identified followed by identification of their orthologs. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, MAPCAX genes (i. e. , nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective APC and axin signaling pathway, such as cancer. Table 1 (Example II) lists the modifiers and their orthologs.

In vitro and in vivo methods of assessing MAPCAX function are provided herein.

Modulation of the MAPCAX or their respective binding partners is useful for understanding the association of the APC and axin pathways and their members in normal and disease conditions and for developing diagnostics and therapeutic modalities for APC and axin related pathologies. MAPCAX-modulating agents that act by inhibiting or enhancing MAPCAX expression, directly or indirectly, for example, by affecting a MAPCAX function such as enzymatic (e. g. , catalytic) or binding activity, can be identified using methods provided herein. MAPCAX modulating agents are useful in diagnosis, therapy and pharmaceutical development. nucleic acids and potypeptides of the mvendon Sequences related to MAPCAX nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) or RefSeq number), shown in Table 1 and in the sequence listing.

The term"MAPCAX polypeptide"refers to a full-length MAPCAX protein or a functionally active fragment or derivative thereof. A"functionally active"MAPCAX fragment or derivative exhibits one or more functional activities associated with a full- length, wild-type MAPCAX protein, such as antigenic or immunogenic activity,

enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of MAPCAX proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds. , John Wiley & Sons, Inc., Somerset, New Jersey) and as further discussed below. In one embodiment, a functionally active MAPCAX polypeptide is a MAPCAX derivative capable of rescuing defective endogenous MAPCAX activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. For purposes herein, functionally active fragments also include those fragments that comprise one or more structural domains of a MAPCAX, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A. , et al., Nucleic Acids Res, 1999,27 : 260-2). Methods for obtaining MAPCAX polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of a MAPCAX. In further preferred embodiments, the fragment comprises the entire functionally active domain.

The term"MAPCAX nucleic acid"refers to a DNA or RNA molecule that encodes a MAPCAX polypeptide. Preferably, the MAPCAX polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human MAPCAX. Methods of identifying orthlogs are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95: 5849-5856 ; Huynen MA et al., Genome Research (2000) 10: 1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22: 4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e. g. , retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two

species. Structural threading or other analysis of protein folding (e. g. , using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as C. elega71s, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term"orthologs"encompasses paralogs. As used herein, "percent (%) sequence identity"with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2. 0al9 (Altschul et al., J. Mol. Biol. (1997) 215: 403-410) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity"is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.

A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine ; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine ; interchangeable basic amino acids are arginine, lysine and histidine ; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.

Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2: 482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Molec. Biol. , 147: 195-197; Nicholas et al. , 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www. psc. edu) and references cited therein.; W. R. Pearson, 1991, Genomics 11: 635-650). This algorithm can

be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed. , 5 suppl. 3: 353- 358, National Biomedical Research Foundation, Washington, D. C. , USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14 (6): 6745-6763). The Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the "Match"value reflects"sequence identity." Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of a MAPCAX. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e. g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989) ). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of a MAPCAX under high stringency hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at ros° C in a solution comprising 6X single strength citrate (SSC) (1X SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 ug/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, 1X Denhardt's solution, 100 jUg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for lh in a solution containing 0. 1X SSC and 0. 1% SDS (sodium dodecyl sulfate).

In other embodiments, moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 yg/ml denatured salmon sperm DNA ; hybridization for 18-20h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7. 5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 yglml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.

Alternatively, low stringency conditions can be used that are: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 yg/ml

denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.

Isolation, Production, Expression, and Mis-expression of MAPCAX Nucleic Acids and Polypeptides MAPCAX nucleic acids and polypeptides are useful for identifying and testing agents that modulate MAPCAX function and for other applications related to the involvement of MAPCAX in the APC and axin pathways. MAPCAX nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art. In general, the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (e. g., generation of fusion proteins). Overexpression of a MAPCAX protein for assays used to assess MAPCAX function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art ; any suitable means therefore may be used (e. g. , Higgins SJ and Hames BD (eds. ) Protein Expression: A Practical Approach, Oxford University Press Inc. , New York 1999; Stanbury PF et al. , Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S (ed. ) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein Science <BR> <BR> <BR> <BR> (eds. ), 1999, John Wiley & Sons, New York). In particular embodiments, recombinant MAPCAX is expressed in a cell line known to have defective APC or axin function. The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.

The nucleotide sequence encoding a MAPCAX polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native MAPCAX gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems

may be utilized, such as mammalian cell systems infected with virus (e. g. vaccina virus, adenovirus, etc.) ; insect cell systems infected with virus (e. g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. An isolated host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.

To detect expression of the MAPCAX gene product, the expression vector can comprise a promoter operably linked to a MAPCAX gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e. g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the MAPCAX gene product based on the physical or functional properties of the MAPCAX protein in in vitro assay systems (e. g. immunoassays).

The MAPCAX protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i. e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection.

A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e. g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310: 105-111).

Once a recombinant cell that expresses the MAPCAX gene sequence is identified, the gene product can be isolated and purified using standard methods (e. g. ion exchange, affinity, and gel exclusion chromatography; centrifugation ; differential solubility; electrophoresis). Alternatively, native MAPCAX proteins can be purified from natural sources, by standard methods (e. g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.

The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of MAPCAX or other genes associated with the APC and axin pathways. As used herein, mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e. g. by gene knock-out or blocking expression that would otherwise normally occur).

Genetically modified animals Animal models that have been genetically modified to alter MAPCAX expression may be used in in vivo assays to test for activity of a candidate APC and axin modulating agent, or to further assess the role of MAPCAX in a APC and axin pathways process such as apoptosis or cell proliferation. Preferably, the altered MAPCAX expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal MAPCAX expression. The genetically modified animal may additionally have altered APC and axin expression (e. g. APC and axin knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice or rats), among others. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i. e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4: 761- 763. ) or stably integrated into its germ line DNA (i. e. , in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.

Methods of making transgenic animals are well-known in the art (for transgenic mice see Brinster et al. , Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U. S. Pat. Nos.

4,736, 866 and 4,870, 009, both by Leder et al. , U. S. Pat. No. 4,873, 191 by Wagner et al., and Hogan, B. , Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. , (1986); for particle bombardment see U. S. Pat. No. , 4,945, 050, by Sandford et al. ; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218: 348-53 and U. S. Pat. No. 4,670, 388; for transgenic insects see Berghammer A. J. et al., A Universal Marker for Transgenic Insects (1999) Nature 402: 370-371; for transgenic Zebrafish see Lin S. , Transgenic Zebrafish, Methods Mol Biol. (2000); 136: 375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47: 897-905; for transgenic rats see Hammer et al., Cell (1990) 63: 1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e. g. , Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed. , IRL Press (1987) ). Clones of the nonhuman transgenic animals can be produced

according to available methods (see Wilmut, 1. et al. (1997) Nature 385: 810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).

In one embodiment, the transgenic animal is a"knock-out"animal having a heterozygous or homozygous alteration in the sequence of an endogenous MAPCAX gene that results in a decrease of MAPCAX function, preferably such that MAPCAX expression is undetectable or insignificant. Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it. The transgene can be a human gene (e. g. , from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species. For example, a mouse MAPCAX gene is used to construct a homologous recombination vector suitable for altering an endogenous MAPCAX gene in the mouse genome. Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244: 1288-1292; Joyner et al., Nature (1989) 338: 153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra ; Pursel et al., Science (1989) 244: 1281-1288; Simms et al., Bio/Technology (1988) 6: 179-183). In a preferred embodiment, knock-out animals, such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson MH et al. , (1994) Scan J Immunol 40: 257-264; Declerck PJ et al., (1995) J Biol Chem. 270: 8397-400).

In another embodiment, the transgenic animal is a"knock-in"animal having an alteration in its genome that results in altered expression (e. g. , increased (including ectopic) or decreased expression) of the MAPCAX gene, e. g. , by introduction of additional copies of MAPCAX, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the MAPCAX gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.

Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene. One example of such a system that may be produced is the cre/loxP recombinase system of bacteriophage PI (Lakso et al., PNAS (1992) 89: 6232-6236; U. S. Pat. No. 4,959, 317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be

provided through the construction of"double"transgenic animals, e. g. , by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.

(1991) Science 251: 1351-1355; U. S. Pat. No. 5,654, 182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25: 83-6).

The genetically modified animals can be used in genetic studies to further elucidate the APC and axin pathways, as animal models of disease and disorders implicating defective APC and axin function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below. The candidate therapeutic agents are administered to a genetically modified animal having altered MAPCAX function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered MAPCAX expression that receive candidate therapeutic agent.

In addition to the above-described genetically modified animals having altered MAPCAX function, animal models having defective APC and axin function (and otherwise normal MAPCAX function), can be used in the methods of the present invention. For example, a APC and axin knockout mouse can be used to assess, in vivo, the activity of a candidate APC and axin modulating agent identified in one of the in vitro assays described below. Preferably, the candidate APC and axin modulating agent when administered to a model system with cells defective in APC and axin function, produces a detectable phenotypic change in the model system indicating that the APC and axin function is restored, i. e. , the cells exhibit normal cell cycle progression.

Modulating Agent$ The invention provides methods to identify agents that interact with and/or modulate the function of MAPCAX and/or the APC and axin pathways. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the APC and axin pathways, as well as in further analysis of the MAPCAX protein and its contribution to the APC and axin pathways. Accordingly, the invention also provides methods for

modulating the APC and axin pathways comprising the step of specifically modulating MAPCAX activity by administering a MAPCAX-interacting or-modulating agent.

As used herein, an"MAPCAX-modulating agent"is any agent that modulates MAPCAX function, for example, an agent that interacts with MAPCAX to inhibit or enhance MAPCAX activity or otherwise affect normal MAPCAX function. MAPCAX function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the MAPCAX-modulating agent specifically modulates the function of the MAPCAX. The phrases"specific modulating agent", "specifically modulates", etc. , are used herein to refer to modulating agents that directly bind to the MAPCAX polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the MAPCAX. These phrases also encompass modulating agents that alter the interaction of the MAPCAX with a binding partner, substrate, or cofactor (e. g. by binding to a binding partner of a MAPCAX, or to a protein/binding partner complex, and altering MAPCAX function). In a further preferred embodiment, the MAPCAX-modulating agent is a modulator of the APC and axin pathways (e. g. it restores and/or upregulates APC and axin function) and thus is also an APC and axin-modulating agent.

Preferred MAPCAX-modulating agents include small molecule compounds; MAPCAX-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in"Remington's Pharmaceutical Sciences"Mack Publishing Co. , Easton, PA, l9th edition.

SmaU moleeute modulators Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains. Chemical agents, referred to in the art as"small molecule"compounds are typically organic, non-peptide molecules, having a molecular weight up to 10,000, preferably up to 5,000, more preferably up to 1,000, and most preferably up to 500 daltons. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based

on known or inferred properties of the MAPCAX protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for MAPCAX-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151: 1947-1948).

Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the APC and axin pathways. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

Protein Modulators Specific MAPCAX-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the APC and axin pathways and related disorders, as well as in validation assays for other MAPCAX-modulating agents. In a preferred embodiment, MAPCAX-interacting proteins affect normal MAPCAX function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, MAPCAX-interacting proteins are useful in detecting and providing information about the function of MAPCAX proteins, as is relevant to APC and axin related disorders, such as cancer (e. gO, for diagnostic means).

A MAPCAX-interacting protein may be endogenous, i. e. one that naturally interacts genetically or biochemically with a MAPCAX, such as a member of the MAPCAX pathway that modulates MAPCAX expression, localization, and/or activity.

MAPCAX-modulators include dominant negative forms of MAPCAX-interacting proteins and of MAPCAX proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous MAPCAX-interacting proteins (Finley, R.

L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover

D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al. , Gene (2000) 250: 1-14; Drees BL Curr Opin Chem Biol (1999) 3: 64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27: 919-29; and U. S. Pat. No. 5,928, 868). Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e. g. , Pandley A and Mann M, Nature (2000) 405: 837-846; Yates JR 3rd, Trends Genet (2000) 16: 5-8).

A MAPCAX-interacting protein may be an exogenous protein, such as a MAPCAX-specific antibody or a T-cell antigen receptor (see, e. g. , Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). MAPCAX antibodies are further discussed below.

In preferred embodiments, a MAPCAX-interacting protein specifically binds a MAPCAX protein. In alternative preferred embodiments, a MAPCAX-modulating agent binds a MAPCAX substrate, binding partner, or cofactor.

Antibodies In another embodiment, the protein modulator is a MAPCAX specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify MAPCAX modulators. The antibodies can also be used in dissecting the portions of the MAPCAX pathway responsible for various cellular responses and in the general processing and maturation of the MAPCAX.

Antibodies that specifically bind MAPCAX polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of MAPCAX polypeptide, and more preferably, to human MAPCAX. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab'). sub. 2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Epitopes of MAPCAX which are particularly antigenic can be selected, for example, by routine screening of MAPCAX polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U. S. A. 78: 3824-28; Hopp and Wood, (1983) Mol. Immunol.

20: 483-89; Sutcliffe et al. , (1983) Science 219: 660-66) to the amino acid sequence of a MAPCAX. Monoclonal antibodies with affinities of 108 Dol preferably 109 M-1 to 101° M- , or stronger can be made by standard procedures as described (Harlow and Lane, supra ;

Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U. S. Pat. Nos. 4,381, 292; 4,451, 570; and 4,618, 577). Antibodies may be generated against crude cell extracts of MAPCAX or substantially purified fragments thereof. If MAPCAX fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of a MAPCAX protein. In a particular embodiment, MAPCAX-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.

The presence of MAPCAX-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding MAPCAX polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.

Chimeric antibodies specific to MAPCAX polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81: 6851-6855; Neuberger et al. , Nature (1984) 312: 604-608 ; Takeda et al., Nature (1985) 31: 452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CORs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84: 2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann LM, et al., 1988 Nature 323 : 323-327). Humanized antibodies contain-10% murine sequences and-90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co MS, and Queen C. 1991 Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. Immun.

10: 239-265). Humanized antibodies and methods of their production are well-known in the art (U. S. Pat. Nos. 5,530, 101,5, 585,089, 5,693, 762, and 6,180, 370).

MAPCAX-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via

an amino acid bridge, can be produced by methods known in the art (U. S. Pat. No.

4,946, 778; Bird, Science (1988) 242: 423-426; Huston et al. , Proc. Natl. Acad. Sci. USA (1988) 85: 5879-5883; and Ward et al., Nature (1989) 334: 544-546).

Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al. , Science (1989) 246: 1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al. , Int J. Biol Markers (1989) 4: 131-134).

A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U. S. Pat. Nos. 3,817, 837; 3,850, 752; 3,939, 350; 3,996, 345; 4,277, 437; 4,275, 149; and 4,366, 241). Also, recombinant immunoglobulins may be produced (U. S. Pat. No. 4,816, 567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U. S. Pat. No. 6,086, 900).

When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies.

Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e. g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies'concentrations in such vehicles are typically in the range of about 1 mg/ml to aboutl0 mg/ml.

Immunotherapeutic methods are further described in the literature (US Pat. No. 5,859, 206; W00073469).

Nucleic Acid Modulators Other preferred MAPCAX-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit MAPCAX activity. Preferred nucleic acid modulators interfere with the function of the MAPCAX nucleic acid such as DNA replication, transcription, translocation of the MAPCAX RNA to the site of protein translation, translation of protein from the MAPCAX RNA, splicing of the MAPCAX RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the MAPCAX RNA.

In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a MAPCAX mRNA to bind to and prevent translation, preferably by binding to the 5'untranslated region. MAPCAX-specific antisense oligonucleotides, preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10,15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50,40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.

In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, O, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e. g. see W099/18193 ; Probst JC, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22 (3): 271-281 ; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7: 187-95 ; US Pat. No.

5,235, 033; and US Pat No. 5,378, 841).

Alternative preferred MAPCAX nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific,

post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al. , 1998 Nature 391: 806-811; Fire, A. Trends Genet. 15,358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15,485-490 (2001); Hammond, S. M. , et al. , Nature Rev. Genet. 2,110-1119 (2001); Tuschl, T. Chem. Biochem. 2,239- 245 (2001); Hamilton, A. et al. , Science 286,950-952 (1999); Hammond, S. M. , et al., Nature 404,293-296 (2000); Zamore, P. D. , et al. , Cell 101,25-33 (2000); Bernstein, E., et al. , Nature 409,363-366 (2001); Elbashir, S. M. , et al., Genes Dev. 15,188-200 (2001); W00129058 ; W09932619 ; Elbashir SM, et al., 2001 Nature 411: 494-498).

Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used to elucidate the function of particular genes (see, for example, U. S. Pat. No. 6,165, 790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway.

For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan JF, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36: 1923-1937; Tonkinson JL et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14: 54-65).

Accordingly, in one aspect of the invention, a MAPCAX-specific nucleic acid modulator is used in an assay to further elucidate the role of the MAPCAX in the APC and axin pathways, and/or its relationship to other members of the pathway. In another aspect of the invention, a MAPCAX-specific antisense oligomer is used as a therapeutic agent for treatment of APC and axin-related disease states.

Assay Systems The invention provides assay systems and screening methods for identifying specific modulators of MAPCAX activity. As used herein, an"assay system" encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the MAPCAX nucleic acid or protein. In general, secondary assays further assess the activity of a MAPCAX modulating agent identified by a primary assay and may confirm

that the modulating agent affects MAPCAX in a manner relevant to the APC and axin pathways. In some cases, MAPCAX modulators will be directly tested in a secondary assay.

In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising a MAPCAX polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e. g. binding activity), which is based on the particular molecular event the screening method detects. A statistically significant difference between the agent- biased activity and the reference activity indicates that the candidate agent modulates MAPCAX activity, and hence the APC and axin pathways. The MAPCAX polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.

Primary Assays The type of modulator tested generally determines the type of primary assay.

Primary assays for small molecule modulators For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam GS et al., Curr Opin Chem Biol (1997) 1: 384-91 and accompanying references). As used herein the term"cell-based"refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term"cell free"encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (ego7 receptor-ligand binding), transcriptional activity (e. g., using a reporter gene), enzymatic activity (e. gO, via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.

Cell-based screening assays usually require systems for recombinant expression of MAPCAX and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when MAPCAX-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the MAPCAX protein may be assayed by various known methods such as substrate processing (e. g. ability of the candidate MAPCAX-specific binding agents to function as negative effectors in MAPCAX-expressing cells), binding equilibrium constants (usually at least about 107 M-1, preferably at least about 108 M-1, more preferably at least about 109 M-1), and immunogenicity (e. g. ability to elicit MAPCAX specific antibody in a heterologous host such as a mouse, rat, goat or rabbit).

For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.

The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a MAPCAX polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The MAPCAX polypeptide can be full length or a fragment thereof that retains functional MAPCAX activity. The MAPCAX polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The MAPCAX polypeptide is preferably human MAPCAX, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of MAPCAX interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has MAPCAX-specific binding activity, and can be used to assess normal MAPCAX gene function.

Suitable assay formats that may be adapted to screen for MAPCAX modulators are known in the art. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes PB, Curr Opin Chem Biol (1998) 2: 597-603; Sundberg SA, Curr Opin Biotechnol 2000,11 : 47-53). In one preferred embodiment, screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to

monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e. g., Selvin PR, Nat Struct Biol (2000) 7: 730-4; Fernandes PB, supra ; Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4: 445-451).

A variety of suitable assay systems may be used to identify candidate MAPCAX and APC and axin pathways modulators (e. g. U. S. Pat. Nos. 5,550, 019 and 6,133, 437 (apoptosis assays); U. S. Pat. No. 6,114, 132 (phosphatase and protease assays), U. S. Pat.

Nos. 5,976, 782,6, 225,118 and 6,444, 434 (angiogenesis assays), among others). Specific preferred assays are described in more detail below.

Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays measure the same parameters as kinase assays. In one example, the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M et al., Biochem J (1999) 343: 35-391). In another example, fluorescence polarization (FP), a solution-based, homogeneous technique requiring no immobilization or separation of reaction components, is used to develop high throughput screening (HTS) assays for protein phosphatases. This assay uses direct binding of the phosphatase with the target, and increasing concentrations of target-phosphatase increase the rate of dephosphorylation, leading to a change in polarization (Parker GJ et al. , (2000) J Biomol Screen 5: 77-88).

Glycosyltransferases mediate changes in glycosylation patterns that, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. An assay for glycosyltransferase uses scintillation methods to measure the transfer of carbohydrate from radiolabeled sugar-nuecleotide donor to a synthetic glycopolymer acceptor that is coupled to polyacrylamide and coated on plastic microtiter plates (Donovan RS et al., Glycoconj J (1999) 16 : 607-615).

Assays for ATPase activity are well-known in the art, such as described in Blackburn et al (Blackburn CL, et al. , (1999) J Org Chem 64: 5565-5570). The ATPase assay is performed using the EnzCheck ATPase kit (Molecular Probes). The assays are performed using an Ultraspec spectrophotometer (Pharmacia), and the progress of the reaction are monitored by absorbance increase at 360 nm. Microtubules (1.7 mM final), kinesin (0.11 mM final), inhibitor (or DMSO blank at 5% final), and the EnzCheck

components (purine nucleotide phosphorylase and MESG substrate) are premixed in the cuvette in a reaction buffer (40 mM PIPES pH 6.8, 5 mM paclitaxel, 1 mM EGTA, 5 mM MgC12). The reaction is initiated by addition of MgATP (1 mM final).

Apoptosis assays. Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature 371,346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169,1747). Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al. , 1998, Blood 15: 4730-41). Other cell-based apoptosis assays include the caspase-3/7 assay and the cell death nucleosome ELISA assay. The caspase 3/7 assay is based on the activation of the caspase cleavage activity as part of a cascade of events that occur during programmed cell death in many apoptotic pathways. In the caspase 3/7 assay (commercially available Apo- NE Homogeneous Caspase-3/7 assay from Promega, cat# 67790), lysis buffer and caspase substrate are mixed and added to cells. The caspase substrate becomes fluorescent when cleaved by active caspase 3/7. The nucleosome ELISA assay is a general cell death assay known to those skilled in the art, and available commercially (Roche, Cat# 1774425). This assay is a quantitative sandwich-enzyme-immunoassay which uses monoclonal antibodies directed against DNA and histones respectively, thus specifically determining amount of mono-and oligonucleosomes in the cytoplasmic fraction of cell lysates. Mono and oligonucleosomes are enriched in the cytoplasm during apoptosis due to the fact that DNA fragmentation occurs several hours before the plasma membrane breaks down, allowing for accumalation in the cytoplasm. Nucleosomes are not present in the cytoplasmic fraction of cells that are not undergoing apoptosis. An apoptosis assay system may comprise a cell that expresses a MAPCAX, and that optionally has defective APC and axin function (e. g. APC and axin is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate APC and axin modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate APC and axin modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether MAPCAX function plays a direct role in apoptosis.

For example, an apoptosis assay may be performed on cells that over-or under-express

MAPCAX relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the MAPCAX plays a direct role in the apoptotic response.

Apoptosis assays are described further in US Pat. No. 6,133, 437.

Cell proliferation and cell cycle assays. Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA.

Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107,79), or by other means.

Cell proliferation is also assayed via phospho-histone H3 staining, which identifies a cell population undergoing mitosis by phosphorylation of histone H3. Phosphorylation of histone H3 at serine 10 is detected using an antibody specfic to the phosphorylated form of the serine 10 residue of histone H3. (Chadlee, D. N. 1995, J. Biol. Chem 270: 20098- 105). Cell Proliferation may also be examined using [3H]-thynidine incorporation (Chen, J. , 1996, Oncogene 13: 1395-403; Jeoung, J. , 1995, J. Biol. Chem. 270: 18367-73). This assay allows for quantitative characterization of S-phase DNA syntheses. In this assay, cells synthesizing DNA will incorporate [3H]-thymidine into newly synthesized DNA.

Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e. g. , Beckman LS 3800 Liquid Scintillation Counter). Another proliferation assay uses the dye Alamar Blue (available from Biosource International), which fluoresces when reduced in living cells and provides an indirect measurement of cell number (Voytik-Harbin SL et al. , 1998, In Vitro Cell Dev Biol Anim 34: 239-46). Yet another proliferation assay, the MTS assay, is based on in vitro cytotoxicity assessment of industrial chemicals, and uses the soluble tetrazolium salt, MTS. MTS assays are commercially available, for example, the Promega CellTiter 96'D AQueous Non-Radioactive Cell Proliferation Assay (Cat. &num G5421).

Cell proliferation may also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989) ). For example, cells transformed with MAPCAX are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.

Cell proliferation may also be assayed by measuring ATP levels as indicator of metabolically active cells. Such assays are commercially available, for example Cell Titer-GloTM, which is a luminescent homogeneous assay available from Promega.

Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray JW et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49: 237-55). Cells transfected with a MAPCAX may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson), which indicates accumulation of cells in different stages of the cell cycle.

Accordingly, a cell proliferation or cell cycle assay system may comprise a cell that expresses a MAPCAX, and that optionally has defective APC and axin function (e. g.

APC and axin is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate APC and axin modulating agents. In some embodiments of the invention, the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate APC and axin modulating agents that is initially identified using another assay system such as a cell-free assay system. A cell proliferation assay may also be used to test whether MAPCAX function plays a direct role in cell proliferation or cell cycle. For example, a cell proliferation or cell cycle assay may be performed on cells that over-or under-express MAPCAX relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the MAPCAX plays a direct role in cell proliferation or cell cycle.

Angiogenesis. Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HIS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Hatrigel (Becton Dickinson). Accordingly, an angiogenesis assay system may comprise a cell that expresses a MAPCAX, and that optionally has defective APC and axin function (e. g. APC and axin is over-expressed or under-expressed relative to wild- type cells). A test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate APC and axin modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate APC and axin modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used

to test whether MAPCAX function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over-or under-express MAPCAX relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the MAPCAX plays a direct role in angiogenesis. U. S. Pat. Nos. 5,976, 782, 6,225, 118 and 6,444, 434, among others, describe various angiogenesis assays.

Hypoxic induction. The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIP-1), is upregulated in tumor cells following exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF.

Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with MAPCAX in hypoxic conditions (such as with 0. 1% 02, 5% C02, and balance N2, generated in a Napco 7001 incubator (Precision Scientific) ) and normoxic conditions, followed by assessment of gene activity or expression by Taqman (E). For example, a hypoxic induction assay system may comprise a cell that expresses a MAPCAX, and that optionally has defective APC and axin function (e. g. APC and axin is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate APC and axin modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate APC and axin modulating agents that is initially identified using another assay system. A hypoxic induction assay may also be used to test whether MAPCAX function plays a direct role in the hypoxic response. For example, a hypoxic induction assay may be performed on cells that over-or under-express MAPCAX relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the MAPCAX plays a direct role in hypoxic induction.

Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2x final test concentration

and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.

Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice. In an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents.

Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.

High-throughput cell adhesion assays have also been described. In one such assay, small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off. In this assay, not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey JR et al. , Bioconjug Chem. 2001 May-Jun; 12 (3): 346-53).

Tubulogenesis. Tubulogenesis assays monitor the ability of cultured cells, generally endothelial cells, to form tubular structures on a matrix substrate, which generally simulates the environment of the extracellular matrix. Exemplary substrates include Matrigel (Becton Dickinson), an extract of basement membrane proteins containing laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at 4° C and forms a solid gel at 37'C. Other suitable matrices comprise extracellular components such as collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-angiogenic stimulant, and their ability to form tubules is detected by imaging. Tubules can generally be detected after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Tube formation assays are well known in the art (e. g. , Jones MK et al., 1999, Nature Medicine 5: 1418-1423). These assays have traditionally involved stimulation with serum or with the growth factors FGF or VEGF. Serum represents an undefined source of growth factors. In a preferred embodiment, the assay is performed with cells cultured in serum free medium, in order to control which process or pathway a

candidate agent modulates. Moreover, we have found that different target genes respond differently to stimulation with different pro-angiogenic agents, including inflammatory angiogenic factors such as TNF-alpa. Thus, in a further preferred embodiment, a tubulogenesis assay system comprises testing a MAPCAX's response to a variety of factors, such as FGF, VEGF, phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.

Cell Migration. An invasion/migration assay (also called a migration assay) tests the ability of cells to overcome a physical barrier and to migrate towards pro-angiogenic signals. Migration assays are known in the art (e. g. , Paik JH et al. , 2001, J Biol Chem 276: 11830-11837). In a typical experimental set-up, cultured endothelial cells are seeded onto a matrix-coated porous lamina, with pore sizes generally smaller than typical cell size. The matrix generally simulates the environment of the extracellular matrix, as described above. The lamina is typically a membrane, such as the transwell polycarbonate membrane (Corning Costar Corporation, Cambridge, MA), and is generally part of an upper chamber that is in fluid contact with a lower chamber containing pro-angiogenic stimuli. Migration is generally assayed after an overnight incubation with stimuli, but longer or shorter time frames may also be used. Migration is assessed as the number of cells that crossed the lamina, and may be detected by staining cells with hemotoxylin solution (VWR Scientific, South San Francisco, CA), or by any other method for determining cell number. In another exemplary set up, cells are fluorescently labeled and migration is detected using fluorescent readings, for instance using the Falcon HTS FluoroBlok (Becton Dickinson). While some migration is observed in the absence of stimulus, migration is greatly increased in response to pro-angiogenic factors. As described above, a preferred assay system for migration/invasion assays comprises testing a MAPCAX's response to a variety of pro-angiogenic factors, including tumor angiogenic and inflammatory angiogenic agents, and culturing the cells in serum free medium.

Sprouting assay. A sprouting assay is a three-dimensional in vitro angiogenesis assay that uses a cell-number defined spheroid aggregation of endothelial cells ("spheroid"), embedded in a collagen gel-based matrix. The spheroid can serve as a starting point for the sprouting of capillary-like structures by invasion into the extracellular matrix (termed"cell sprouting") and the subsequent formation of complex anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112: 3249-58). In an exemplary experimental set-up, spheroids are prepared by pipetting 400 human umbilical

vein endothelial cells into individual wells of a nonadhesive 96-well plates to allow overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341-52, 1998).

Spheroids are harvested and seeded in 900, ul of methocel-collagen solution and pipetted into individual wells of a 24 well plate to allow collagen gel polymerization. Test agents are added after 30 min by pipetting 100 y1 of 10-fold concentrated working dilution of the test substances on top of the gel. Plates are incubated at 37°C for 24h. Dishes are fixed at the end of the experimental incubation period by addition of paraformaldehyde. Sprouting intensity of endothelial cells can be quantitated by an automated image analysis system to determine the cumulative sprout length per spheroid.

Primary assays for antibody inodulators For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the MAPCAX protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting MAPCAX-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.

In some cases, screening assays described for small molecule modulators may also be used to test antibody modulators.

Primary assays for nucleic acid modula : tors For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance MAPCAX gene expression, preferably mRNA expression.

In general, expression analysis comprises comparing MAPCAX expression in like populations of cells (e. g., two pools of cells that endogenously or recombinantly express MAPCAX) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e. g., using the TaqMan@, PE Applied Biosystems), or microarray analysis may be used to confirm that MAPCA : S mRNA expression is reduced in cells treated with the nucleic acid modulator (e. g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc. , chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001,33 : 142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol 2001,12 : 41-47). Protein expression may also be monitored. Proteins are most

commonly detected with specific antibodies or antisera directed against either the MAPCAX protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).

In some cases, screening assays described for small molecule modulators, particularly in assay systems that involve MAPCAX mRNA expression, may also be used to test nucleic acid modulators.

Secondary Assays Secondary assays may be used to further assess the activity of MAPCAX- modulating agent identified by any of the above methods to confirm that the modulating agent affects MAPCAX in a manner relevant to the APC and axin pathways. As used herein, MAPCAX-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with MAPCAX.

Secondary assays generally compare like populations of cells or animals (e. g., two pools of cells or animals that endogenously or recombinantly express MAPCAX) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate MAPCAX-modulating agent results in changes in the APC and axin pathways in comparison to untreated (or mock-or placebo- treated) cells or animals. Certain assays use"sensitized genetic backgrounds", which, as used herein, describe cells or animals engineered for altered expression of genes in the APC and axin or interacting pathways.

Cell-based assays Cell based assays may detect endogenous APC and axin pathways activity or may rely on recombinant expression of APC and axin pathways components. Any of the aforementioned assays may be used in this cell-based format. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.

Animal Assays A variety of non-human animal models of normal or defective APC and axin pathways may be used to test candidate MAPCAX modulators. Models for defective APC

and axin pathways typically use genetically modified animals that have been engineered to mis-express (e. g., over-express or lack expression in) genes involved in the APC and axin pathways. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.

In a preferred embodiment, APC and axin pathways activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal APC and axin are used to test the candidate modulator's affect on MAPCAX in Matrigel (D assays. Matrigel (E) is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C, but rapidly forms a solid gel at 37° C. Liquid Matrigel (E) is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the MAPCAX. The mixture is then injected subcutaneously (SC) into female athymic nude mice (Taconic, Germantown, NY) to support an intense vascular response. Mice with Matrigel@ pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel@ pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.

In another preferred embodiment, the effect of the candidate modulator on MAPCAX is assessed via tumorigenicity assays. Tumor xenograft assays are known in the art (see, e. g. , Ogawa K et al. , 2000, Oncogene 19: 6043-6052). Xenografts are typically implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre-existing tumor or from in vitro culture. The tumors which express the MAPCAX endogenously are injected in the flank, 1 x 105 to 1 x 107 cells per mouse in a volume of 100 u, L using a 27gauge needle. Mice are then ear tagged and tumors are measured twice weekly. Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus administration. Depending upon the pharmacokinetics of each unique candidate modulator, dosing can be performed multiple times per day. The tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. At the end of the experiment, the excised tumors maybe utilized for biomarker identification or further analyses. For immunohistochemistry staining, xenograft tumors are fixed in 4% paraformaldehyde,

0. 1M phosphate, pH 7.2, for 6 hours at 4°C, immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.

In another preferred embodiment, tumorogenicity is monitored using a hollow fiber assay, which is described in U. S. Pat No. US 5,698, 413. Briefly, the method comprises implanting into a laboratory animal a biocompatible, semi-permeable encapsulation device containing target cells, treating the laboratory animal with a candidate modulating agent, and evaluating the target cells for reaction to the candidate modulator. Implanted cells are generally human cells from a pre-existing tumor or a tumor cell line. After an appropriate period of time, generally around six days, the implanted samples are harvested for evaluation of the candidate modulator. Tumorogenicity and modulator efficacy may be evaluated by assaying the quantity of viable cells present in the macrocapsule, which can be determined by tests known in the art, for example, MTT dye conversion assay, neutral red dye uptake, trypan blue staining, viable cell counts, the number of colonies formed in soft agar, the capacity of the cells to recover and replicate in vitro, etc.

In another preferred embodiment, a tumorogenicity assay use a transgenic animal, usually a mouse, carrying a dominant oncogene or tumor suppressor gene knockout under the control of tissue specific regulatory sequences; these assays are generally referred to as transgenic tumor assays. In a preferred application, tumor development in the transgenic model is well characterized or is controlled. In an exemplary model, the"RIPl-Tag2" transgene, comprising the SV40 large T-antigen oncogene under control of the insulin gene regulatory regions is expressed in pancreatic beta cells and results in islet cell carcinomas (Hanahan D, 1985, Nature 315: 115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93: 2002-2007; Bergers G et al, 1999, Science 284: 808-812). An"angiogenic switch, "occurs at approximately five weeks, as normally quiescent capillaries in a subset of hyperproliferative islets become angiogenic. The RIP1-TAG2 mice die by age 14 weeks. Candidate modulators may be administered at a variety of stages, including just prior to the angiogenic switch (e. g., for a model of tumor prevention), during the growth of small tumors (e. g. , for a model of intervention), or during the growth of large and/or invasive tumors (e. g. , for a model of regression). Tumorogenicity and modulator efficacy can be evaluating life-span extension and/or tumor characteristics, including number of tumors, tumor size, tumor morphology, vessel density, apoptotic index, etc.

Diagnostic and therapeutic uses Specific MAPCAX-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the APC and axin pathways, such as angiogenic, apoptotic, or cell proliferation disorders.

Accordingly, the invention also provides methods for modulating the APC and axin pathways in a cell, preferably a cell pre-determined to have defective or impaired APC and axin function (e. g. due to overexpression, underexpression, or misexpression of APC and axin, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates MAPCAX activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the APC and axin function is restored. The phrase"function is restored", and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored APC and axin function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells. The invention also provides methods for treating disorders or disease associated with impaired APC and axin function by administering a therapeutically effective amount of a MAPCAX-modulating agent that modulates the APC and axin pathways. The invention further provides methods for modulating MAPCAX function in a cell, preferably a cell pre-determined to have defective or impaired MAPCAX function, by administering a MAPCAX-modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired MAPCAX function by administering a therapeutically effective amount of a MAPCAX-modulating agent.

The discovery that MAPCAX is implicated in APC and axin pathways provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders involving defects in the APC and axin pathways and for the identification of subjects having a predisposition to such diseases and disorders.

Various expression analysis methods can be used to diagnose whether MAPCAX expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis. (e. g., Current Protocols in Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman WM et al., Biotechniques (1999) 26: 112-125; Kallioniemi OP, Ann Med 2001,33 : 142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,12 : 41-47).

Tissues having a disease or disorder implicating defective APC and axin signaling that express a MAPCAX, are identified as amenable to treatment with a MAPCAX modulating

agent. In a preferred application, the APC and axin defective tissue overexpresses a MAPCAX relative to normal tissue. For example, a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial MAPCAX cDNA sequences as probes, can determine whether particular tumors express or overexpress MAPCAX. Alternatively, the TaqMan (3 is used for quantitative RT-PCR analysis of MAPCAX expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).

Various other diagnostic methods may be performed, for example, utilizing reagents such as the MAPCAX oligonucleotides, and antibodies directed against a MAPCAX, as described above for: (1) the detection of the presence of MAPCAX gene mutations, or the detection of either over-or under-expression of MAPCAX mRNA relative to the non-disorder state ; (2) the detection of either an over-or an under- abundance of MAPCAX gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by MAPCAX.

Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in MAPCAX expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for MAPCAX expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. Preferably, the disease is cancer. The probe may be either DNA or protein, including an antibody.

EXAMPLES The following experimental section and examples are offered by way of illustration and not by way of limitation.

I. C. elegans Axin/APC Suppressor Screen We have discovered that while RNAi of ape-1 in a wildtype background does not produce a Muv phenotype, apr-l inactivation enhances the penetrance of the Muv phenotype of the pry-1 mutant to 95% (see also Gleason et al., supra). This enhancement of the pry-1 Muv phenotype requires wildtype bar-1/beta-catenin and pop-1/TCF activity, suggesting that apr-l normally negatively regulates beta-catenin. beta-catenin-specific suppressor genes, when inactivated, likely suppress beta-catenin's inappropriate

transcriptional activation of target genes and, therefore, may be relevant for cancer therapy.

We designed a genetic screen to identify genes in addition to bar-l/beta-catenin and pop-1/TCF that act positively in beta-catenin signaling and, when inactivated, suppress the Muv mutant phenotype of pry-1 (mu38) ; apr-1 (RNAi). The function of individual genes was inactivated by RNAi in pry-1 mutant Ll larvae, in combination with apr-1 RNAi, and suppression of the Muv phenotype was scored as a statistically significant increase in the proportion of adults that did not display the Muv phenotype.

Suppressor genes were subsequently counterscreened to eliminate those that appeared to suppress the pry-1 (rrzu38) ; apr-1 (RNAi) mutant non-specifically, rather than those that specifically function in beta-catenin signaling. Suppressor genes that passed two specificity assays were considered to be beta-catenin-specific suppressors. First, these suppressors, like bar-l/beta-catenin, do not suppress the Muv phenotype of three mutations in genes unrelated to beta-catenin signaling (let-60/Etas, lin-12/Notch, and lin- 15). Second, these suppressors are not generally defective in the RNAi response, as determined by co-RNAi with genes unrelated to beta-catenin signaling.

II. Analysis of Table 1 BLAST analysis (Altschul et al., supra) was employed to identify orthologs of C. elegans modifiers. The columns"MAPCAX symbol", and"MAPCAX name aliases" provide a symbol and the known name abbreviations for the Targets, where available, from Genbank."MAPCAX RefSeq_NA or GINA","MAPCAX GIAA","MAPCAX NAME", and"MAPCAX Description"provide the reference DNA sequences for the MAPCAXs as available from National Center for Biology Information (NCBI), MAPCAX protein Genbank identifier number (GI#), MAPCAX name, and MAPCAX description, all available from Genbank, respectively. The length of each amino acid is in the"MAPCAX Protein Length"column.

Names and Protein sequences of C. elegans modifiers of APC and axin from screen (Example I), are represented in the"Modifier Name"and"Modifier GIAA" column by GI&num , respectively.

Tablel MAPCA MAPCAX MAPCAX NA MAPCA AA MAPCAX MAPCAX MAP Modifi Modifi X symbol name aliases RefSeq_N SE X SE name description CAX er er A or Q GIAA Q protename GIAA GI_NA ID or ID in NO RefSeq_ NO lengt AA : h LOC2561 LOC256129 XM_17195 1 22051164 27 similar to UDP-na 397 T15D6 175090 29 similar to UDP-5.1 GlcNAc : betaGa. 5 57 GlcNAc : betaGa 1 beta-1, 3-N- 1 beta-1,3-N- acetylglucosami acetylglucosami nyltransferase nyltransferase 1 ; beta-1, 3-N- 1 ; beta-1, 3-N- acetylglucosami acetylglucosami nyltransferase; nyltransferase; beta-1, 3-N- beta-1, 3-N- acetylglucosami acetylglucosami nyltransferase nyltransferase 1; UDP- 1; UDP-Gal : betaGlcNA Gal: betaGlcNA c beta 1,3- c beta 1, 3- galactosyltransf galactosyltransf erase, erase, polypeptide 6 polypeptide 6 na MGC4655 MGC4655 1 NM_03330 2 1520863128 hypothetical transferase ; 377 T15D6 175090 hypothetical 9.1 protein transferase;. 5 57 protein MGC4655 UDP-galactose MGC4655 beta-N- acetylglucosa mine beta-1,3- galactosyltrans ferase B3GNT4 B3GNT4 1 NM_03076 3 13540527 29 UDP-transferase, 378 T15D6 175090 B3GN-T4J 5. 1XM03 GlcNAc : betaGa transferring. 5 57 beta3Gn-T4 9199.1 1 beta-1,3-N- glycosyl beta-1, 3-N- acetylglucosami groups; acetylglucosami nyltransferase 4 acetylglucosa nyltransferase minyltransfera bGn-T4 | UDP- se GlcNAc : betaGa 1 beta-1, 3-N- acetylglucosami nyltransferase 4 B3GNT3 B3GNT3 NM_01425 4 7657172 30 UDP-acetylglucosa 372 T15D6 175090 TMEM31 6.2 GlcNAc : betaGa minyltransfera. 5 57 B3GN-T3 1 beta-1,3-N- se B3GNT-3 acetylglucosami HP10328 nyltransferase 3 B3GAL-T8 beta3Gn-T3 transmembrane protein 3 | putative type II membrane protein | beta- 1,3-N- acetylglucosami nyltransferase bGnT-3 UDP- GlcNAc : betaGa 1 beta-1, 3-N- acetylglucosami nyltransferase 3 B3GNT7 B3GNT7 NM_14523 5 21687139 31 UDP- transferase ; 401 T15D6 175090 hypothetical 6. 1 XM_04 GlcNAc : betaGa UDP-galactose. 5 57 gene supported 8735. 1 1 beta-1,3-N- beta-N- by AK000770 acetylglucosami acetylglucosa UDP-nyltransferase 7 mine beta-1, 3- GIcNAc : betaGa galactosyltrans 1 beta-1,3-N- ferase acetylglucosami nyltransferase 7 B3GNT1 B3GNT1 NM_00657 6 9845238 32 UDP-acetylglucosa 397 T15D6 175090 B3GNT l 7. 3 NM_03 GIcNAc : betaGa minyltransfera. 5 57 B3GN-T1 l 3252. 1 1 beta-1,3-N- se ; N- B3GN-T21 acetylglucosami acetyllactosam B3GNT-2 {yltransferase 1 ine synthase BETA3GNT beta3gal-T5 gene j beta-1,3- N- acetylglucosami nyltransferase bGnT-1 beta- 1,3-N- acetylglucosami nyltransferase bGnT-2 | UDP- GlcNAc : betaGa 1 beta-1, 3-N- acetylglucosami nyltransferase 1 IMAGE : 4 IMAGE: 490709 NM_13870 7 2016257633 beta-1, 3-N- acetylglucosa 384. T15D6 175090 907098 8 B3Gn-T6 6. 1|bd_16 acetylglucosami minyltransfera. 5 57 beta-1, 3-N- 6247. 1 nyltransferase se acetylglucosami protein nyltransferase protein CHL1 CHL1 | CALL NM-00661 8 5729767 34 cell adhesion cell adhesion 1224 lad-1 175387 L1CAM2 | cell 4.1 molecule with molecule 00 adhesion homology to molecule with L1CAM (close homology to homolog of Ll) L1CAM (close homologue of L1) | cell adhesion molecule with homology to L1CAM (close homolog of Ll) L1CAM L1CAM | NM_00042 9 13435353 35 L1 cell cell adhesion 1253 lad-1 175387 HSAS MASA 5.2 NM_02 adhesion molecule; cell 00 MIC5SPG1) 4003.1 molecule adhesion CAML1 (hydrocephalus, molecule; cell CD171 stenosis of adhesion HSAS1 | N-aqueduct of molecule; CAML1) LI Sylviusl, cytoskeletal cell adhesion MASA (mental protein molecule | retardation, binding; neural cell aphasia, integrin adhesion shuffling gait binding molecule Ll and adducted Ll cell adhesion thumbs) molecule syndrome, (hydrocephalus, spastic stenosis of paraplegia 1) aqueduct of Sylvius 1, MASA (mental retardation, aphasia, shuffling gait and adducted thumbs) syndrome, spastic paraplegia 1) NFASC NFASC XM_04680 10 27478636 36 neurofascin cell adhesion 1066 lad-1 175387 KIAA0756 8.8 molecule; cell 00 neurofascin adhesion molecule; protein binding; cytoskeletal protein binding; transmembran e receptor HUS1 HUS1 IHUS1 NM_00450 11 4758576 37 HUS1 DNAbinding ; 280 hus-1 175079 (S. pombe) 7.1 checkpoint protein 89 checkpoint homolog (S. binding ; ATP homolog pombe) binding ; DNA JUS1 clamp loader checkpoint homolog (S. pombe) HUSlb HUSlb | similar NM_14895 12 2250737438 similar to HUS1 protein binding 278 hus-1 175079 to HUS1 9.1 checkpoint 89 checkpoint homolog (S. homolog (S. pombe); HUS1 pombe); HUS1 (S. pombe) (S. pombe) checkpoint checkpoint homolog homolog FLJ12735 FLJ12735 NM_02485 13 26080431 39 hypothetical ATP binding; 1844 rfc-1 175632 hypothetical 7.3 protein DNA clamp 26 protein FLJ12735 loader FLJ12735 RFC1 RFC1 A1 NM_00291 14 15011931 40 rerplication DNA 1148 rfc-1 175632 RFC PO-GA 3. 21NM_00 factor C dependent 26 RECC1 | 6081 (activator 1) 1, adenosinetriph MHCBFB 145kDa osphatase; RFC140 enzyme replication activator; factor Cl 1 enzyme MHC binding activator; ATP factor, beta binding ; DNA replication clamp loader factor C (activator 1) 1 (145kD) | replication factor C (activator 1) 1, 145kDa PPP4C PPP4C | PP4 1 NM_00272 15 4506027 41 protein protein 307 ph-175543 PPX l Protein 0 phosphatase 4 serine/threonin 4.2 98 phosphatase 4, (formerly X), e phosphatase; catalytic subunit catalytic subunit protein protein phosphatase; phosphatase 4 protein (formerly X), phosphatase catalytic subunit YME1L1 YME1L1 NM_01426 16 21327685 42 YME1-like 1 ATPase ; 773 3L509 175542 FTSH MEG4 3 NM_139 (S. cerevisiae) ATPase; ATP 64 YME1L ATP- 312 NM_1 binding ; ATP dependent 39313 binding; metalloprotease metallopeptida FtsHl homolog se; | YMEl-like 1 metallopeptida (S. cerevisiae) se; chaperone; peptidase ; zinc binding EHD1 EHD1 PAST NM_00679 17 5803009 43 EH-domain protein 534 rme-1 175651 HAST) H-5 containing 1 binding ; 30 PAST | testilin | insulin-like EH domain growth factor containing 1 receptor homolog of binding Drosophila past EH-domain containing 1 EHD2 EHD2 EH NM_01460 18 21361462 44 EH-domain nucleotide 543 rme-1 175651 domain 1 containing 2 binding ; 30 containing 2 1 protein binding EH-domain containing 2 EHD3 EHD3 l EH NM_01460 19 7657056 45 EH-domain nucleotide 535 rme-1 175651 domain 0 containing 3 binding 30 containing 3 EH-domain containing 3 EHD4 EHD4 EH NM_13926 20 21264315 46 EH-domain collagen 541 rme-1 175651 domain 5 containing 4 binding ; 30 containing 4 l nucleotide ortholog of rat binding ; pincher | EH-calcium ion domain binding containing 4 KIAA096 KIAA0963 | NM_01496 21 7662410 47 KIAA0963 na 1366 nsh-1 175530 3 FLJ00173 l 3 protein 78 KIAA0963 protein MOP3 MOP3 NM_01818 22 11990420 48 MOP-3 na 1392 nsh-1 175530 FLJ10701 3 78 FLJ10833 MOP-3 TNKS TNKS TIN1 NM_00374 23 4507613 49 tankyrase, NAD+ ADP-1327 Ce_pm 251460 PARPL TINF1 7 TRF1- ribosyltransfer e-5 18 TNKS1 interacting ase; protein TANKYRASEI ankyrin-related binding tankyrase, ADP-ribose TRF1-polymerase interacting ankyrin-related ADP-ribose polymerase TNKS2 TNKS2 TNKL NM_02523 24 13376842 50 tankyrase, NAD+ ADP-1166 Ce_pm 251460 TANK2) 5 TRF1-ribosyltransfer e-5 18 tankyrase 2 interacting ase ; protein tankyrase, ankyrin-related binding TRF1-ADP-ribose interacting polymerase 2 ankyrin-related ADP-ribose polymerase 2 KIAA043 KIAA0433 1 NM_01521 25 7662118 51 KIAA0433 establishment 1243 1G205 251441 3 KIAA0433 6 protein and/or 24 protein maintenance of cell polarity KIAA037 KIAA0377 | NM_01465 26 7662084 52 KIAA0377 establishment 1406 1G205 251441 7 KIAA0377 9 gene product and/or 24 gene product maintenance of cell polarity NRCAM NRCAM 0 NM_00501 53 4826864 54 neuronal cell cell adhesion 1180 Lad-1 175387 KIAA0343 | 0.1 adhesion molecule 00 Bravo) molecule neuronal cell adhesion molecule

III. High-Throughput In Vitro Fluorescence Polarization Assay Fluorescently-labeled MAPCAX peptide/substrate are added to each well of a 96- well microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of MAPCAX activity.

IV. High-Throughput In Vitro Binding Assay.

33P-labeled MAPCAX peptide is added in an assay buffer (100 mM KC1, 20 mM HEPES pH 7.6, 1 mM MgCl2, 1% glycerol, 0.5% NP-40,50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25°C for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate APC and axin modulating agents.

Ve Immunoprecipitations and Immunoblotting For coprecipitation of transfected proteins, 3 x 106 appropriate recombinant cells containing the MAPCAX proteins are plated on 10-cm dishes and transfected on the following day with expression constructs. The total amount of DNA is kept constant in each transfection by adding empty vector. After 24 h, cells are collected, washed once with phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer

containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease inhibitors (complete, Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removed by centrifugation twice at 15,000 x g for 15 min. The cell lysate is incubated with 25 ul of M2 beads (Sigma) for 2 h at 4 °C with gentle rocking.

After extensive washing with lysis buffer, proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and blotted with the indicated antibodies. The reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).

VI. Expression analysis All cell lines used in the following experiments are NCI (National Cancer Institute) lines, and are available from ATCC (American Type Culture Collection, Manassas, VA 20110-2209). Normal and tumor tissues are obtained from Impath, UC Davis, Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.

TaqMan (X3 analysis is used to assess expression levels of the disclosed genes in various samples.

RNA is extracted from each tissue sample using Qiagen (Valencia, CA) RNeasy kits, following manufacturer's protocols, to a final concentration of 50ng/1lL Single stranded cDNA is then synthesized by reverse transcribing the RNA samples using random hexamers and 500ng of total RNA per reaction, following protocol 4304965 of Applied Biosystems (Foster City, CA).

Primers for expression analysis using TaqMan@ assay (Applied Biosystems, Foster City, CA) are prepared according to the Tasman@ protocols, and the following criteria : a) primer pairs are designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product. Expression analysis is performed using a 7900HT instrument.

TaqMan (íg) reactions are carried out following manufacturer's protocols, in 25 gel total volume for 96-well plates and 10 1ll total volume for 384-well plates, using 300nM primer and 250 nM probe, and approximately 25ng of cDNA. The standard curve for result analysis is prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be

present in appreciable amounts is good. The raw data are normalized using 18S rRNA (universally expressed in all tissues and cells).

For each expression analysis, tumor tissue samples are compared with matched normal tissues from the same patient. A gene is considered overexpressed in a tumor when the level of expression of the gene is 2 fold or higher in the tumor compared with its matched normal sample. In cases where normal tissue is not available, a universal pool of cDNA samples is used instead. In these cases, a gene is considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type is greater than 2 times the standard deviation of all normal samples (i. e. , Tumor-average (all normal samples) > 2 x STDEV (all normal samples) ).

A modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the modulator, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator. The expression data for the gene (s) can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.