WO2004074441A2

WO2004074441A2 - Amplification or overexpression of mll septin-like fusion (msf) and septin9 and methods related thereto

Info

Publication number: WO2004074441A2
Application number: PCT/US2004/004468
Authority: WO
Inventors: Cristina Montagna; Kent Hunter; Thomas Ried
Original assignee: Government Of The United States Of America Represented By The Secretary Department Of Health And Human Services
Priority date: 2003-02-19
Filing date: 2004-02-18
Publication date: 2004-09-02
Also published as: WO2004074441A3

Abstract

Methods of detecting cancer in a mammal, which methods comprise determining whether or not the mammal has (i) an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, or (ii) an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, wherein amplification of the gene or overexpression of the protein or of the nucleic acid molecule is indicative of cancer; a method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing; a method of inducing apoptosis in a cell; methods of testing a candidate drug for efficacy as an anti-cancer drug; and methods for evaluating the progression of cancer in a mammal.

Description

AMPLIFICATION OR OVEREXPRESSION OF MLL SEPTIN-LIKE FUSION (MSF) AND SEPTLN9 AND METHODS RELATED THERETO

FIELD OF THE INVENTION [0001] This invention pertains to methods of detecting cancer, a method of inhibiting a protein, oligonucleotides for use therein, a method of inducing apoptosis, methods of testing a candidate drug for efficacy as an anti-cancer drug, and methods for evaluating the progression of cancer.

BACKGROUND OF THE INVENTION [0002] Mouse models of human breast cancer are widely used to study molecular pathways of tumorigenesis (Henninghausen, On cogene Reviews 19: 157 (2000)). The overexpression of oncogenes known to be involved in human breast cancer, such as c-Myc, Herceptin-2/Neu (HER2/Neu), and Cyclin Dl (Ccndl), with tissue-specific promoters induces tumors in the mammary gland of transgenic mice (Guy et al., Proc. Natl. Acad. Sci. USA 89: 10578-10582 (1992); and Stewart et al., Cell 38: 627-637 (1984)). Likewise, the inactivation of tumor suppressor genes via introduction of viral oncogenes or by conditional knockout using CreLox technology can promote tumorigenesis (Xu et al., Nat. Genet. 22: 37-43 (1999); and Maroulakou et al, Proc. Natl. Acad. Sci. USA 91: 11236-11240 (1994)). [0003] Human breast cancers, like other epithelial cancers, are characterized by a recurrent and conserved pattern of genomic imbalances (Ried et al., Genes Chrom. Cancer 25: 195-204 (1999)). These imbalances result in up- or down-regulation of oncogenes and tumor suppressor genes, respectively, located on these chromosomes or chromosomal regions. The strong conservation of specific chromosomal imbalances in human solid tumors suggests that these aneuploidies are crucial events in tumorigenesis. [0004] The detection of recurring regions of genomic imbalances also can provide entry points for the molecular cloning of cancer-associated genes. The advent of molecular cytogenetic tools has allowed the screening of the entire murine genome for such genetic alterations. In particular, comparative genomic hybridization (CGH) and spectral karyotyping (SKY) have facilitated the analysis of such changes in mouse tumors (Weaver et al, Genes Chrom. Cancer 25: 251-260 (1999); and Liyanage et al., Nat. Genet. 14: 312- 315 (1996)). These techniques have been utilized to characterize genomic imbalances in several models of human breast cancer. For example, the overexpression of c-Myc or HER2/Neu oncogenes under mouse mammary tumor virus (MMTV) or endogenous promoters results in tumorigenesis; however, cytogenetic analysis has revealed that secondary genomic alterations are required, most notably deletions that map to mouse chromosome 4 (band C-E), a region that is orthologous to human lp32-36 (Weaver et al., Oncogene 21: 5097-5107 (2002); and Montagna et al, Oncogene 21: 890-898 (2002)). Conditional inactivation of the breast cancer susceptibility gene Brcal, however, requires amplification of terminal mouse chromosome 11, which, in some cases, includes the HER2/Neu oncogene in addition to losses on mouse chromosome 4 (Xu et al., Nat. Genet. 22: 37-43 (1999); Weaver et al., Genes Chrom. Cancer 25: 251-260 (1999); and Liyanage et al, Nat. Genet. 14: 312-315 (1996)).

[0005] It is an object of the present invention to provide new methods of detecting cancer, materials for use therein, and related methods. This and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION [0006] Applicants have demonstrated that the MSF gene in humans and the Septin9 gene in mice are amplified in cancer models. Furthermore, Applicants have shown that the products encoded by these genes are overexpressed in cancer. In this regard, the present invention provides methods of detecting cancer in a mammal. One method comprises determining whether or not the mammal has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. In this method, amplification of the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing is indicative of cancer. Another method comprises determining whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. In this method, overexpression of the protein or of the nucleic acid molecule is indicative of cancer.

[0007] The present invention also provides a method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell. The method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein.

[0008] Further provided are isolated or purified oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9. These oligonucleotides are suitable for use in the foregoing methods, as described herein. [0009] The present invention also provides a method of inducing apoptosis in a cell, which expresses a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. The method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein. [0010] Methods of testing a candidate drug for efficacy as an anti-cancer drug are also provided by the present invention. One method comprises comparing (i) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which has an amplification in a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, before administration of the candidate drug to the cell to (ii) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal after administration of the candidate drug to the cell. In this method, a decrease in the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug. Another method comprises comparing (i) the concentration of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a cell before administration of the candidate drug to the cell to (ii) the concentration of the protein or the nucleic acid molecule after administration of the candidate drug to the cell. In this method, a decrease in the concentration of the protein or the nucleic acid molecule upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.

[0011] The present invention further provides methods for evaluating the progression of cancer in a mammal. One method comprises monitoring the copy number of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal, which has cancer, for a period of time. In this method, an increase in the copy number over the period of time indicates a progression of cancer in the mammal, whereas a decrease in the copy number over the period of time indicates a regression of cancer in the mammal. Another method comprises monitoring the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a mammal, which has cancer, for a period of time. In this method, an increase in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a progression of cancer in the mammal, whereas a decrease in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a regression of cancer in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS [0012] Figure 1 represents a listing of the nucleotide and amino acid sequences discussed herein. DETAILED DESCRIPTION OF THE INVENTION [0013] The present invention provides methods of detecting cancer in a mammal. One method comprises determining whether or not the mammal has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, h this method, amplification of the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing is indicative of cancer. Another method comprises determining whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. In this method, overexpression of the protein or the nucleic acid molecule is indicative of cancer.

[0014] The Septin9 (Sept9) gene, also known in the art as the SL3-3 integration site-1 (Sintl) gene, was first described in Sorensen et al, J Virol. 74(5): 2161-2168 (2000), as a gene in mouse that encodes a protein (Septin9; Sept9; Sintl) of 334 amino acids. The coding sequence of the Septin9 gene and the amino acid sequence of the encoded protein are publicly available at the National Center for Biotechnology Information (NCBI) website as GenBank Accession No. NM_017380 and NP_059076, respectively, and are disclosed herein as SEQ ID NO: 3 and SEQ ID NO: 5, respectively. Septin9 is a member of the Septin protein family, which comprises several proteins known to function in a variety of cellular functions, including cytokinesis, exocytosis, and other processes involving the cytoskeleton (See Field et al., Trends Cell Biol. 9(10): 387-394 (1999); and Cooper et al., J. Cell. Biol. 134(6): 1345-1348 (1996)). Like its family members, Septin9 is evolutionarily conserved, having orthologous proteins in a variety of species, including Aspergillus nidulans, C intestinalis, catfish, cattle, Crytococcus, Drosophila melanogaster, frog, Gallus gallus, Homo sapiens, M. Grisea, Medaka, Neurospora crassa, pig, rat, Schizosaccharomyces pombe, and zebrafish. The term "orthologous" as used herein means deriving from a common ancestor gene but present in different species. In this regard, the term "orthologs" as used herein refers to genes, nucleic acid molecules encoded thereby, i.e., mRNA, or proteins encoded thereby that are derived from a common ancestor gene but are present in different species.

[0015] The MSF gene, also known as AF17q25, is the human ortholog of mouse Septin9. MSF was first described in Osaka et al, Proc. Natl. Acad. Sci. USA 96: 6428-6433 (1999), as a gene that encodes a protein of 568 amino acids. The coding sequence of the MSF gene and the amino acid sequence of the protein encoded thereby are publicly available at the NCBI website as GenBank Accession No. XM_113892 and XP_113892, respectively, and are disclosed herein as SEQ ID NO: 4 and SEQ LD NO: 6, respectively. [0016] In one of the present inventive methods of detecting cancer in a mammal, a determination is made as to whether or not the mammal has an amplification of the MSF gene, of the Septin9 gene, or of an ortholog of either of the foregoing. The term "amplification" as used herein refers to an increase in the copy number of chromosomal sequences, i.e., genes.

[0017] Methods of determining whether or not a mammal has an amplification of a particular gene are known in the art. Suitable methods include, for instance, Polymerase Chain Reaction (PCR), microarray analysis, in situ hybridization, and Southern blotting, some of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2^nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989. In such methods, an oligonucleotide probe designed to hybridize selectively to the gene of which an amplification is being determined (i.e., the MSF gene, the Seρtin9 gene, or the ortholog of either of the foregoing) is added to a sample containing genomic DNA obtained from the mammal. The oligonucleotide probe and the genomic DNA of the sample are incubated under conditions that permit selective hybridization. Preferably, the hybridization is done under high stringency conditions. By "high stringency conditions," it is meant that the probe specifically hybridizes to target sequences of the genomic DNA in an amount that is detectably stronger than non-specific hybridization. High stringency conditions, then, would be conditions, which would distinguish a polynucleotide with an exact complementary sequence of the target sequences of the genomic DNA from those sequences containing only a few small regions (e.g., 3-10 bases) with exact complementary sequence of the targets of the genomic DNA. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the target sequences of the genomic DNA and are particularly suitable for detecting amplifications of genomic sequences. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. [0018] After incubating the oligonucleotide probe and the genomic DNA obtained from the mammal, the complex comprising the oligonucleotide probe hybridized to the genomic DNA, or portion thereof, is amplified before detection. The way in which amplification of the complex is achieved can be through template-dependent amplification of the genomic DNA sequence that is adjacent to the nucleotide sequence to which the oligonucleotide probe hybridizes. Various template-dependent processes for amplifying such DNA sequence are known in the art, a number of which are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. One of the best-known processes is PCR. In this method, the complex - is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Preferred enzymes include, for example, DNA polymerases, such as T4 DNA polymerase and TaQMan DNA polymerase (Applied Biosystems, Foster City, CA). Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product, or amplicons, is produced. [0019] Other methods for amplification of the genomic DNA sequence include the ligase chain reaction (LCR), which is disclosed in U.S. Patent No. 4,883,750; isothermal amplification, in which restriction endonucleases and ligases are used to achieve the amplification of molecules that contain nucleotide 5'-[α -thio]-triphosphates in one strand (Walker et al., Proc. Natl Acad. Sci. USA 89: 392-396 (1992)); strand displacement amplification (SDA), which involves multiple rounds of strand displacement and synthesis, i.e., nick translation, and repair chain reaction (RCR), which involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. Target-specific sequences also can be detected using a cyclic probe reaction (CPR). hi CPR, a probe having 3' and 5' sequences of nonspecific DNA and a middle sequence of specific RNA is hybridized to DNA, which is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe are identified as distinctive products, which are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. A number of other amplification processes are contemplated; however, the invention is not limited as to which method is used.

[0020] Following amplification of the genomic DNA sequence, it can be desirable to separate the amplicons from the oligonucleotide probe for the purpose of determining whether specific amplification has occurred. In one embodiment, the amplicons are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al. (1989), supra. Alternatively, chromatographic techniques can be employed to effect separation. There are many kinds of chromatography that can be used in the context of the present inventive methods, e.g., adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas cliromatography (Freifelder, Physical Biochemistry Applications to Biochemistry and Molecular Biology, 2ⁿ Ed., Wm. Freeman and Co., New York, N.Y. (1982)). [0021] Amplicons must be visualized in order to confirm that hybridization of the oligonucleotide probe with the genomic DNA occurred. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplicons are integrally labeled with radio-, colorimetrically-, or fluorometrically-labeled nucleotides, the amplicons then can be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. The oligonucleotide probe that hybridizes can, alternatively, be radio-, colorimetrically-, or fluorometrically-labeled.

[0022] Alternatively, visualization of the amplicons can be achieved indirectly. Following separation of the amplicons from the oligonucleotide probe, another oligonucleotide probe is brought into contact with the amplicons. This other probe can be conjugated to a chromophore or can be radiolabeled. In another embodiment, the other probe is conjugated to a binding partner, such as an antibody or biotin, where the other member of the binding pair carries a detectable moiety (i.e., a label). [0023] One example of the foregoing is described in U.S. Patent No. 5,279,721, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention. [0024] It will be understood that the oligonucleotide probes described above are limited inasmuch as any oligonucleotide having any nucleotide sequence can be used as long as the oligonucleotide is hybridizable to the MSF gene, the Septin9 gene, or the ortholog of either of the foregoing of the genomic DNA.

[0025] In the foregoing methods of determining whether or not a mammal has an amplification of the MSF gene, Septin9 gene, or ortholog of either of the foregoing, it may be desirable to carry out the methods with a control, wherein the control is a sample containing genomic DNA of a mammal that is known not to have cancer. In this manner, the copy number of the genes of the test mammal can be directly compared to that of the control.

[0026] hi another method of detecting cancer in a mammal, a determination is made as to whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, either of which is encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. The term "overexpression" as used herein means an increase in the level of protein or nucleic acid molecule. The term "nucleic acid molecule" can be any nucleic acid molecule, e.g., RNA (e.g., mRNA) and cDNA, as long as it is encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. [0027] Methods of determining whether or not a mammal has an overexpression of a protein or a nucleic acid molecule are known in the art. Suitable methods include, for instance, Western blotting, in the case that an overexpression of a protein is being determined, and Northern blotting, Reverse transcription-PCR (RT-PCR), and Real-Time PCR, in the case that an overexpression of a RNA or cDNA is being determined. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. [0028] In a preferred embodiment of the invention, real-time PCR is used when determining whether or not a mammal has an overexpression of a nucleic acid molecule encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, hi real-time PCR, which is described in Bustin, J. Mol. Endocrinology 25: 169-193 (2000), PCRs are carried out in 96-well plates in the presence of a fluorogenic oligonucleotide probe that hybridizes to the amplicons. The fluorescent probes are double-labeled with a reporter fluorochrome and a quencher fluorochrome. When the probe anneals to the complementary sequence of the amplicon during PCR, the Taq polymerase, which possesses 5' nuclease activity, cleaves the probe such that the quencher fluorochrome is displaced from the reporter fluorochrome, thereby allowing the latter to emit fluorescence. The resulting increase in emission, which is directly proportional to the level of amplicons, is monitored by a spectrophotometer. The cycle of amplification at which a particular level of fluorescence is detected by the spectrophotometer is called the threshold cycle, C_T. It is this value that is used to compare levels of amplicons. With respect to the present inventive methods, it is desirable that the real-time PCR is carried out as in Example 2. [0029] When determining whether or not a mammal has an overexpression of a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, various assays (i.e., immunobinding assays) are contemplated. The various useful immunodetection assays have been described in Nakamura et al., Handbook of Experimental Immunology, 4^th ed., Wol. 1, Chapter 27, Blackwell Scientific Publ., Oxford, 1987 and include Western blotting, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay.

[0030] In general, the immunobinding assays involve obtaining a sample containing the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, a peptide fragment thereof, or an antibody that specifically binds to the protein or peptide fragment thereof, and contacting the sample with an antibody that specifically binds to the protein, peptide or antibody under conditions effective to allow the formation of immunocomplexes. Any suitable antibody can be used in conjunction with the present invention such that the antibody is specific for the protein or peptide fragment thereof encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing or antibody thereto. Such antibodies can be made in accordance with those methods of making antibodies known in the art. See, for instance, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publishers, Cold Spring Harbor, NY, 1988. Alternatively, fragments of the antibody can be used as long as the fragment specifically binds to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. Such fragments are known in the art to include, for instance, F(ab)₂' fragments, single chain antibody variable region fragment (ScFv) chains, and the like. [0031] The immunobinding assays for use in the present invention include methods of detecting or quantitating the immune complexes formed upon incubating the sample with the antibody. In general, the detection of immune complexes is well-known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. Patents concerning the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, additional advantages can be realized by using a secondary binding ligand, such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.

[0032] The antibody used to form the immune complexes can, itself, be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the presence of or the amount of the primary immune complexes to be determined. [0033] Alternatively, the first added component that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the first antibody. In these cases, the second binding ligand is, itself, often an antibody, which can be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0034] Further methods include the detection of primary immune complexes by a two- step approach. A second binding ligand, such as an antibody, that has binding affinity for the first antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. A number of other assays are contemplated; however, the invention is not limited as to which method is used. [0035] For purposes of the present inventive methods, the cancer detected by the present inventive methods can be any cancer, including, but not limited to, lung cancer, brain cancer, ovarian cancer, uterine cancer, testicular cancer, lymphoma, leukemia, stomach cancer, pancreatic cancer, skin cancer, breast cancer, adenocarcinoma, glio a, bone cancer, and the like. The present inventive methods of detecting cancer are particularly useful for detecting breast cancer or adenocarcinoma.

[0036] For purposes of the present inventive methods, the mammal can be any mammal, including, but not limited to, mammals of the order Rodentia, such as mice, the order Logomorpha, such as rabbits, the order Carnivora, including Felines (cats) and Canines (dogs), the order Artiodactyla, including Bovines (cows) and Swines (pigs), the order Perssodactyla, including Equines (horses), the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0037] The present invention also provides a method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell. The method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein.

[0038] Further provided by the present invention is a method of inducing apoptosis in a cell, which expresses a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. The method comprises administering to the cell an inhibitor of the protein in an amount that is sufficient to inhibit the protein.

[0039] For purposes of the present invention, the phrase "inhibitor of the protein" refers to any chemical compound, natural or synthetic, that inhibits the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. As used herein, the term "inhibit," and words stemming therefrom, do not necessarily imply 100% or complete inhibition. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. Jh this regard, inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can induce any level of inhibition. Desirably, the inhibitors of the protein can inhibit at least 10% of the function or activity of the protein in the absence of any inhibitors of the protein. It is more preferred that the inhibitors of the protein achieve at least 50% inhibition. Most preferably, the inhibitor of the protein inhibits 90% or more of the activity of the protein in the absence of any inliibitors of the protein. As generally known by one of ordinary skill in the art, the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, is to hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In other words, the proteins encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing have GTPase activity.

[0040] For purposes of the present inventive method of inhibiting the protein and of the present inventive method of inducing apoptosis in a cell, any inhibitor of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be employed. The inhibitor of the protein can be, for instance, an inhibitor that inhibits the enzymatic activity of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. In this regard, the inhibitor can be one that is known to inhibit guanosine triphosphatase (GTPase) activity, i.e., a GTPase inhibitor. Such inhibitors are known in the art and include non-hydrolyzable GTP analogs. [0041] Alternatively, the inhibitor of the protein can be an isolated or purified oligonucleotide that can hybridize to a nucleic acid molecule encoding the protein, such that administration of the oligonucleotide will result in the inhibition of the expression of the protein. The oligonucleotide can be of any length, comprising any number of nucleotides, as long as it can hybridize to the nucleic acid molecule encoding the protein. Preferably, the oligonucleotide that can hybridize is at least 18 nucleotides in length. Furthermore, the oligonucleotide can be of any nucleotide sequence as long as it can hybridize to the nucleic acid molecule in a manner sufficient to inhibit the expression of the protein. While it is likely that many other oligonucleotides having different sequences are suitable for use in the present inventive methods, the oligonucleotide preferably comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9.

[0042] In this regard, the present invention also provides the isolated or purified oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 8, or SEQ ID NO: 9. The term "isolated" as used herein is defined as having been removed from its natural environment. The term "purified" as used herein is defined as having removed some or all other constituents. The term "oligonucleotide" as used herein is defined as a polymer of DNA or RNA, (i.e., a polynucleotide), which can be single-stranded or double-stranded, synthesized or obtained from natural sources, and which can contain natural, non-natural or altered nucleotides and can contain natural, non-natural or altered internucleotide linkages. With respect to the isolated or purified oligonucleotides of the present invention, it is preferred that no insertions, deletions, inversions, and/or substitutions are present in the oligonucleotide. However, it may be suitable in some instances for the isolated oligonucleotides of the present invention to comprise one or more insertions, deletions, and or substitutions. It is, furthermore, preferred that the isolated oligonucleotides of the present invention are synthesized, single-stranded polymers of DNA.

[0043] A variety of techniques used to synthesize the present inventive oligonucleotides are known in the art. See, for example, Sambrook et al, 1989, supra; and Lemaitre et al, Proc. Natl. Acad. Sci. USA 84: 648-652 (1987). The oligonucleotides can alternatively by synthesized commercially by companies, such as Eurogentec, Belgium. [0044] With respect to the method of inducing apoptosis in a cell and to the method of inhibiting a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, the inhibitor of the protein can, alternatively, be an antibody, or fragment thereof, that binds specifically to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. Antibodies suitable for use in the present inventive methods of inhibiting a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be synthesized by methods of making antibodies that are known in the art. See, for example, Harlow et al, Antibodies: A Laboratory Manual Cold Spring Harbor Publishers, Cold Spring Harbor, NY, 1988. Fragments of antibodies that bind to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing are also suitable for use. The fragment can be any fragment that binds specifically to the protein. The fragment can include, for instance, an F(ab₂)' fragment. One of ordinary skill in the art recognizes that, in general, the antibodies, and fragments thereof, will bind to the protein and prevent its activity by preventing a substrate or another protein from binding to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, wherein the binding of the substrate or other protein is necessary for the function of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing.

[0045] Inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing that are useful in the present inventive methods can be in the form of a salt, which is preferably a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example j3-toluenesulphonic acid. [0046] Inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing that can be used in the present inventive methods, can be formed as a composition, such as a pharmaceutical composition. Pharmaceutical compositions containing the inhibitor of the protein can comprise more than one active ingredient, such as more than one type of inhibitor of the protein, e.g. a composition comprising a GTPase inhibitor and an isolated or purified oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9. The pharmaceutical composition can alternatively comprise an inhibitor of the protein in combination with other pharmaceutically active agents or drugs.

[0047] In the present inventive method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, or in the present inventive method of inducing apoptosis, an isolated or purified oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9 can be co- administered with a chemotherapeutic agent. Also, an antibody that binds specifically to the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can be co-administered with a chemotherapeutic agent. The term "chemotherapeutic agent" as used herein refers to chemical agents or drugs that are destructive of malignant cells and tissues or are toxic to the causative agent of the cancer being treated, such as a virus, bacterium, or other microorganism. Such chemotherapeutic agents include, for example, paclitaxel, cisplatin, vincristine, vinblastine, camptothecin, bleomycin, emetine, thioguanine, 5-azacytidine, hydroxyurea, cytosine arabinoside, and the like.

[0048] The carrier can be any suitable carrier. Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. It will be appreciated by one of ordinary skill in the art that, in addition to the following described pharmaceutical composition, the compounds and inhibitors of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0049] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0050] The choice of carrier will be determined in part by the particular inhibitor of the protein, as well as by the particular method used to administer the inhibitor of the protein. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present inventive methods. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the compound comprising the inhibitor of the protein are known, and, although more than one route can be used to administer a particular inhibitor of the protein, a particular route can provide a more immediate and more effective response than another route.

[0051] Injectable formulations are among those formulations that are preferred in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0052] Topical formulations are well-known to those of skill in the art. Such formulations are particularly suitable in the context of the present invention for application to the skin.

[0053] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0054] The inhibitor of the protein, alone or in combination with each other and or with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa. [0055] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inhibitor of the protein can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose,^' hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0056] Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0057] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0058] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers can be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0059] Additionally, the inhibitor of the protein, or compositions comprising such an inhibitor of the protein, can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0060] One of ordinary skill in the art will readily appreciate that the inhibitor of the protein of the present inventive methods can be modified in any number of ways, such that the therapeutic efficacy of the inhibitor is increased through the modification. For instance, the inhibitor of the protein could be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating inhibitors to targeting moieties is known in the art. See, for instance, Wadwa et al, J Drug Targeting 3: 111 (1995), and U.S. Patent No. 5,087,616. The term "targeting moiety" as used herein, refers to any molecule or agent that specifically recognizes and binds to a cell-surface receptor, such that the targeting moiety directs the delivery of the inhibitor to a population of cells on which surface the receptor is expressed. Targeting moieties include, but are not limited to, antibodies, or fragments thereof, peptides, hormones, growth factors, cytokines, and any other naturally- or non-naturally-existing ligands, which bind to cell surface receptors. The term "linker" as used herein, refers to any agent or molecule that bridges the inhibitor of the protein to the targeting moiety. One of ordinary skill in the art recognizes that sites on the inhibitor of the protein, which are not necessary for the function of the inhibitor of the protein, are ideal sites for attaching a linker and/or a targeting moiety, provided that the linker and/or targeting moiety, once attached to the inhibitor of the protein, do(es) not interfere with the function of the inhibitor of the protein, i.e., the ability to inhibit the protein encoded by the MSF gene, the Septin9 gene or an ortholog of either of the foregoing. [0061] Alternatively, the inhibitor of the protein can be modified into a depot form, such that the manner in which the inhibitor of the protein is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of inhibitors can be, for example, an implantable composition comprising the inhibitor of the protein and a porous material, such as a polymer, wherein the inhibitor is encapsulated by or diffused throughout the porous material. The depot is then implanted into the desired location within the body and the inhibitor of the protein is released from the implant at a predetermined rate by diffusing through the porous material.

[0062] Furthermore, the present inventive methods can comprise the administration of the inhibitor of the protein, in the presence or absence of an agent that enhances its efficacy. [0063] For purposes of all of the present inventive methods, the amount or dose of the compound or inhibitor administered should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. Particularly, the dose of the inhibitor of the protein should be sufficient to inhibit the protein encoded by the MSF gene, the Septin9 gene or an ortholog of either of the foregoing in a cell within about 1-2 hours, if not 3-4 hours, from the time of administration. The dose will be determined by the efficacy of the particular inhibitor and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated. Many assays for determining an administered dose are known in the art. For purposes of the present invention, an assay, which comprises comparing the extent to which the protein is inhibited in a cell upon administration of a given dose of an inhibitor of the protein to a mammal among a set of mammals of which is each given a different dose of the inhibitor, could be used to determine a starting dose to be administered to a mammal. The extent to which the protein is inhibited upon administration of a certain dose can be assayed by measuring GTPase activity as described in Tu et al, J Biol. Chem. 276: 20160-20166 (2001).

[0064] The dose also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inhibitor. Ultimately, the attending physician will decide the dosage of the inhibitor of the protein with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inhibitor to be administered, route of administration, and the severity of the condition being treated.

[0065] With respect to the present inventive method of inducing apoptosis and to the present inventive method of inhibiting a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, the protein is in a cell For purposes of the present inventive methods, the cell can be any cell, which expresses or contains a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. Preferably, the cell has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. Preferably, the cell also or alternatively has an overexpression of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing. [0066] The cell of the present inventive methods also can be a cell of any tissue type, normal or diseased, such as breast tissue, carcinoma tissue, and the like. The cell can be a cultured cell (in vitro) or a cell of a cultured tissue or organ (in situ). Alternatively, the cell can be in a living system (in vivo) or can be the living system, such as when the cell is a unicellular organism. Preferably, the cell is in a multicellular host. More preferably, the host is a mammal. For purposes of the present inventive methods mammals include those that are discussed herein. Most preferably, the mammal is a human.

[0067] In a preferred embodiment of the present inventive method of inducing apoptosis in a cell and of the method of inhibiting a protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing, the cell is in a host that has cancer. In this embodiment, the cancer is treated upon inhibiting the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing. The term "treat," and words stemming therefrom, as used herein, can be any level of treatment having a potential benefit or therapeutic effect. In this regard, the inhibitors of the protein encoded by the MSF gene, the Septin9 gene, or an ortholog of either of the foregoing can treat cancer to any degree. [0068] Methods of testing the induction of apoptosis are known in the art. One such method includes use of the "In Situ Cell Death Detection Kit, Fluorescein" (Roche, Indianapolis, IN).

[0069] Methods of testing a candidate drug for efficacy as an anti-cancer drug are also provided by the present invention. One method comprises comparing (i) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which has an amplification in a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, before administration of the candidate drug to the cell to (ii) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal after administration of the candidate drug to the cell. In this method, a decrease in the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug. Another method comprises comparing (i) the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a cell before administration of the candidate drug to the cell to (ii) the concentration of the protein or the nucleic acid molecule after administration of the candidate drug to the cell. In this method, a decrease in the concentration of the protein or the nucleic acid molecule upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.

[0070] The present invention further provides methods for evaluating the progression of cancer in a mammal One method comprises monitoring the copy number of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal, which has cancer, for a period of time. In this method, an increase in the copy number over the period of time indicates a progression of cancer in the mammal, whereas a decrease in the copy number over the period of time indicates a regression of cancer in the mammal. Another method comprises monitoring the concentration of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a mammal, which has cancer, for a period of time. In this method, an increase in the concentration of the protein or the nucleic acid molecule over the period of time indicates a progression of cancer in the mammal, whereas a decrease in the concentration of the protein or the nucleic acid molecule over the period of time indicates a regression of cancer in the mammal.

[0071] For purposes of the present inventive methods of testing a candidate drug and methods for evaluating the progression of cancer, one of ordinary skill in the art understands that the level of amplification of a MSF gene, a Septin 9 gene or an ortholog of either of the foregoing, as well as the concentration of a protein or a nucleic acid molecule encoded thereby, can be determined through any of the methods of detecting an amplification of a gene or an overexpression of a protein or a nucleic acid molecule described herein. Furthermore, the cancer and the cell of the methods of testing a candidate drug and of the methods for evaluating the progression of cancer can be any cancer and any cell, respectively, as described herein.

EXAMPLES

[0072] Abbreviations. For convenience, the following abbreviations are used herein: HER2/NEU, herceptin-2.Neu; Ccndl, Cyclier Dl; CGH, comparative genomic hybridization; SKY, spectral karyotyping; MMTV, mouse mammary tumor virus; PyVmt, Polyoma virus middle T; FISH, fluorescene in situ hybridization; MSF, MLL Septin-like Fusion; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; LCR, ligase chain reaction; SDA, strand displacement amplification; RPR, repair chain reaction; CPR, cyclic probe reaction; RT-PCR, reverse transcription - PCR; ELISA, enzyme - linked immunosorbent assay; ScFv, single chain antibody variable region fragment; GTP, guanosine triphosphates; GDP, guanosine diphosphate; ATCC, American Type Culture Collection; Dmin, double minute; BAC, bacterial artificial chromosome; FAM, flourescein; TAMRA, 6-carboxy-tetramethyl-rhodamine; Si, small interference; and GTPase, guanosine triphosphatase.

[0073] Tumors and cell lines. Mammary epithelial cell lines were derived from 15 different MMTV- PyVmt primary tumors and 11 cell lines were derived from their lung metastases. C-myc 83, brt-1, brt-5, brt-10 and pbrt-1 were derived form the MMTV-c-Myc transgenic model and the Brcal conditional knock-out as previously reported (Weaver et al, Chromosomes Cancer 25: 251-260 (1999); and Weaver et al, Oncogene 21: 5097-5107 (2002)). FF2, 4, 5, 7, 8, 9, 10, 12 and 21 cell lines were derived from the Notch4 transgenic mice described earlier (Gallahan et al, Cancer Res. 56: 175-1785 (1996)). Metaphase chromosomes were prepared from the cell lines at passages 15-20 after colcemid arrest (1 hour, final concentration 0.1 mg/ml) and standard hypotonic treatment and fixation in methanol/acetic acid. The human breast cancer cell lines BT 549, UACC 812, Zr 75-30, UACC 893, T47D, Pc3 and MDA 157 (American Type Culture Collection, (ATCC) Manassas, VA), SUM159 (University of Michigan) and MPE 600 (Vysis, Downers Grove, IL) were cultured as described on the internet at the website for the ATCC. [0074] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0075] Example 1

[0076] This example demonstrates a method of detecting an amplification of a Septin 9 gene and of a MSF gene.

[0077] In order to identify secondary genetic events required for PyVmt- associated carcinogenesis in the mammary gland, 26 cell lines established from primary tumors or tumor metastases were analyzed by CGH and SKY. SKY and CGH were performed essentially as described before. Details of the protocols can be found on the internet at the website for the laboratory of Thomas Reid at the National Institutes of Health. The following mouse BAC clones for FISH analysis were selected using the website for the NCBI: Mouse Genome Resources and the website for the European Bioinformatics Institute for mouse: RP23-147O23, 284L12, 101J23, 341C5, 202G21, 369E6, 382B19, 99M13, 84C12, 2814, 354K2, 333B13, 48A17, and 480Bll (Research Genetics, Carlsbad, CA). The clones were labeled by nick-translation and hybridized to tumor metaphase chromosomes according to standard procedures. Images were acquired using a DMRXA microscope (Leica, Wetzlar, Germany), connected to a Sensys CCD camera (Roper Scientific, Tucson, AZ) using Q-FISH software (Leica Microsystems, Cambridge, UK). Complete results of the SKY and CGH analysis and the derived tumor karyotypes can be retrieved from the National Cancer Institute/NCBI SKY and CGH website. Total mRNA from the human breast cancer cell lines SUM159, BT 549, UACC 812, MPE 600, Zr 75-30, UACC 893, T47D, Pc3, and MDA 157 was extracted using Qiagen mini kit (Qiagen, Valencia, CA).

[0078] The screening tests for genomic imbalances and chromosomal translocations revealed three hotspots of genetic instability in this tumor model. Frequent losses of the distal part of chromosome 4 (10 of 26 cases), and copy number increases on chromosomes 11 and 15 (17 and 10 cases, respectively) were detected. The chromosome 11 gain was particularly striking because high-level copy number increases (amplifications) were focused to just chromosome band 11E2. A summary of the genomic imbalances can be viewed at the NCI/NCBI SKY/CGH website. SKY analysis revealed that different chromosomal events led to the genomic amplification of chromosome 11 band E2. Nine tumors showed jumping translocations (recipient chromosomes were 1, 4, 5, 6, 7, and X) and 4 tumors showed duplications of distal chromosome 11. Two tumors (158mt, 2571mt) revealed numerous double minute chromosomes (dmin), a cytogenetic feature reflecting the amplification of oncogenes in solid tumors (8-20 dmin per cell in 50% of the cells). Mouse chromosome 11 contains a plethora of breast cancer-associated genes, including the tumor suppressor genes Trp53 and Brcal and the HER2/Neu oncogene, a member of the Egf receptor family. To determine whether these genes were deleted or amplified, FISH analysis with gene-specific BAC clones was performed. In each case the copy numbers were equal to the ploidy of the cell. c-Myc, another oncogene commonly upregulated in breast carcinomas, was also present in the normal copy number on mouse chromosome 15. CGH analyses of human carcinomas suggested a second region of frequent amplification that maps to chromosome band 17q23 (Ried et al, Cancer Res. 55: 5415-5423 (1995); and Kallioniemi et al, Proc. Natl. Acad. Sci. USA 91: 2156-2160 (1994)). Candidate genes in this amplicon include Rad51c and Tbx2. Neither of these genes was involved in the copy number increases on the orthologous region of mouse chromosome 11. In order to create additional probes for band 11E2, a physical map of BAC clones specific for the cytogenetically identified region of copy number gain on chromosome 11E2 was established. Eleven BAC clones spanning a region of 17 Mbp were selected and hybridized to tumor metaphase chromosomes derived from five different tumors representing the diversity of cytogenetic abnormalities observed. In some tumors (157mt, 404mt), the BAC clones (RP23-284L12 and 84C12), containing sequence homology for Sept9 and Rac3 were present in additional copy numbers, h other tumors (158mt, 2571mt), only the Sept9- specific clone showed copy number increases. Of note, only this clone specifically labeled the double minute chromosomes identified in a subset of tumors with gain on chromosome 11E2. Therefore, the minimally amplified region comprises exclusively BAC clone RP23- 284L12, which is the clone that contains the Sept9 gene. Sequence analysis revealed that no other known genes are present in this genomic clone.

[0079] This example demonstrated that the MSF gene is amplified in human cancer, while the Septin9 gene is amplified in mouse tumors.

[0080] Example 2

[0081] This example demonstrates the quantitative analysis of gene expression. [0082] It was important to determine whether the Sept9 expression level was increased as a result of its genomic amplification. Primers for quantitative real-time PCR were designed for this purpose. Due to the genomic co-amplification of the Rac3 gene in two tumors, primers specific for this gene were included in the analysis. Primers for the Grb2 gene were included as a positive control, as this gene is specifically upregulated by PyVmt. For real-time PCR experiments, cDNA was reverse-transcribed from 2 mg of total RNA by using a first-strand cDNA synthesis kit (Invitrogen, Grand Island, NY) and analyzed with the ABI PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and fluorescein/6-carboxy-tetramethyl-rhodamine (FAM/TAMRA) real-time PCR probes (Synthegen, Houston, TX) for the cDNA-specific real-time quantitative PCR assay were designed using Primer express 1.5 software for human GRB2, MSF, and RAC3 genes, and for the mouse Bax, Grb2, HER2/Neu, Rac3, Sept9 and Thbsl genes (see Table 1). The custom designed primers were used along with commercially available primers for mouse glyceraldehyde-3 -phosphate dehydrogenase mRNA and 18S rRNA housekeeping controls (Applied Biosystems, Foster City, CA) to determine relative mRNA expression levels. cDNA derived from normal mouse brain and normal mammary glands were used for generating a standard curve and normalizing data.- The normal mammary gland standard was arbitrarily designated as 1.0. cDNA extracted from samples 143mt, 404mt, 257 lmt and pbrt-5 were hybridized to 96-gene cDNA array filters from the mouse cancer pathway finder and cell-cycle series (SuperArray, Bethesda, MD) following the supplier's protocol.

[0084] The results showed that Sept9, but not Rac3, is consistently present at substantially increased expression levels in all cases with a genomic amplification of 11E2, when compared to the expression level in the normal mammary gland. In order to assess whether Sept9 gene amplification and overexpression is restricted to the PyVmt model, the transcription profiling was extended to four additional models of human breast cancer, where gain of chromosome 11 or amplification of the distal region of it are frequently observed. Myc 83, a tumor from a mouse model that overexpresses c-Myc under the control of the MMTV-promoter, also revealed increased expression levels. Sept9 levels were increased in two of four mammary gland adenocarcinomas from Brcal conditional knockouts as well. Additionally, nine tumors from transgenic mice were analyzed for Notch4. Increased transcript levels of Sept9 defined the majority of the tumors, and in some cases, in the absence of Sept9 genomic amplification. These results indicate that Sept9, but not Grb2 or Rac 3, is the common target for mRNA overexpression in the region of genomic amplification, and suggest that cellular pathways that promote tumorigenesis independent of the oncogenic stimulus are dependent on, or at least benefit from, increased Sept9 levels. Human chromosome 17q, which is orthologous to mouse chromosome 11, is frequently gained in human breast carcinomas. In fact, this chromosome arm contains several amplicons, with either known (HER2/Neu on chromosome band 17ql3) or suspected oncogenes (Monni et al, Proc. Natl. Acad. Sci. U.S.A. 98: 5711-5716 (2001)). However, in cytogenetic studies of human breast cancer, increased copy numbers distal to chromosome band 17q23, also have been frequently observed (Orsetti et al, Oncogene 18: 6262-6270 (1999)). The MSF gene maps to band 17q25. RNA was extracted from a series of breast cancer cell lines, some of which showed gain or amplification on band 17q25 (Forozan et al, Cancer Res 60: 4519-4525 (2000)). The quantitative PCR clearly established that MSF/Sept9 is highly expressed in six of nine cell lines, and that the overexpression of this gene can occur in the absence of genomic copy number increase. In two cell lines (MDA157, MPE600), MSF/Sept9 overexpression was accompanied by an increase in RAC3 expression. GRB2 mRNA levels were not increased. This finding establishes that abnormal expression of the MSF/Sept9 gene is not only involved in murine tumorigenesis, but in human carcinomas as well.

[0085] This example demonstrated that the nucleic acid molecule encoded by the MSF gene and that of the Septin9 gene is overexpressed in human and mouse tumors, respectively.

[0086] Example 3

[0087] This example demonstrates a method of inhibiting a protein encoded by a Septin9 gene and a protein encoded by a MSF gene.

[0088] In order to determine the consequences of Sept9 gene inactivation, small interference RNA primers (siRNA) were designed and were transiently transfected into cells with and without Sept9 overexpression. siRNAs corresponding to pEGFP reporter gene and to Sept9 mRNAs were designed as recommended (Elbashir et al, Embo J, 20: 6877-6888 (2001)), with two base overhangs (Xeragon, Germantown, MD). The following gene- specific sequences were used successfully: si-GFP antisense 5'- GAACUUCAGGGUCAGCUUGCCG-dTT-3' (SEQ ID NO: 7); Sept9 siRNA sense 5'- GUCCACUUUAAUCAAUACC dTT-3' (SEQ ID NO: 8) and antisense 5'- GGUAUUGAUUAAAGUGGAC dTT-3' (SEQ ID NO:9). Annealing for duplex siRNA formation was performed in annealing buffer (100 mM potassium acetate, 30 mM HEPES- KOH at pH 7.4, 2 mM magnesium acetate) for 1 min at 90°C, followed by 1 hr at 37°C. Transfections of reporter plasmid and siRNA were performed with Oligofectamine (Invitrogen, Grand Island, NY) in 6-well plates. Briefly, 3 ml of oligofectamine diluted in Opti-MEM were applied to the siRNA duplex mixture (60 pmole in 3 ml annealing buffer) and incubated for 25 min at room temperature. The siRNA duplex oligofectamine mixture was added to cultured cells (40-50% confluent). The cells were seeded the previous day in tissue culture media with 10% FBS but without antibiotics. Transfection was carried out for 48 hr. mRNA was extracted with Qiagen mini kit (Qiagen, Valencia, CA) and expression level for Bax, Sept9 and Thspl was determined by real-time PCR. [0089] Successful transfection was determined by real-time PCR as the levels of detectable RNA decreased. A mRNA expression decrease (28.8-58%) was observed in the three cell lines with Sept9 overexpression. Independent of that, transfection efficiency using GFP siRNA on the same cells in combination with a GFP vector was determined. While GFP transfection alone revealed 40% positive cells, none of the cells showed green fluorescence when the GFP vector was combined with siGFP, indicating that transfection was successful. Under the conditions used here, the inactivation of Sept9 expression induced no visible morphological changes, or changes on the growth kinetics of the cell populations, nor was DNA synthesis impaired as measured by BrdU incorporation. Reduction of Sept9 expression by means of siRNA, however, resulted in the upregulation of Bax message in cell lines 404mt, 257 lmt and brt-5, whereas the expression levels in the cell line that did not overexpress Sept9 were slightly reduced. Treatment of the cells with Sept9 siRNA also caused the upregulation of Thspl in cell lines 404mt, 2571mt and brt-5, all three belonging to the Sept9 overexpressing group. Thspl levels were downregulated in cell line 143mt, which showed no a priori increase of Sept9 levels.

[0090] This example demonstrated that the protein encoded by the Septin9 gene or by the MSF gene can be inhibited by the siRNAs described herein.

[0091] Example 4

[0092] This example demonstrates that Thspl and Bax are increased in the absence of

Sept9 overexpression.

[0093] Septins play a role in multiple cellular functions ranging from vesicle transport to cytokinesis. In order to identify in which cancer-related pathway the overexpression observed here exerts its function, pathway finder array filters that allow one to query the expression levels of 96 genes simultaneously were utilized. One array contained genes involved in cell-cycle regulation, and the second array contained genes that affect different cancer pathways. Four cell lines were tested, three of which were derived from the group of tumors induced by PyVmt overexpression. One tumor, pbrt-5, was derived from conditional Brcal knockouts. The latter and two of the PyVmt models showed 2.8-12.3- fold increased expression levels of Sept9. The two groups of tumors could be distinguished by the expression levels of Thspl and Bax, which increased only in the absence of Sept9 overexpression, i.e., the one PyVmt cell line that did not overexpress Sept9 showed increased levels of Thspl and Bax. The downregulation of Thspl and Bax was confirmed in a larger series of tumors using quantitative reverse transcriptase PCR. It was concluded that the downregulation of Thspl and Bax is linked to the overexpression of Sept9. Upregulation of both of Thspl and Bax induces apoptosis (de Fraipont et al, Trends Mol Med, 7: 401-407 (2001); and Mitchell et al, Cancer Res 60: 6318-6325 (2000)). [0094] This example suggests that inhibition of Septin9 leads to the upregulation of Thspl and Bax, which, in turn, leads to the induction of apoptosis.

[0095] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0096] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention. [0097] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications' and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. An isolated or purified oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

2. An isolated or purified oligonucleotide consisting of the nucleotide sequence of SEQ TD NO: 8 or SEQ ID NO: 9.

3. A method of detecting cancer in a mammal, which method comprises determining whether or not the mammal has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, wherein amplification of the MSF gene, the Septin-9 gene, or the ortholog of either of the foregoing is indicative of cancer.

4. The method of claim 3, wherein the cancer is an adenocarcinoma or a breast cancer.

5. The method of claim 3 or 4, wherein the mammal is a human.

6. A method of detecting cancer in a mammal, which method comprises determining whether or not the mammal has an overexpression of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, wherein overexpression of the protein or of the nucleic acid molecule is indicative of cancer.

7. The method of claim 4, wherein the cancer is an adenocarcinoma or a breast cancer.

8. The method of claim 6 or 7, wherein the mammal is a human.

9. A method of inhibiting a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein, whereupon the protein in the cell is inhibited.

10. The method of claim 9, wherein the cell has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, or the cell has an overexpression of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.

11. The method of claim 9 or 10, wherein the cell is in a host.

12. The method of claim 11 , wherein the host is a mammal.

13. The method of claim 12, wherein the mammal is a human.

14. The method of claim 11, wherein the host has cancer and wherein the cancer is treated in the host by way of the method.

15. The method of claim 14, wherein the cancer is an adenocarcinoma or a breast cancer.

16. The method of claim 9 or 10, wherein the inliibitor of the protein is an isolated or purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ JD NO: 9.

17. The method of claim 16, wherein the isolated or purified nucleic acid molecule is co-administered with a chemotherapeutic agent.

18. The method of claim 9 or 10, wherein the inhibitor of the protein is an antibody that binds specifically to the protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.

19. The method of claim 18, wherein the antibody is co-administered with a chemotherapeutic agent.

20. A method of inducing apoptosis in a cell, which expresses a protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, which method comprises administering to the cell an inhibitor of the protein in an amount sufficient to inhibit the protein, whereupon apopotosis in the cell is induced.

21. The method of claim 20, wherein the cell has an amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, or the cell has an overexpression of a protein or a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.

22. The method of claim 20 or 21, wherein the cell is in a host.

23. The method of claim 22, wherein the host is a mammal.

24. The method of claim 23, wherein the mammal is a human.

25. The method of claim 22, wherein the host has cancer and wherein the cancer is treated in the host by way of the method.

26. The method of claim 25, wherein the cancer is an adenocarcinoma or a breast cancer.

27. The method of claim 20 or 21, wherein the inhibitor of the protein is an isolated or purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9.

28. The method of claim 27, wherein the isolated or purified nucleic acid molecule is co-administered with a chemotherapeutic agent.

29. The method of claim 20 or 21 , wherein the inhibitor of the protein is an antibody that binds specifically to the protein encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing.

30. The method of claim 29, wherein the antibody is co-administered with a chemotherapeutic agent.

31. A method of testing a candidate drug for efficacy as an anti-cancer drug, which method comprises comparing (i) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a cell, which has an amplification in a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, before administration of the candidate drug to the cell to (ii) the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal after administration of the candidate drug to the cell, wherein a decrease in the level of amplification of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.

32. A method of testing a candidate drug for efficacy as an anti-cancer drug, which method comprises comparing (i) the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a cell before administration of the candidate drug to the cell to (ii) the concentration of the protein or of the nucleic acid molecule after administration of the candidate drug to the cell, wherein a decrease in the concentration of the protein or of the nucleic acid molecule upon administration of the candidate drug is indicative of the efficacy of the candidate drug as an anti-cancer drug.

33. A method for evaluating the progression of cancer in a mammal, which method comprises monitoring the copy number of a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing in a mammal, which has cancer, for a period of time, wherein an increase in the copy number over the period of time indicates a progression of cancer in the mammal and a decrease in the copy number over the period of time indicates a regression of cancer in the mammal.

34. A method for evaluating the progression of cancer in a mammal, which method comprises monitoring the concentration of a protein or of a nucleic acid molecule, wherein the protein or the nucleic acid molecule is encoded by a MSF gene, a Septin9 gene, or an ortholog of either of the foregoing, in a mammal, which has cancer, for a period of time, wherein an increase in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a progression of cancer in the mammal and a decrease in the concentration of the protein or of the nucleic acid molecule over the period of time indicates a regression of cancer in the mammal.