WO2007013574A1 - Compositions and methods for treating breast cancer - Google Patents

Abstract

The invention features a method for inhibiting growth of a cancer cell by contacting the cell with a composition of a siRNA that inhibits expression of A2254 or A5623. Methods of treating cancer are also within the invention. The invention also features products, including nucleic acid sequences and vectors as well as to compositions comprising them, useful in the provided methods. The invention also provides a method for inhibiting growth of a tumor cell, for example a breast cancer cell, as well as a method for identifying compounds that reduce or prevent the binding between A5623 and A2254. Moreover, the present invention relates to methods of treatment or prevention of cancer, particularly breast cancer, comprising administering a compound that inhibits binding between A2254 and A5623 as well as to compositions comprising such binding inhibitors.

Description

DESCRIPTION

COMPOSITIONS AND METHODS FOR TREATING BREAST CANCER

This application claims the benefit of U.S. Provisional Application Serial No. 60/702,446 filed July 25 2005, the contents of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to the field of biological science, more specifically to the field of cancer research. In particular, the present invention relates a composition comprising a nucleic acid capable of inhibiting expression of the genes encoding A2254 or A5623. In some embodiments, the compound is a small interfering RNA (siRNA) corresponding to a subsequence from these genes. The present invention also relates to methods and kits for identifying compounds useful in the treatment and prevention of cancer. Moreover, the present invention relates to methods of treatment or prevention of cancer, particularly breast cancer, comprising administering a compound that inhibits binding between A2254 and A5623 as well as to compositions comprising such binding inhibitors.

Background Art

Breast cancer (BRC) is a complex disease characterized by accumulation of genetic and epigenetic changes in a large number of genes (Nathanson KL., et al, Nat Med, 7(5): 552-6, 2001). A multi-step model of mammary carcinogenesis, i.e., transformation of normal cells through stages of atypical ductal hyperplasia, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC), is similar to the stages of carcinogenesis in other tissues, but the precise molecular mechanisms driving breast cancer remain unknown. Obviously, molecular factors leading to development of primary breast cancer, its progression, and its metastasis would be likely targets for development of better tools for prevention and treatment of this disease.

Gene-expression profiles obtained by cDNA microarray analysis can provide a considerable amount of information for characterizing the nature of individual cancers; the promise of such information lies in its potential for improving clinical strategies for treating neoplastic diseases through development of novel drugs (Petricoin, E. F., 3rd, et al, Nat Genet, 32 Suppl: 474-479, 2002; Bange J,., et al, Nat Med, 7 (5): 548-52, 2001). With that goal in mind, we have been analyzing the expression profiles of 77 breast tumors, including 12 DCISs and 69 IDCs purified by means of a combination of laser-mi crobeam microdissection (LMM) and a cDNA microarray representing 27,648 genes (Nishidate T, et al, Int J Oncol. 2004 Oct;25(4)797-819). The data from these experiments not only should provide important information about breast tumorigenesis, but should be valuable for identifying candidate genes whose products might serve as diagnostic markers and/or as molecular targets for treatment of breast cancer.

Disclosure of the Invention

The present invention based on the discovery that A2254 and A5623 were significantly overexpressed in breast cancer cells and inhibiting expression of A2254 or A5623 is effective in inhibiting the cellular growth of various cancer cells, including those involved in BRC. The inventions described in this application are based in part on this discovery.

To isolate novel molecular targets for treatments of breast cancer, the present inventors investigated precise genome-wide expression profiles of 77 cases with premenopausal breast cancer by using a combination of cDNA microarray and laser microbeam microdissection. Among the up-regulated genes, the present inventors identified A2254, designated kinesin family member 2C (KIF2C) and A5623 protein regulator of cytokinesis 1 (PRCl), whose expression showed to be up-regulated in 44 of 60 and 37 of 58 breast cancer cases from which the inventors were able to obtain expression data, respectively. Subsequent semi-quantitative RT-PCR and Northern blot analyses confirmed that A2254 and A5623 were significantly overexpressed in clinical breast cancer samples and breast cancer cell lines, compared to normal human tissues, including breast ductal cells or normal breast. Immunocytochemical staining shows that exogenous A2254 and A5623 localized to the cytoplasmic and/or cytoplasmic apparatus in COS7 cells. In particular, exogenous A5623 was observed in the intermediate filament network in COS7 cells.

Treatment of breast cancer cells with small interfering RNAs (siRNAs) effectively inhibited expression of A2254 and A5623 and suppressed cell/tumor growth of breast cancer cells, T47D and HBC5, respectively. In addition, the present inventors found that both of A2254 [and A5623] transient overexpression in HT1080 cells promotes cell migration in scratch assay, suggesting that these genes play key roles in cell migration (data not shown). These findings suggest that overexpression of A2254 and A5623 might be involved in breast tumorigenesis or metastasis and might be promising strategies for specific treatment for breast cancer patients.

Furthermore, the present inventors demonstrated that A5623 can bind to A2254 through a part of its N terminal side, and that A5623 and A2254 colocalized on the midbody during anaphase and cytokinesis. Immunocytochemical staining shows that endogenous A2254 localized to mitotic spindle poles in prophase, and then to the entire mitotic spindle through metaphase into the early stage of anaphase in breast cancer cells. These findings suggest that the complex of A2254 and A5623 might be involved in breast tumorigenesis and be promising strategies for specific treatment for breast cancer patients. A2254, which is designated Kinesin family member 2C, has two different transcriptional variants consisting of 21 and 20 exons, corresponding to A2254V1 (GenBank Accession NO: AB264115, SEQ ID NO; 35, 36) and A2254V2 (GenBank Accession NO; AY026505, SEQ ID NO; 37, 38), respectively (Figure 3A, upper panel). Therefore, in the present invention, A2254 includes A2254V1 and A2254V2. Exon 1 and 2 of the Vl variant was 185bp and 94bp, respectively, while V2 variant has no exon 1 and 2 of Vl, but a novel exon consisting of 346bp as exon 1. Last exon (exon 20) of V2 variant was 537bp shorter than the 3' end of last exon (exon 21) of Vl variant. The full-length cDNA sequences of A2254V1 and A2254V2 variants contained 2886, and 2401 nucleotides, respectively. The ORF of these variants start at within each exon 1. Eventually, Vl and V2 transcripts encode 725 and 671 amino acids, respectively.

A5623, which is designated Protein regulator of cytokinesis 1, has also three different transcriptional variants consisting of 15, 14 and 14 exons, corresponding to A5623V1 (GenBank Accession NO; NM__003981; SEQ ID NO; 39, 40), A5623V2 (GenBank Accession NO; NMJ99413; SEQ ID NO; 41, 42) and 5623V3 (GenBank Accession NO; NM_199414; SEQ ID NO; 43, 44) respectively (Figure 3B, upper panel). Therefore, in the present invention, A5623 includes A5623V1, A5623V2 and A5623V3. There were alternative variations in exon 13 and 14 of Vl, and the other remaining exons were common to all variants. V2 variant has no exon 14 of the Vl, generating a novel early stop codon within last exon. Exon 14 of the V3 variant was completely deleted, and exon 13 of V3 was 77bp shorter than that of the Vl at the 3' end, generating a novel early stop codon within last exon as well. The full-length cDNA sequences of A5623V1, A5623V2 and 5623V3 variants consist of 3128, 3091 and 3011 nucleotides, respectively. The ORF of these variants start at within each exon 1. Eventually, Vl, V2 and V3 transcripts encode 620, 606 and 566 amino acids, respectively.

The invention provides methods for inhibiting cell growth. Among the methods provided are those comprising contacting a cell with a composition comprise a small interfering RNA (siRNA) that inhibits expression of A2254 or A5623. The invention also provides methods for inhibiting tumor cell growth in a subject. Such methods include administering to a subject a composition comprising a small interfering RNA (siRNA) that hybridizes specifically to a sequence from A2254 or A5623. Another aspect of the invention provides methods for inhibiting the expression of the A2254 or A5623 gene in a cell of a biological sample. Expression of the gene may be inhibited by introduction of a double stranded ribonucleic acid (RNA) molecule into the cell in an amount sufficient to inhibit expression of the A2254 or A5623 gene. Another aspect of the invention relates to products including nucleic acid sequences and vectors as well as to compositions comprising them, useful, for example, in the provided methods. Among the products provided are siRNA molecules having the property to inhibit expression of the A2254 or A5623 gene when introduced into a cell expressing said gene. Among such molecules are those that comprise a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to an A2254 or A5623 target sequence, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand. The sense and the antisense strands of the molecule hybridize to each other to form a double- stranded molecule.

A further aspect of the present invention is a method of screening for a compound useful in treating or preventing cancer, in particular breast cancer, said method comprising the steps of:

(a) contacting a polypeptide comprising an A2254-binding domain of an A5623 polypeptide with a polypeptide comprising an A5623-binding domain of an A2254 polypeptide in the presence of a test compound;

(b) detecting binding between the polypeptides; and

(c) selecting a test compound that inhibits binding between the polypeptides.

The skilled person can determine an A2254-binding domain of an A5623 polypeptide or an A5623-binding domain of an A2254 polypeptide by methods known in the art for determining protein-interaction domains. For example, an interaction domain can be identified by interaction domain mapping. For example, different parts of the A2254 or A5623 polypeptide, respectively, are expressed and screened for interaction by applying, e.g., the yeast two-hybrid system. An interaction identified by the yeast two-hybrid system can be verified, for example, by applying a biochemical assay, e.g., immunoprecipitation or interaction on a column. The afore mentioned methods for identifying and/or mapping an interaction domain are in no way limiting since the skilled person can readily apply other methods known in the art for identification and mapping of interaction domains.

Preferably, the polypeptide comprising the A2254-binding domain which is applied in the method of scrrening an inhibitor of the binding between an A5623 polypeptide and an A2254 polypeptide comprises an A5623 polypeptide, such as A5623V1 (SEQ ID NO: 40), A5623V2 (SEQ ID NO: 42) or A5623V3 (SEQ ID NO: 44). In another preferred aspect, the polypeptide comprising the A5623-binding domain which is applied in the method of scrrening an inhibitor of the binding between an A5623 polypeptide and an A2254 polypeptide comprises an A2254 polypeptide, such as A2254V1 (SEQ ID NO: 36) or A2254V1 (SEQ ID NO: 38).

A still further aspect of the present invention is a kit for screening for a compound for treating or preventing breast cancer, wherein the kit comprises:

(a) a polypeptide comprising an A2254-binding domain of an A5623 polypeptide;

(b) a polypeptide comprising an A5623-binding domain of an A2254 polypeptide, and

(c) a reagent to detect the interaction between the polypeptides.

Preferably, the polypeptide comprising the A2254-binding domain comprises an A5623 polypeptide or the polypeptide comprising the A5623-binding domain comprises an A2254 polypeptide.

In yet another aspect, the present invention relates to a method for treating or preventing breast cancer in a subject, wherein the method comprises the step of administering a pharmaceutically effective amount of a compound that inhibits the binding between an A5623 polypeptide and an A2254 polypeptide.

Moreover, the present invention also relates to a composition for treating or preventing breast cancer, wherein the composition comprises a pharmaceutically effective amount of a compound that inhibits the binding between an A5623 polypeptide and an A2254 polypeptide, and a pharmaceutically acceptable carrier. As used herein, the term "organism" refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal, including a human being. As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g. bodily fluids, including but not limited to blood,' mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules. The invention features methods of inhibiting cell growth. Cell growth is inhibited by contacting a cell with a composition of a small interfering RNA (siRNA) of A2254 or A5623. The cell is further contacted with a transfection-enhancing agent. The cell is provided in vitro, in vivo or ex vivo. The subject is a mammal, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The cell is a breast ductal cell. Alternatively, the cell is a tumor cell (i.e., cancer cell) such as a carcinoma cell or an adenocarcinoma cell. For example, the cell is a breast cancer cell. By inhibiting cell growth is meant that the treated cell proliferates at a lower rate or has decreased viability than an untreated cell. Cell growth is measured by proliferation assays known in the art.

By the term "siRNA" is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a. sense A2254 or A5623 nucleic acid sequence, an anti-sense A2254 or A5623 nucleic acid sequence or both. The siRNA may comprise two complementary molecules or may be constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin, which, in some embodiments, leads to production of micro RNA (miRNA).

Binding of the siRNA to an A2254 or A5623 transcript in the target cell results in a reduction in A2254 or A5623 production by the cell. The length of the oligonucleotide is at least about 10 nucleotides and may be as long as the naturally-occurring A2254 or A5623 transcript. Preferably, the oligonucleotide is about 19 to about 25 nucleotides in length. Most preferably, the oligonucleotide is less than about 75, about 50, or about 25 nucleotides in length. Examples of siRNA oligonucleotides of A2254 or A5623 which inhibit A2254 or A5623 expression in mammalian cells include oligonucleotides containing target sequences, for example, nucleotides of SEQ ID NOs: 21 or 25, or 29 or 33, respectively.

Methods for designing double stranded RNA having the ability to inhibit gene expression in a target cell are known. (See for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website

(http://www.ambion.co.jp/techlib/tb/tb_506.html). The computer program available from Ambion, Inc. selects nucleotide sequences for siRNA synthesis based on the following protocol.

Selection of siRNA Target Sites

1. Beginning with the AUG start codon of the transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et a/., Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13(24): 3191-7 (1999), don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75bases) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.

2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. It is suggested to use BLAST (Altschul SF, et.al, Nucleic Acids Res. 1997;25: 3389-402; J MoI Biol. 1990;215:403 -10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/

3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.

Also included in the invention are isolated nucleic acid molecules that include the nucleic acid sequence of target sequences, for example, nucleotides of SEQ ID NOs: 21, 25, 29 and 33 or a nucleic acid molecule that is complementary to the nucleic acid sequence of nucleotides of SEQ ID NOs: 21, 25, 29 and 33. As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment {e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the present invention, isolated nucleic acid includes DNA, RNA, and derivatives thereof. When the isolated nucleic acid is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two nucleic acids or compounds or associated nucleic acids or compounds or combinations thereof. Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. For the purposes of this invention, two sequences having 5 or fewer mismatches are considered to be complementary. Furthermore, the sense strand and antisense strand of the isolated nucleotide of the present invention can form double stranded nucleotide or hairpin loop structure by the hybridization. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches. The nucleic acid molecule is less than 2886 or 3128 nucleotides in length for A2254 or A5623, respectively. For example, the nucleic acid molecule is less than about 500, about 200, or about 75 nucleotides in length. Also included in the invention is a vector containing one or more of the nucleic acids described herein, and a cell containing the vectors. The isolated nucleic acids of the present invention are useful for siRNA against A2254 or A5623, or DNA encoding the siRNA. When the nucleic acids are used for siRNA or coding DNA thereof, the sense strand is preferably longer than about 19 nucleotides, and more preferably longer than 21 nucleotides.

The invention is based in part on the discovery that the gene encoding A2254 or A5623 is over-expressed in breast cancer (BRC) compared to non-cancerous breast tissue. The cDNA of A2254V1 and A2254V2 are 2886 and 2401 nucleotides in length respectively. The cDNA of A5623V1, A5623V2 and A5623V3 are 3128, 3091 and 3011 nucleotides in length respectively. The nucleic acid and polypeptide sequences of A2254V1 and A2254V2 are shown in SEQ ID NO: 35 and 36, and SEQ ID NO: 37 and 38, respectively. The nucleic acid and polypeptide sequences of A5623V1, A5623V2 and A5623V3 are shown in SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42 and SEQ ID NO: 43 and 44, respectively. The sequence data are also available via following accession numbers. A2254: AY026505

A5623: NM_003981, NMJ 99413, NMJ 99414

Methods of inhibiting cell growth The present invention relates to inhibiting cell growth, i.e., cancer cell growth by inhibiting expression of A2254 or A5623. Alternatively, the present invention relates to inhibiting cell growth, i.e. cancer cell growth, in particular breast cancer growth, by inhibiting the binding between an A5623 polypeptide and an A2254 polypeptide since it was found that both polypeptides can interact and their interaction is believed to play a crucial role carcinogenesis, in particular in breast cancer carcinogenesis. Accordingly, the present invention relates to means and methods for treating or preventing breast cancer in a subject by inhibiting the binding of an A5623 polypeptide and an A2254 polypeptide. Furthermore, the present invention envisages an inhibitor of the binding between an A5623 polypeptide and an A2254 polypeptide for use as a medicament for treating or preventing cancer, in particular for treating or preventing breast cancer.

Expression of A2254 or A5623 is inhibited, for example, by small interfering RNA (siRNA) that specifically target the A2254 or A5623 gene. A2254 or A5623 targets include, for example, nucleotides of SEQ ID NOs: 21, 25, 29, and 33. In non-mammalian cells, double-stranded RNA (dsRNA) has been shown to exert a strong and specific silencing effect on gene expression, which is referred as RNA interference (RNAi) (Sharp PA. Genes Dev. 1999;13(2):139-41.). dsRNA is processed into 20-23 nucleotides dsRNA called small interfering RNA (siRNA) by an enzyme containing RNase III motif. The siRNA specifically targets complementary mRNA with a multicomponent nuclease complex (Hammond SM et ah, Nature. 2000; 404(6775):293- 6; Hannon GJ. Nature. 2002;418(6894):244-51.). In mammalian cells, siRNA composed of 20 or 21-mer dsRNA with 19 complementary nucleotides and 3' terminal noncomplementary dimmers of thymidine or uridine, have been shown to have a gene specific knock-down effect without inducing global changes in gene expression (Elbashir SM et ah, Nature. 2001 ;411(6836):494-8.). In addition, plasmids containing small nuclear RNA (snRNA) U6 or polymerase III Hl-RNA promoter effectively produce such short RNA recruiting type III class of RNA polymerase III and thus can constitutively suppress its target mRNA (Miyagishi M. et ah, Nat Biotechnol. 2002;20(5):497-500.; Brummelkamp TR, et al, Science. 296(5567):550-3, 2002.).

The growth of cells is inhibited by contacting a cell, with a composition containing an siRNA of A2254 or A5623. The cell is further contacted with a transfection agent. Suitable transfection agents are known in the art. By inhibition of cell growth is meant the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the composition. Cell growth is measured by methods known in the art such as, the MTT cell proliferation assay.

The siRNA of A2254 or A5623 is directed to a single target of A2254 or A5623 gene sequence. Alternatively, the siRNA is directed to multiple targets of A2254 or A5623 gene sequences. For example, the composition contains siRNA of A2254 or A5623 directed to two, three, four, or five or more target sequences of A2254 or A5623. By A2254 or A5623 target sequence is meant a nucleotide sequence that is identical to a portion of the A2254 or A5623 gene. The target sequence can include the 5' untranslated (UT) region, the open reading frame (ORF) or the 3' untranslated region of the human A2254 or A5623 gene. Alternatively, the siRNA is a nucleic acid sequence complementary to an upstream or downstream modulator of A2254 or A5623 gene expression. Examples of upstream and downstream modulators include a transcription factor that binds the A2254 or A5623 gene promoter, a kinase or phosphatase that interacts with the A2254 or A5623 polypeptide, an A2254 or A5623 promoter or enhancer. siRNA of A2254 or A5623 which hybridize to target mRNA decrease or inhibit production of the A2254 or A5623 polypeptide product encoded by the A2254 or A5623 gene by associating with the normally single-stranded mRNA transcript, thereby interfering with translation and thus, expression of the protein. Thus, siRNA molecules of the invention can be defined by their ability to hybridize specifically to mRNA or cDNA from an A2254 or A5623 gene under stringent conditions. For the purposes of this invention the terms "hybridize" or "hybridize specifically" are used to refer the ability of two nucleic acid molecules to hybridize under "stringent hybridization conditions". The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-1O⁰C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42⁰C, or, 5x SSC, 1% SDS, incubating at 65⁰C, with wash in 0.2x SSC, and 0.1% SDS at 5O⁰C.

The siRNA of the invention is less than about 500, about 200, about 100, about 50, or about 25 nucleotides in length. Preferably the siRNA is about 19 to about 25 nucleotides in length. Exemplary nucleic acid sequence for the production of A2254 or A5623 siRNA include the sequences of nucleotides of SEQ ID NOs: 21 or 25, or 29 or 33 as the target sequence, respectively. Furthermore, in order to enhance the inhibition activity of the siRNA, nucleotide "u" can be added to 3 'end of the antisense strand of the target sequence. The number of "u"s to be added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. The added "u"s form single strand at the 3 'end of the antisense strand of the siRNA.

The cell is any cell that expresses or over-expresses A2254 or A5623. The cell is an epithelial cell such as a breast ductal cell. Alternatively, the cell is a tumor cell such as a carcinoma, adenocarcinoma, blastoma, leukemia, myeloma, or sarcoma. The cell is a breast cancer. An siRNA of A2254 or A5623 is directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. Alternatively, the DNA encoding the siRNA of A2254 or A5623 is in a vector.

Vectors are produced for example by cloning an A2254 or A5623 target sequence into an expression vector operatively-linked regulatory sequences flanking the A2254 or A5623 sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee, N.S., et al, (2002) Nature Biotechnology 20: 500-5). An RNA molecule that is antisense to A2254 or A5623 mRNA is transcribed by a first promoter {e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the A2254 or A5623 mRNA is transcribed by a second promoter {e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands hybridize in vivo to generate siRNA constructs -for silencing of the A2254 or A5623 gene. Alternatively, two constructs are utilized to create the sense and anti-sense strands of a siRNA construct. Cloned A2254 or A5623 can encode a construct having secondary structure, e.g., hairpins, wherein a single transcript has both the sense and complementary antisense sequences from the target gene.

A loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Thus, the present invention also provides siRNA having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to an mRNA or a cDNA from A2254 or A5623. In preferred embodiments, [A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of nucleotides of SEQ ID NOs: 21, 25, 29 and 33, [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and

[A'] is a ribonucleotide sequence consisting of the complementary sequence of [A] The region [A] hybridizes to [A'], and then a loop consisting of region [B] is formed. The loop sequence may be preferably about 3 to about 23 nucleotides in length. The loop sequence, for example, can be selected from group consisting of following sequences (http://www.ambion.com/techlib/tb/tb_506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.M. et al, (2002) Nature 418: 435-8.).

CCC, CCACC or CCACACC: Jacque, J.M. et al, (2002) Nature, 418: 435-8. UUCG: Lee, N. S. et al, (2002) Nature Biotechnology 20 : 500-5; Fruscoloni, P. et al, (2003) Proc. Natl. Acad. Sci. USA 100(4): 1639-44. UUCAAGAGA: Dykxhoorn, D. M. et al, (2003) Nature Reviews Molecular Cell

Biology 4: 457-67.

For example, preferable siRNAs having hairpin loop structure of the present invention are shown below. In the following structure, the loop sequence can be selected from group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. Preferable loop sequence is UUCAAGAGA ("ttcaagaga" in DNA).

GAGAAGAAGGCCCAGAACU-[B]-AGUUCUGGGCCUUCUUCUC (for target sequence of SEQ JD NO:21)

CUCUAGGACUUGCAUGAUU-[B]-AAUCAUGCAAGUCCUAGAG (for target sequence of SEQ ID NO:25) GGAAAGACUCAUCAAAAGC-[B]-GCUUUUGAUGAGUCUUUCC (for target sequence of SEQ JD NO:29) GCAUAUCCGUCUGUCAGAA-[B]-UUCUGACAGACGGAUAUGC (for target sequence of SEQ ID NO:33)

The regulatory sequences flanking the A2254 or A5623 sequence are identical or are different, such that their expression can be modulated independently, or in a temporal or spatial manner. siRNAs are transcribed intracellularly by cloning the A2254 or A5623 gene templates into a vector containing, e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human Hl RNA promoter. For introducing the vector into the cell, transfection-enhancing agent can be used. FuGENE (Roche Diagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent.

Oligonucleotides and oligonucleotides complementary to various portions of A2254 or A5623 mRNA were tested in vitro for their ability to decrease production of A2254 or A5623 in tumor cells {e.g., using the breast cell line such as breast cancer (BRC) cell line) according to standard methods. A reduction in A2254 or A5623 gene product in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition is detected using specific antibodies of A2254 or A5623 or other detection strategies. Sequences which decrease production of A2254 or A5623 in in vitro cell-based or cell-free assays are then tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assay are test in vivo in rats or mice to confirm decreased A2254 or A5623 production and decreased tumor cell growth in animals with malignant neoplasms.

Methods of treating malignant tumors

Patients with tumors characterized as over-expressing A2254 or A5623 are treated by administering siRNA of A2254 or A5623. siRNA therapy is used to inhibit expression of A2254 or A5623 in patients suffering from or at risk of developing, for example, breast cancer (BRC). Such patients are identified by standard methods of the particular tumor type.

Breast cancer (BRC) is diagnosed for example, by CT, MRI, ERCP, MRCP, computer tomography, or ultrasound. Treatment is efficacious if the treatment leads to clinical benefit such as, a reduction in expression of A2254 or A5623, or a decrease in size, prevalence, or metastatic potential of the tumor in the subject. When treatment is applied prophylactically,

"efficacious" means that the treatment retards or prevents tumors from forming or prevents or alleviates a symptom of clinical symptom of the tumor. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type. siRNA therapy is carried out by administering to a patient a siRNA by standard vectors encoding the siRNAs of the invention and/or gene delivery systems such as by delivering the synthetic siRNA molecules. Typically, synthetic siRNA molecules are chemically stabilized to prevent nuclease degradation in vivo. Methods for preparing chemically stabilized RNA molecules are well known in the art. Typically, such molecules comprise modified backbones and nucleotides to prevent the action of ribonucleases. Other modifications are also possible, for example, cholesterol-conjugated siRNAs have shown improved pharmacological properties (Song et al. Nature Med. 9:347-51 (2003)), Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, or viral vectors such as herpes viruses, retroviruses, adenoviruses and adeno-associated viruses, among others. A therapeutic nucleic acid composition is formulated in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal, e.g., physiological saline. A therapeutically effective amount of a compound is an amount which is capable of producing a medically desirable result such as reduced production of an A2254 or A5623 gene product, reduction of cell growth, e.g., proliferation, or a reduction in tumor growth in a treated animal. Parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes, may be used to deliver siRNA compositions of A2254 or A5623. For treatment of breast tumors, direct infusion the celiac artery, splenic artery, or common hepatic artery, is useful.

Dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular nucleic acid to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosage for intravenous administration of nucleic acids is from approximately 10⁶ to 10²² copies of the nucleic acid molecule.

The polynucleotides are administered by standard methods, such as by injection into the interstitial space of tissues such as muscles or skin, introduction into the circulation or into body cavities or by inhalation or insufflation. Polynucleotides are injected or otherwise delivered to the animal with a pharmaceutically acceptable liquid carrier, e.g., a liquid carrier, which is aqueous or partly aqueous. The polynucleotides are associated with a liposome (e.g., a cationic or anionic liposome). The polynucleotide includes genetic information necessary for expression by a target cell, such as promoters.

Alternatively, patients with tumors, in particular breast tumors can be treated by administering a compound that inhibits the binding between an A5623 polypeptide and an A2254 polypeptide. Without being bound by theory, it is believed that an inhibitor of the interaction between an A5623 polypeptide and an A2254 polypeptide alters, interacts, modulates, interferes or abolishes the binding of an A5623 polypeptide to an A2254 polypeptide or the binding of an A2254 polypeptide to an A5623 polypeptide, respectively. By that, both proteins are believed to be no longer capable of interacting in the way they do in cancer cells. Accordingly, it is further believed that the crucial role of both proteins in carcinogenesis, in particular in breast cancer carcinogenesis, as is suggested by the observations made by the persent inventors, can be intercepted and, thus, prevention or treatment of cancer, in particular breast cancer may be achieved.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Producing and identifying compounds that reduce or inhibit the binding between A5623 and A2254

In view of the evidence provided in the examples, one aspect of the invention involves identifying test compounds that reduce or prevent the binding between A5623 and A2254.

Methods for determining A5623/A2254 binding include any methods for determining the interaction of two proteins. Further methods are described hereinbelow. Such assays include, but are not limited to, traditional approaches, such as, cross-linking, co- immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature 340:245-6 (1989); Chien et ah, Proc. Natl. Acad. Sd. USA 88, 9578-82 (1991)) and as disclosed by Chevray and Nathans (Proc. Natl. Acad. ScL USA 89:5789-93 (1992)). Many transcriptional activators, such as yeast GALA, consist of two physically discrete modular domains, one acting as the DNA- binding domain, while the other one functioning as the transcription activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-lacZ reporter gene under control of a GALA-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. While the application refers to "A5623 " or "A2254," it is understood that where the interaction of the two is analyzed or manipulated, it is possible to use the binding portions of one or both of the proteins in place of the full-length copies of the proteins. Fragments of A5623 that bind to A2254 may be readily identified using standard deletion analysis and/or mutagenesis of A5623 to identify fragments that bind to A2254. Similar analysis may be used to identify A5623-binding fragments of A2254.

As disclosed herein, any test compounds, including, e.g., proteins (including antibodies), muteins, polynucleotides, nucleic acid aptamers, and peptide and nonpeptide small organic molecules, may serve as the test compounds of the present invention. Test compounds may be isolated from natural sources, prepared synthetically or recombinantly, or any combination of the same.

For example, peptides may be produced synthetically, using solid phase techniques as described in "Solid Phase Peptide Synthesis" by G. Barany and R. B. Merrifield in Peptides, Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York, N. Y., pp. 100-118 (1980). Similarly, nucleic acids can also be synthesized using the solid phase techniques, as described in Beaucage, S.L., & Iyer, R.P. (1992) Tetrahedron, 48, 2223-311; and Matthes et al, EMBO J., 3:801-5 (1984).

Where inhibitory peptides are identified, modifications of peptides of the present invention, with various amino acid mimetics or unnatural amino acids, are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al, Eur. J. DrugMetab PharmacoMnet. 11 :291-302 (1986). Other useful peptide modifications known in the art include glycosylation and acetylation.

Both recombinant and chemical synthesis techniques may be used to produce test compounds of the present invention. For example, a nucleic acid of test compound may be produced by insertion into an appropriate vector, which may be expressed when transfected into a competent cell. Alternatively, nucleic acids may be amplified using PCR techniques or expression in suitable hosts {see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA).

Peptides and proteins may also be expressed using recombinant techniques well known in the art, e.g., by transforming suitable host cells with recombinant DNA constructs as described in Morrison, J. Bact, 132:349-51 (1977); and Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wu et al, eds, 1983).

Anti-A5623 and anti-A2254 antibodies

In some aspects of the present invention, test compounds are anti-A5623 or anti- A2254 antibodies. In some embodiments, the antibodies are chimeric, including but not limited to, humanized antibodies. In some cases, antibody embodiments of the present invention will bind either A5623 or A2254 at the interface where one of these proteins associates with the other. In some embodiments, these antibodies bind A5623 or A2254 with a K_a of at least about 10⁵ mol^'1, 10⁶ mol^'1 or greater, 10⁷ mol^"1 or greater, 10⁸ mol^"1 or greater, or 10⁹ mol^"1 or greater under physiological conditions. Such antibodies can be purchased from a commercial source, for example, Chemicon, Inc. (Temecula Calif), or can be raised using as an immunogen, such as a substantially purified A5623 or A2254 protein, e.g., a human protein, or a fragment thereof. Methods of preparing both monoclonal and polyclonal antibodies from provided immunogens are well-known in the art. For purification techniques and methods for identifying antibodies to specific immunogens, see e.g., PCT/US02/07144 (WO/03/077838), the contents of which are incorporated by reference herein. Methods for purifying antibodies using, for example, antibody affinity matrices to form an affinity column are also well known in the art and available commercially (AntibodyShop, Copenhagen, Denmark). Identification of antibodies capable of disrupting A5623/A2254 association is performed using the same test assays detailed below for test compounds in general.

Converting enzymes

Converting enzymes may act as test compounds of the present invention. In the context of the present invention, converting enzymes are molecular catalysts that perform covalent post-translational modifications to either A5623 or A2254, or both of them. Converting enzymes of the present invention will covalently modify one or more amino acid residues of A5623 and/or A2254 in a manner that causes either an allosteric alteration in the structure of the modified protein, or alters the A5623/A2254 molecular binding site chemistry or structure of the modified protein in a manner that interferes with binding between A5623 and A2254. Interference with binding between the two molecules refers to a decrease in the K_a of binding by at least 25%, 30%, 40%, 50%, 60%, 70% or more relative to the Ka of binding between the proteins measured at 3O⁰C and an ionic strength of 0.1 in the absence of detergents. Exemplary converting enzymes of the invention include kinases, phosphatases^ amidases, acetylases, glycosidase and the like.

Constructing test compound libraries

Although the construction of test compound libraries is well known in the art, the present section provides additional guidance in identifying test compounds and construction libraries of such compounds for screening for effective inhibitors of A5623/A2254 interaction. Further guidance on compound libraries is provided herein below.

Molecular modeling

Construction of test compound libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of the target molecules to be inhibited, i.e., A5623 and A2254. One approach to preliminary screening of test compounds suitable for further evaluation is computer modeling of the interaction between the test compound and its target. In the present invention, modeling the interaction between A5623 and A2254 provides insight into both the details of the interaction itself, and suggests possible strategies for disrupting the interaction, including potential molecular inhibitors of the interaction. Computer modeling technology allows the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user. An example of the molecular modeling system described generally above consists of the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen, et al. Acta Pharmaceutica Fennica 97, 159-166 (1988); Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinlay and Rossmann, Anmi. Rev. Pharmacol. Toxiciol. 29, 111-122 (1989); Perry and Davies, Prog Clin Biol Res.291 : 189-93(1989); Lewis and Dean, Proc. R. Soc. LondB Biol Sci. 236, 125-40 and 141-62 (1989); and, with respect to a model receptor for nucleic acid components, Askew, et al., J. Am. Chem. Soc. I l l, 1082-90 (1989).

Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif, Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al. (1988) J. Med. Chem. 31:722-9; Meng et al. (1992) J. Computer Chem. 13:505-24; Meng et al. (1993) Proteins 17:266-78; Shoichet et al. (1993) Science 259:1445-50.

Once a putative inhibitor of A5623/A2254 interaction has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors, or "test compounds" may be screened using the methods of the present invention to identify test compounds of the library that disrupt A5623/A2254 association. Combinatorial chemical synthesis

Combinatorial libraries of test compounds may be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors of the A5623/A2254 interaction. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries may be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.

Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-93 (1991) and Houghten et al, Nature 354:84-6 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al, Proc. Natl. Acad. Sci. USA 90:6909-13 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568- 70 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-8 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)); nucleic acid libraries (see Ausubel, and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al, Nature Biotechnology, 14(3):309-14 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-2 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1; 6(6):624-31.; isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like). Further guidance on combinatorial chemical synthesis is provided herin below. Phase display

Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott and Smith, Science 249:386-90, 1990; Cwirla, et al, Proc. Natl. Acad. ScL, 87:6378-82, 1990; Devlin et al, Science, 249:404-6, 1990), very large libraries can be constructed (e.g., 10⁶ -10⁸ chemical entities). A second approach uses primarily chemical methods, of which the Gey sen method (Geysen et al. , Molecular Immunology 23 : 709- 15 , 1986; Geysen et al. J. Immunologic Method 102:259-74, 1987; and the method of Fodor et al (Science 251:767-73, 1991) are examples. Furka et al. (14th International Congress of Biochemistry, Volume #5, Abstract FR:013, 1988; Furka, Int. J. Peptide Protein Res. 37:487- 493, 1991), Houghten (U.S. Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N. J., Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

Screening test compound libraries Screening methods of the present invention provide efficient and rapid identification of test compounds that have a high probability of interfering with A5623/A2254 association. Generally, any method that determines the ability of a test compound to interfere with A5623/A2254 association is suitable for use with the present invention. For example, competitive and non-competitive inhibition assays in an ELISA format may be utilized. Control experiments should be performed to determine maximal binding capacity of system (e.g., contacting bound A5623 with A2254 and determining the amount of A2254 that binds to A5623 in the examples below). Further guidance on screening test compound libraries is provided herein below.

Competitive assay format Competitive assays may be used for screening test compounds of the present invention.

By way of example, a competitive ELISA format may include A5623 (or A2254) bound to a solid support. The bound A5623 (or A2254) would be incubated with A2254 (or A5623) and a test compound. After sufficient time to allow the test compound and/or A2254 (or A5623) to bind A5623 (or A2254), the substrate would be washed to remove unbound material. The amount of A2254 bound to A5623 is then determined. This may be accomplished in any of a variety of ways known in the art, for example, by using an A2254 (or A5623) species tagged with a detectable label, or by contacting the washed substrate with a labeled anti-A2254 (or A5623) antibody. The amount of A2254 (or A5623) bound to A5623 (or A2254) will be inversely proportional to the ability of the test compound to interfere with the A2254/A5623 association. Protein, including but not limited to, antibody, labeling is described in Harlow & Lane, Antibodies, A Laboratory Manual (1988). In a variation, A5623 (or A2254) is labeled with an affinity tag. Labeled A5623 (or

A2254) is then incubated with a test compound and A2254 (or A5623), then immunoprecipitated. The immunoprecipitate is then subjected to Western blotting using an anti-A2254 (or A5623) antibody. As with the previous competitive assay format, the amount of A2254 (or A5623) found associated with A5623 (or A2254) is inversely proportional to the ability of the test compound to interfere with the A5623/A2254 association.

Non-competitive assay format

Non-competitive binding assays may also find utility as an initial screen for testing compound libraries constructed in a format that is not readily amenable to screening using competitive assays, such as those described herein. An example of such a library is a phage display library (See, e.g., Barret, et al (1992) Anal. Biochem 204, 357-364).

Phage libraries find utility in being able to produce quickly working quantities of large numbers of different recombinant peptides. Phage libraries do not lend themselves to competitive assays of the invention, but can be efficiently screened in a non-competitive format to determine which recombinant peptide test compounds bind A5623 or A2254. Test compounds identified as binding can then be produced and screened using a competitive assay format. Production and screening of phage and cell display libraries is well-known in the art and discussed in, for example, Ladner et al, WO 88/06630; Fuchs et al. (1991) Biotechnology 9: 1369-72; Goward et al. (1993) TIBS 18:136-40; Charbit et al. (1986) EMBO J 5, 3029-37; Cull et al. (1992) PNAS USA 89:1865-9; Cwirla, et al (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6378-82.

An exemplary non-competitive assay would follow an analogous procedure to the one described for the competitive assay, without the addition of one of the components (A5623 or A2254). However, as non-competitive formats determine test compound binding to A5623 or A2254, the ability of test compound to bind both A5623 and A2254 needs to be determined for each candidate. Thus, by way of example, binding of the test compound to immobilized A5623 may be determined by washing away unbound test compound; eluting bound test compound from the support, followed by analysis of the eluate; e.g., by mass spectroscopy, protein determination (Bradford or Lowry assay, or Abs. at 280nm determination.). Alternatively, the elution step may be eliminated and binding of test compound determined by monitoring changes in the spectroscopic properties of the organic layer at the support surface. Methods for monitoring spectroscopic properties of surfaces include, but are not limited to, absorbance, reflectance, transmittance, birefringence, refractive index, diffraction, surface plasmon resonance, ellipsometry, resonant mirror techniques, grating coupled waveguide techniques and multipolar resonance spectroscopy, all of which are known to those of skill in the art. A labeled test compound may also be used in the assay to eliminate need for an elution step. In this instance, the amount of label associated with the support after washing away unbound material is directly proportional to test compound binding.

A number of well-known robotic systems have been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N. J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

Screening converting enzymes

Test compounds that are converting enzymes may be assayed in a noncompetitive format, using co-factors and auxiliary substrates specific for the converting enzyme being assayed. Such co-factors and auxiliary substrates are known to one of skill in the art, given the type of converting enzyme to be investigated. One exemplary screening procedure for converting enzymes involves first contacting A5623 and/or A2254 with the converting enzyme in the presence of co-factors and auxiliary substrates necessary to perform covalent modification of the protein characteristic of the converting enzyme, preferably under physiologic conditions. The modified protein(s) is then tested for its ability to bind to its binding partner {i.e., binding of A5623 to A2254). Binding of the modified protein to its binding partner is then compared to binding of unmodified control pairs to determine if the requisite change in K₃ noted above has been achieved.

To facilitate detection of proteins in performing the assay, one or more proteins may be labeled with a detectable label as described above, using techniques well known to those of skill in the art.

Methods for Screens

The screening embodiments described above are suitable for high through-put determination of test compounds suitable for further investigation. In particular, the screening of the present invention preferably comprises the following detection steps: (a) contacting a polypeptide comprising an A2254-binding domain of an A5623 polypeptide with a polypeptide comprising an A5623-binding domain of an A2254 polypeptide in the presence of a test compound;

(b) detecting binding between the polypeptides; and

(c) selecting a test compound that inhibits binding between the polypeptides. Alternatively, the test compound under investigation may be added to proliferating cells and proliferation of the treated cells monitored relative to proliferation of a control population not supplemented with the test compound. Cell lines suitable for screening test compounds will be obvious to one of skill in the art provided with the teachings presented herein. For in vivo testing, the test compound may be administered to an accepted animal model. The introduction of the gene into animal cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu, et ah, Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, MoI Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et a/., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, MoI Cell Biol 4: 1641-3 (1984)), the

Lipofectin method (Derijard B, et a\. Cell 7: 1025-37 (1994); Lamb et al, Nature Genetics 5: 22-30 (1993): Rabindran et a!., Science 259: 230-4 (1993)), and so on. The genes can express protein fused with tag (e.g. HA or Myc).

The subcellular localization of A5623 and A2254 is investigated by immunohistochemical staining. Cells are transfected with tagged-A5623, tagged-A2254, or their combination. Image of the localization can be obtained using microscope such as fluorescence microscope.

In the preferred embodiment, cell expressing both of A2254 and A5623 may be obtained by transfection with expression vector of A2254 and A5623 gene into suitable cell line. For example, HEK293, SW480 or COS7 cells may be used as the cell line. Furthermore, the detection reagent is preferably an antibody which recognizes A2254. Alternatively, when the A2254 or A5623 is expressed as fused protein with tag, an antibody recognizing the tag fused with the protein may also be used as the detection reagent. In the kit of the present invention, the antibody may be labeled with fluorescence agent (e.g., FITC, TAMRA, or GFP). In particular, as mentioned herein, the present inventors have observed that an A5623 polypeptide and an A2254 polypeptide interact with each other. Accordingly, it is believed that the interaction of both polypeptides plays a crucial role in carcinogenesis, in particular breast cancer carcinogenesis. Hence, it is intended to screen for a compound useful in treating or preventing cancer, in particular breast cancer, that inhibits an interaction between an A5623 polypeptide and an A2254 polypeptide or a vice versa interaction. Namely, a polypeptide comprising an A2254-binding domain of an A5623 polypeptide is contacted with a polypeptide comprising an A5623-binding domain of an A2254 polypeptide in the presence of a test compound, the binding of the two polypeptides is detected and, a test compound is selected which inhibits binding between the two polypeptides. Of course, in the alternative, it is also possible to contact a polypeptide comprising an A5623-binding domain of an A2254 polypeptide with a polypeptide comprising an A2254-binding domain of an A5623 polypeptide in the presence of a test compound.

Preferably, the polypeptide comprising the A2254-binding domain comprises an A5623 polypeptide and the polypeptide comprising the A5623-binding domain comprises an A2254 polypeptide.

The term "contacting" encompasses that a polypeptide comprising an A2254- binding domain of an A5623 polypeptide is contacted with a polypeptide comprising an A5623-binding domain of an A2254 polypeptide or that a polypeptide comprising an A5623- binding domain of an A2254 polypeptide is contacted with a polypeptide comprising an A2254-binding domain of an A5623 polypeptide in the presence of a test compound by any known means and methods in the art with a test compound. For example, a cell-based assay or a cell-free assay may be applied for screening an inhibitor of the binding between an A5623 polypeptide and an A2254 polypeptide. In an example of a cell-based assay, cells expressing a polypeptide comprising an A5623-binding domain and a polypeptide comprising an A2254-binding domain may be used for screening. In an example of a cell-free assay, a partially or completely purified polypeptide comprising an A5623-binding domain anda partially or completely purified polypeptide comprising an A2254-binding domain may be used. Of course, it is also possible to use cell extracts of cells expressing a polypeptide comprising an A5623-binding domain and a polypeptide comprising an A2254-binding domain.

Further examples of test assays or methods are described herein above. The term "test compound" or "compound to be tested" refers to a molecule or substance or compound or composition or agent or any combination thereof to be tested by one or more screening method(s) of the invention as a putative inhibitor of the interaction between A5623 and A2254. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof or any of the compounds, compositions or agents described herein. It is to be understood that the term "test compound" when used in the context of the present invention is interchangeable with the terms "test molecule", "test substance", "potential candidate", "candidate" or the terms mentioned hereinabove.

Accordingly, small peptides or peptide-like molecules are envisaged to be used in the screening methods for inhibitor(s) of the interaction. Such small peptides or peptide-like molecules bind to and occupy the interaction domain of either an A5623-binding domain or an A2254-binding domain or of both, thereby making the A5623- or A2254-binding domain inaccessible to the A2254 polypeptide or A5623 polypeptide, respectively. Moreover, any biological or chemical composition(s) or substance(s) may be envisaged as interaction inhibitor. The inhibitory function of the inhibitor can be measured by methods known in the art. Such methods comprise interaction assays, like immunoprecipitation assays, ELISAs, RIAs. Also preferred potential candidate molecules or candidate mixtures of molecules to be used when screening for an inhibitor of the binding between A5623 and A2254 or a vice versa binding, may be, inter alia, substances, compounds or compositions which are of chemical or biological origin, which are naturally occurring and/or which are synthetically, recombinantly and/or chemically produced. Thus, candidate molecules may be proteins, protein-fragments, peptides, amino acids and/or derivatives thereof or other compounds, such as ions, which bind to and/or interact with an A5623 and/or A2254 polypeptidel. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.

In addition, the generation of chemical libraries is well known in the art. For example, combinatorial chemistry is used to generate a library of compounds to be screened in the assays described herein. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in every possible combination to yield peptides of a given length. Millions of chemical compounds can theoretically be synthesized through such combinatorial mixings of chemical building blocks. For example, one commentator observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. (Gallop A, et al. Journal of Medicinal Chemistry, 37(9) 1233-51 (1994)). Other chemical libraries known to those in the art may also be used, including natural product libraries. Once generated, combinatorial libraries are screened for compounds that possess desirable biological properties.

In the context of the present invention, libraries of compounds are screened to identify compounds that function as inhibitors of the binding between an A5623 and A2254 polypeptide. For example, first, a library of small molecules is generated using methods of combinatorial library formation well known in the art. U. S. Patent NOs. 5,463,564 and 5,574,656 are two such teachings. Then the library compounds are screened to identify those compounds that possess desired structural and functional properties. U. S. Patent No. 5,684,711, discusses a method for screening libraries. To illustrate the screening process, a polypeptide comprising an A2254-binding domain and a polypeptide comprising an A5623- binding domain and chemical compounds of the library are combined and permitted to interact with one another. Further, it is then observed whether the chemical compounds inhibit binding of the two polypeptides. By way of example, FRET technology can be applied insofar as interaction of both polypeptides generates a fluorescent signal which is diminished and/or abolished if a test compound inhibits binding between the polypeptides. Accordingly, both polypeptides may comprise labels suitable for FRET. Such labels are commonly known in the art.

The characteristics of each library compound are encoded so that compounds demonstrating activity in the inhibition of the binding of the polypeptides can be analyzed and features common to the various compounds identified can be isolated and combined into future iterations of libraries. Once a library of compounds is screened, subsequent libraries are generated using those chemical building blocks that possess the features shown in the first round of screen to have activity against the interaction. Using this method, subsequent iterations of candidate compounds will possess more and more of those structural and functional features required for inhibiting the interaction, until a group of inhibitors with high specificity for the inhibition of the binding between the two polypeptides can be found. These compounds can then be further tested for their safety and efficacy as medicaments for use in animals, such as mammals. It will be readily appreciated that this particular screening methodology is exemplary only. Other methods are well known to those skilled in the art.

For example, candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons, preferably less than about 750, more preferably less than about 350 daltons.

Candidate agents may also comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Exemplary classes of candidate agents may include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by functionalizing the amino or carboxylic terminus, e.g. for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidifϊcation, or the like. Other methods of stabilization may include encapsulation, for example, in liposomes, etc. As mentioned above, candidate agents are also found among biomolecules including peptides, amino acids, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Other test compounds may be aptamers, antibodies, affybodies, trinectins, anticalins, or the like compounds. Of course, an aptamers, antibody, affybody, trinectin, or anticalin which is specific for A5623 or A2254 may already he. per se an inhibitor of the binding between A5623 and A2254 and could thus be readily employed in a method for treating or preventing cancer, in particular breast cancer.

A compound identified by the methods for screening an inhibitor of the binding between an A5623 polypeptide and an A2254 polypeptide as described herein may be useful for preventing or treating cancer, in particular breast cancer. Accordingly, an inhibitor has the property of dierectly or indirectly interacting with the A2254-binding domain and/or A5623- binding domain, thereby, as described herein, influencing the interaction of these domains such that they can no longer interact in the way they would do in cancer cells, in particular in breast cancer cells. For example, an inhibitor could be an antibody against the A2256-binding domain or the A5623-binding domain. Another example for such an inhibitor could be an affybody, aptamer, anticalin or trinectin.

Screening and Treatment Kits In one embodiment, the present invention provides an article of manufacture or kit for screening for a compound useful in treating or preventing breast cancer, wherein the kit comprises: (a) an A2254-bind_.ing domain of an A5623 polypeptide; (b) an A5623 -binding domain of an A2254 polypeptide, and (c) a reagent that detects the interaction between the two polypeptides. As discussed above, the polypeptide comprising the A2254-binding domain may comprise a full length A5623 polypeptide or an A2254-binding portion thereof. Likewise, the polypeptide comprising the A5623-binding domain may comprise a full-length A2254 polypeptide or an A5623-binding portion thereof.

In a further embodiment of the invention, articles of manufacture and kits containing materials useful for treating the pathological conditions described herein are provided. The article of manufacture may comprise a container of a medicament as described herein with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. In the context of the present invention, the container holds a composition having an active agent which is effective for treating a cell proliferative disease, for example, breast cancer. In one embodiment, the active agent in the composition is an identified test compound {e.g., antibody, small molecule, etc.) capable of disrupting A5623/A2254 association in vivo. The label on the container should indicate that the composition is used for treating one or more conditions characterized by abnormal cell proliferation. The label may also indicate directions for administration and monitoring techniques, such as those described herein. In addition to the container described above, a kit of the invention may optionally comprise a second container housing a pharmaceutically-acceptable diluent. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

Brief Description of the Drawings Figure 1 shows the results of semi-quantitative RT-PCR for expression of (A) A2254 and (B) A5623 in tumor cells from breast cancer patients (upper panel) (3 T, 3 IT, 149T, 175T, 43 IT, 453T, 491T, 554T, 571T, 709T, 772T and 781T), breast cancer cell lines (HBC4, HBC5, HBLlOO, HCC1937, MCF7, MDA-MB-231, SKBR3, T47D, YMBl) (lower panel), and normal human tissues. Figure 2 shows photographs of Northern blot analyses of the (A) A2254 and (B)

A5623 transcripts in various human tissues (upper panel), and breast cancer cell lines and normal human vital organs (bottom panel).

Figure 3 shows genomic structure of (A) A2254 and (B) A5623. A2254 has two different variants, designed Vl and V2, and A5623 also has three different variants (Vl, V2 and V3). Upper panel; genomic structure, lower panel; each variant-specific semiquantitative RT-PCR results.

Figure 4A shows exogenous expression of A2254 protein by Western blot analysis. Figure 4B shows subcellular localization of A2254 protein. Figure 4C shows exogenous expression of A5623V1, A5623V2 and A5623V3 proteins by Western blot analysis. Figure 4D-F shows subcellular localization of (D) A5623V1, (E) A5623 V2 and (F) A5623 V3 proteins.

Figure 5 shows growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce expression of A2254 in breast cancer cells. Figure 5 A shows semiquantitative RT-PCR showing suppression of endogenous expression of A2254 in breast cancer cell lines, T47D (left panel) and HBC5 (right panel). GAPDH was used as an internal control. Figure 5B shows MTT assay demonstrating a decrease in the numbers of colonies by knockdown of A2254 in T47D (left panel) and HB C5 (right panel) cells. Figure 5 C shows colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of A2254 in T47D (left panel) and HBC5 (right panel) cells. Figure 6 shows growth-inhibitory effects of small-interfering RNAs (siRNAs) designed to reduce expression of A5623 in breast cancer cells. Figure 6A shows semi- quantitative RT-PCR showing suppression of endogenous expression of A5623 in breast cancer cell lines, T47D (left panel) and HBC5 (right panel) cells. GAPDH was used as an internal control. Figure 6B shows MTT assay demonstrating a decrease in the numbers of colonies by knockdown of A5623 in T47D (left panel) and HBC5 (right panel) cells. Figure 6C shows colony-formation assay demonstrating a decrease in the numbers of colonies by knockdown of A5623 in T47D (left panel) and BDBC5 (right panel) cells.

Figure 7. Expression of A5623 and A2254 in breast cancer cell-lines and tissue sections. Figure 7 A, Expression of endogenous A5623 and A2254 protein in breast cancer cell-lines in comparison with HMEC cell-line, examined by Western-blot analysis using anti- A5623 antibody or anti-A2254 antibody. Figure 7B, Subcellular localization of endogenous A5623 protein or A2254 protein in breast cancer cells during cell cycle. HBC4, HBC5, and MCF-7 cells for A5623 and HBC5 cells for A2254 were immunocytochemically stained using affinity-purified anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody (green) and DAPI (blue) to discriminate nucleus {see the Materials and Methods). White arrows indicate localization of A5623 in midbody of telophase cells. Figure 7C, immunohistochemical staining results of breast cancer and normal tissue sections (normal breast tissue, lung, heart, liver, kidney and testis). Endogenous A5623 protein was stained by use of anti-A5623 antibody. The expression was hardly detected from normal breast tissues (sample No. 10441), but cancer cells were intensely stained in all of cancer tissues investigated including sold-tubular (sample No. 234), papillotubular (sample No. 240) and scirrhous (sample No. 179), carcinomas. Representative figures were from microscopic observation with original magnification, x 200.

Figure 8 Interaction between A5623 and A2254. Figure 8A, Expression of A5623 and A2254 in breast cancer cell lines (HBC4, HBC5, HBLlOO, HCC1937, MCF7, MDA-MB- 231, SKBR3, T47D, YMBl), and normal human tissues (N; normal breast ductal cells, MG; mammary gland, LUN; lung, LIV; liver, HEA; heart, KID; kidney and BM; bone marrow) by semi-quantitative RT-PCR. Figure 8B, C, Co-immunoprecipitation of A5623 and A2254. Cell lysates from COS7 cells transfected with HA-tagged A2254 and Flag tagged A5623 proteins were immunoprecipitated with anti-HA or anti-Flag. Immunoprecipitates were immunob lotted using monoclonal anti-HA or anti-Flag antibodies. Figure 8D, Subcellular localization of endogenous A5623 or A2254 in stably expressed cells. Left panel shows endogenous A5623 protein (red) co-localized exogenous A2254 protein (green) in stably A2254-expressed cells, while right panel shows co-localization of endogenous A2254 (red) and exogenous A5623 (green) in stably A5623 cells. Endogenous A2254 protein (red) co- localized exogenous A5623 protein (right panel).

Figure 9 Growth-promoting or invasion effects of exogenous A2254 in NIH3T3 cells. Figure 9A, Western blot analysis of cells expressing exogenous A2254 at high or moderate level or those transfected with mock vector. Exogenous introduction of A2254 expression were validated with anti-HA-tag monoclonal antibody. Beta-actin served as a loading control. Figure 9B, in vitro growth of NIH3T3-A2254 cells. MH3T3 cells transfected with A2254 (NIH3T3-A2254-#1, -#2, -#3, and -#4) and mock (NIH3T3-Mock-#1, -#2, -#3), as measured by MTT assay. Figure 9C, Matrigel invasion assay demonstrating enhancement of invasiveness of Nffl3T3-A2254~#3, and ~#4 and Nffl3T3-Mock-#l. The number of cells migrating through the Matrigel-coated filters was counted. Assays were performed three times in triplicate wells.

Best Mode for Carrying out the Invention The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[General Methods]

Cell lines and clinical materials

Human-breast cancer cell lines HBC4, HBC5, MDA-MB-231 were kindly provided by Dr. Yamori (The Japanese Foundation of Cancer Research, Tokyo), BT-549, MCF-7,

T47D, SKBR3, HCC1937, MDA-MB-435S, YMBl, HBLlOO, and COS7 were obtained from ATCC. All cells were cultured in appropriate media; i.e. RPMI- 1640 (Sigma, St. Louis, MO) for BT-549, HBC4, HBC5, SKBR3, T47D, YMBl, and HCC1937 (with 2mM L-glutamine); Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) for HBLlOO, COS7; EMEM (Sigma) with O.lmM essential amino acid (Roche), ImM sodium pyruvate (Roche), O.Olmg/ml Insulin(Sigma) for MCF-7; L-15 (Roche) for MDA-MB-231 and MDA-MB-435S. Each medium was supplemented with 10% fetal bovine serum (Cansera) and 1% antibiotic/antimycotic solution (Sigma). MDA-MB-231 and MDA-MB-435S cells were maintained at 37⁰C an atmosphere of humidified air without CO₂. Other cell lines were maintained at 37⁰C an atmosphere of humidified air with 5% CO₂. Clinical samples (breast cancer and normal breast duct) were obtained from surgical specimens, concerning which all patients had given informed consent. Isolation of a novel human gene represented by spot A2254 and A5623 on our cDNA microaray

To detect genes that were commonly up-regulated in breast cancer, the overall expression patterns of the 27,648 genes on the microarray were screened to select those with expression ratios >3.0 that were present in >50% of i) all of 77 premenopausal breast cancer cases, ii) 69 invasive ductal carcinomas, iii) 31 well-, iv) 14 moderately-, or v) 24 poorly- differentiated lesions, respectively. Among the total of 493 genes that appeared to up- regulated in tumor cells, we focused on one with in-house identification number, A2254 and A5623 because its expression ratio was greater than 3.0 in more than 50% of the informative breast cancer cases, and showed low expression in normal organs including heart, lung, liver, kidney and bonemarrow through the expression profiles of normal human tissues.

Semi-quantitative RT-PCR analysis

We extracted total RNA from each population of laser-captured cells and then performed T7-based amplification and reverse transcription as described previously (Ono K, et ah, Cancer Res., 60, 5007-11, 2000). We prepared appropriate dilutions of each single- stranded cDNA for subsequent PCR amplification by monitoring the glyceraldehyde-3- phosphate dehydrogenase (GAPDH) as a quantitative internal control. The PCR primer sequences are as follows; 5'-CGACCACTTTGTCAAGCTCA-S' (SEQ ID NO; 1) and 5'-GGTTGAGCACAGGGTACTTTATT-S' (SEQ ID NO; 2) for GAPDH; 5'-AACTTAGAGGTGGGAGCAG-S ' (SEQ ID NO: 45) and 5'-CACAACCATGCCTTACTTTATC-S' (SEQ ID NO: 46) for β '2MG; 5¹-ACTCTAGGACTTGCATGATTGCC-3¹ (SEQ ID NO; 3) and 5¹-TGGGTGTCAAACCAAACAGA-3¹ (SEQ ID NO; 4) for A2254V1, 5'-GTTAGAACTTGTTTCCTCCTCCG-3^• (SEQ ID NO; 5) and

5'-ATCCTCAATGGTATTTCAGC-S' (SEQ ID NO; 6) for A2254V2, 5'-GTGGTCCTAGGAGACTTGGTTTT-S' (SEQ ID NO; 7) and

5'-TACATGCATACCCCCAACAA-S' (SEQ ID NO; 8) for the common region of A5623, S'-GCTTCAGCGAGAACTTTC-S' (SEQ ID NO; 9) and 5¹-CAACTGTAACACTCATTCACATC-3¹ (SEQ ID NO; 10) for A5623V1, 5'-CTATTCTGAGTTTGCGCGAGAAC-S ' (SEQ ID NO; 11) and 5'-CAACTGTAACACTCATTCACATC-S' (SEQ ID NO; 10) for A5623V2, 5^l-CATCCTGAGTGCGAGAACTTTC-3¹ (SEQ ID NO; 12) and 5¹-CAACTGTAACACTCATTCACATC-3¹ (SEQ ID NO; 10) for A5623V3.

Northern-blot analysis

Total RNAs were extracted from all breast cancer cell lines using RNeasy kit (QIAGEN) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated with mRNA purification kit (Amersham Biosciences) following the manufacturer's instructions. A 1-μg aliquot of each mRNA, along with polyA(+) RNAs isolated from normal adult human breast (Biochain), lung, heart, liver, kidney, bone marrow (BD, Clontech, Palo Alto, CA), were separated on 1% denaturing agarose gels and transferred to nylon membranes (Breast cancer-Northern blots). Breast cancer- and Human multiple-tissue Northern blots (Clontech, Palo Alto, CA) were hybridized with an [α³²P]-dCTP-labeled PCR products of A2254 and A5623 prepared by RT-PCR (see below). Pre-hybridization, hybridization and washing were performed according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at - 8O⁰C for 14 days. Specific probes for A2254 (548 bp) and A5623 (454 bp) were prepared by PCR using a primer set as follows; 5^t-ACTCTAGGACTTGCATGATTGCC-3¹ (SEQ TD NO; 3) and

5'-TGGGTGTCAAACCAAACAGA-S ' (SEQ ID NO; 4) for the common region of A2254, 5'- GTGGTCCTAGGAGACTTGGTTTT-S' (SEQ ID NO; 7) and 5'-TACATGCATACCCCCAACAA-3 ' (SEQ ID NO; 8) for the common region of A5623. In addition, specific A2254V1 probe (350 bp) were prepared by PCR using a specific primer set as follows; 5'-CTGGAAGAGC AGGCTAGC AG-3¹ (SEQ ID NO; 13) and 5'-GCTGCGGAGAAAGCTCTATG-3¹ (SEQ ID NO; 14).

Construction of expression vectors For constructing of A5623 and A2254 expression vectors, the entire coding sequence of PRCl cDNA was amplified by the PCR using KOD-Plus DNA polymerase

(Toyobo, Osaka, Japan). Primer sets were

A5623-forward, 5^I-CCGGAATTCTCCGCCATGAGGAGAAGTGA-3^I (SEQ ID NO: 47)

(the underline indicates EcoRI site) and A5623-reverse, 5'-TTGCCGCTCGAGGGACTGGATGTTGGTTGAA-S' (SEQ ID NO: 48)

(the underline indicates Xhol site), A2254-forward, S'-CCGGAATTCATGGCCATGGACTCGTCG-B' (SEQ ID NO: 49) and A2254-reverse, 5'-GCTCCGCICGAGCTGGGGCCGTTTCTT-S' (SEQ ID NO: 50). The PCR products were inserted into the EocRI and Xhol sites of pCAGGS-nHA or pC AGGS- Flag expression vector.

Anti-A5623-specific polyclonal antibodies and anti-A2254-specific polyclonal antibodies.

Plasmids designed to express two fragments of A5623 (179-360 and 234-360 a.a.) or A2254 (86-239 and 124-239 a.a.) with His-tagged epitope at their C-terminus were prepared using pET21 vectors (Novagen, Madison, WI), respectively. The recombinant peptides were expressed in Escherichia coli, BL21 codon-plus strain (Stratagene, La Jolla, CA), respectively, and purified using Ni-NTA resin agarose (Qiagen) according to the supplier's protocols. The purified recombinant proteins were mixed together and then immunized into rabbits. The immune sera were purified on affinity columns according to standard methodology. Affinity- purified anti-A5623 antibodies or anti-A2254 antibodies were used for western blotting, immunoprecipitation, and immunocytostaining as described below. We confirmed that these antibodies could specifically recognize endogenous A5623 proteins or A2254 proteins in MCF7 breast cancer cells by western blot analysis, respectively.

Immvnocytochemical staining

To examine the subcellular localization of endogenous A5623 protein or A2254 protein in breast cancer cell-lines, MCF7, HBC4 and HBC5, we seeded the cells at IxIO⁵ cells per well (Lab-Tek II chamber slide, Nalgen Nunc International, Naperville, DL). 24 hours after incubation, cells were fixed with PBS (-) containing 4% paraformaldehyde for 15 min, and rendered permeable with PBS (-) containing 0.1% Triton X-IOO at 4 oC for 2.5 min. Subsequently, the cells were covered with 3% BSA in PBS (-) at 4 ⁰C for 12 hours to block non-specific hybridization followed by incubation with a rabbit anti-A5623 polyclonal antibody diluted at 1 : 1000 or anti-A2254 polyclonal antibody diluted at 1 : 1000. After washing with PBS (-), the cells were stained by an Alexa488-conjugated anti-rabbit secondary antibody (Molecular Probe, Eugene, OR) diluted at 1: 1000. Nuclei were counter-stained with 4',6'-diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescent images were obtained under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Western blottins analysis We transiently transfected with lμg of ρCAGGS-A2254-HA, pCAGGS-A5623Vl- HA, pCAGGS-A5623V2-HA or pCAGGS-A5623V3-HA into COS7 cells using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, respectively. Cell lysates were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes, then incubated with a mouse anti-HA antibody (Roche) as primary antibody at 1:1000 dilution. After incubation with sheep anti-mouse IgG-HRP as secondary antibody (Amersham Biosciences), signals were visualized with an ECL kit (Amersham Biosciences).

Furthermore, to detect the endogenous A5623 protein or A2254 protein in breast cancer cell lines (BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKB R3, and T47D) and HMEC (human mammary gland epitherial cell), cells were lysed in lysis buffer (5OmM Tris-HCl, pH 8.0/150mM NaCl/0.5% NP-40) including 0.1 % protease inhibitor cocktail III (Calbiochem, San Diego, CA). The amount of total protein was estimated by protein assay kit (Bio-Rad, Hercules, CA), and then proteins were mixed with SDS-sample buffer and boiled before loading at 10% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto nitrocellulose membrane (GE Healthcare). Membranes including proteins were blocked by blocking solution and incubated with anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody for detection of endogenous A5623 protein or A2254 protein. Finally the membrane was incubated with HRP conjugated secondary antibody and protein bands were visualized by ECL detection reagents (GE Healthcare). Beta-actin was examined to serve as a loading control.

Immunocytochemical staining

To examine the sub-cellular localization of A2254, or A5623V1, A5623V2, and A5623V3, we seeded COS7 cells at IxIO⁵ cells per well for all four constructs. After 24 hours, we transiently transfected with lμg of pCAGGS-A2254-HA, pCAGGS-A5623Vl-HA, pCAGGS-A5623V2-HA or pCAGGS-A5623V3-HA into COS7 cells using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, respectively. Then, cells were fixed with PBS containing 4% paraformaldehyde for 15 min, and rendered permeable with PBS containing 0.1% Triton X-100 for 2.5 min at 4⁰C. Subsequently the cells were covered with 3% BSA in PBS for 12 hours at 4⁰C to block non-specific hybridization. Next, each construct-transfected COS7 cells were incubated with a rat anti-HA antibody

(Roche) at 1 : 1000 dilution. After washing with PBS, both transfected-cells were stained by an Alexa594-conjugated anti-rat secondary antibody (Molecular Probe) at 1:1000 dilution. Nuclei were counter-stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI). Fluorescent images were obtained under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Construction of A2254 and A5623 specific-siRNA expression vector using psiU6X3.0

We established a vector-based RNAi system using psiUδBX siRNA expression vector according to the previous report (WO2004/076623). A siRNA expression vector against A2254 (psiU6BX-A2254) and A5623 (psiU6BX-A5623) were prepared by cloning of double- stranded oligonucleotides in Table 1 into the Bbsl site in the psiU6BX3.0 vector. A control plasmid, psiU6BX-Mock, was prepared by cloning double-stranded oligonucleotides of 5 ' -CACCGAAG CAGC ACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-S ' (SEQ ID NO; 15) and

5 '-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-S ' (SEQ ID NO; 16) into the Bbsl site in the psiU6BX3.0 vector.

Oligonucleotide sequences for siRNA

Oligonucleotide sequences used for small interfering RNA of A2254 and A5623 are shown below.

Tablel. Oligonucleotides sequences for small interfering RNA of A2254 and A5623

The underline indicates specific siRNA sequence. Gene-silencing effect ofA2254 andA5623

Human breast cancer cells lines, T47D and HBC5, was plated onto 10-cm dishes (Ix 10⁶ cells/dish) and transfected with psiU6BX-Mock as negative control, psiU6BX-A2254 or psiU6BX-A5623 using FuGENEό regent according to the supplier's recommendations (Roche). Total RNAs were extracted from the cells at 7 days after the transfection of each construct, and then the knockdown effect of siRNAs was confirmed by semi-quantitative RT- PCR using specific primers for common regions of A2254 and A5623 as above mentioned. The primers for GAPDH as internal control is as follows; 5'-CGACCACTTTGTCAAGCTCA-3' (SEQ ID NO; 1) and 5'-GGTTGAGCACAGGGTACTTTATT-S ' (SEQ ID NO; 2). Moreover, transfectants expressing siRNAs using T47D and HBC5 cell lines were grown for 28 days in selective media containing 0.7 mg/ml of neomycin. After fixation with 4% paraformaldehyde, transfected cells were stained with Giemsa solution to assess colony formation. MTT assays were performed to quantify cell viability. After 12 days of culture in the neomycin-containing medium, MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) was added at a concentration of 0.5 mg/ml. Following incubation at 37°C for 2.5 hours, acid-SDS (0.01N HCl/10%SDS) was added; the suspension was mixed vigorously and then incubated overnight at 37°C to dissolve the dark blue crystals. Absorbance at 570nm was measured with a Microplate Reader 550 (BioRad).

Immimoprecipitation and western blotting

Cells were lysed in lysis buffer (50 mM Tris-HCL (pH 8.0), 150 mM NaCL, 0.5% NP-40 and Protease Inhibitor Cocktail Set III (Calbiochem, San Diego, CA)). Equal amounts of total proteins were incubated at 4 ⁰C for 1 hour with 1 mg of a rat anti-HA antibody (Roche) or a mouse anti-Flag antibody (Santa Cruz). Immunocomplexes were incubated with protein G- Sepharose (Zymed Laboratories, South San Francisco, CA) for 1 hour and then washed with lysis buffer. Co-precipitated proteins were separated by SDS-PAGE. Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, and then incubated with a rat anti-HA antibody or a mouse anti-Flag antibody, after incubation with secondary antibody conjugated to HRP; signals were visualized with an ECL kit (Amersham Biosciences).

Establishment ofNIH3T3 cells stably expressing A5623 or A2254. A5623 or A2254 expression vectors or mock vectors were transfected into NIH3T3 cells using FUGENE6 as describe above. Transfected cells were incubated in the culture medium containing 0.9 mg/ml of geneticin (G418) (Invitrogen). Clonal NIH3T3 cells were subcloned by limiting dilution. Expression of HA-tagged A5623 or A2254 were assessed by western blot analysis using anti-HA monoclonal antibody. Eventually, several clones were established and designated as A5623-NIH3T3 or A2254-NIH3T3.

To investigate growth-promoting effect of A2254, we seeded 5000 cells each of four independent A2254-NIH3T3 cells and three independent MOCK-NIH3T3 cells, and counted the number of cells by MTT assay everyday for 6 days. These experiments were done in triplicate.

Furthermore, we investigated invasion through matrigel of A2254-NIH3T3 cells (A2254-MH3T3-3, and -4). In briefly, we seeded 10000 cells each of two independent A2254-NIH3T3 cells (A2254-NIH3T3-3, and -4) and one independent MOCK-NTH3T3 cells, and the cells were grown to the confluent stage in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed in DMEM without the addition of serum or protease inhibitors, and susupended in DMEM at concentrations of IX 10⁵ cells/mL. Before preparing the cell suspensions, the dried layer of Matrigel matrix (Bection Dickinson Labware, Bedford, MA) was rehydrates with DMEM for two hours at room temparature. DMEM containing 10% FBS was added to each lower chamber of 24- well MAtrigel invasion chambers, and 0.5mL (5X10⁴ cells) of cell suspension was added to each insert of the upper chamber. The plates were incubated for 22 hours at 37 ⁰C. After incubation, the chambers were processed and the cells invading through the Matrigel-coated inserts were fixed and stained by Giemsa as directed by supplier (Bection Dickinson Labware).

Immunohistotochemical staining Expression patterns of A5623 protein in breast cancer and normal tissues were investigated using anti-A5623 rabbit polyclonal antibody. Briefly, paraffin-embedded specimens were treated with xylene and ethanol, and were blocked by protein-blocking reagent (Dako Cytomation, Carpinteria, CA). The polyclonal antibody in antibody-diluted solution (1:100) was added and then stained with substrate-chromogen (DAKO liquid DAB chromogen, DakoCytomation). Finally, tissue specimens were stained with hematoxylin to discriminate nucleus from cytoplasm.

[Results] Identification ofA2254 and A5623 as up-regulated genes in breast cancer cells

When we analyzed gene-expression profiles of cancer cells from pre-menopausal 77 breast cancer patients using a cDNA microarray representing 27,648 human genes, we identified 493 genes that were commonly up-regulated in breast cancer cells. Among them, we focused on genes with in-house code A2254, which designed to Kinesin family member 2C (KIF2Q (SEQ ID NO; 35, 36), and with A5623, which designed to Protein regulator of cytokinesis 1 (Genebank accession No. NM_003981; SEQ ID NO; 39, 40). Expression of A2254 and A5623 genes were elevated in 44 of 60 and 37 of 58 breast cancer cases cells on the microarray in comparison with normal breast ductal cells, respectively. To confirm the expression of these up-regulated genes, we performed semi-quantitative RT-PCR analysis to compare the expression level between breast cancer specimens and normal human tissues including normal breast ductal cells. Firstly, we found that A2254 whose expression showed the elevated expression in 7 of 12 clinical breast cancer samples (poorly-differentiated type), compared to normal breast ductal cells and normal human tissues including mammary gland, lung, heart, liver, kidney, and bone marrow (Figure IA, upper panel). In addition, this gene was overexpressed in all of nine breast cancer cell lines as well (Figure IA, lower panel). Next, we also found that A5623 whose expression showed the elevated expression in 7 of 12 clinical breast cancer specimens (poorly-differentiated type) compared to normal human tissues, especially normal breast ductal cells (Figure IB, upper panel), and was overexpressed in all of nine breast cancer cell lines we examined (Figure IB, lower panel).

To further examine the expression pattern of these genes, we performed Northern blot analyses with multiple-human tissues and breast cancer cell lines using cDNA fragments of A2254 and A5623 as probes (see Material and Method). Expression of A2254 was no or undetectable in normal human tissues except testis and thymus (Figure 2A; the upper panel), while was surprisingly overexpressed in all of breast cancer cell lines compared to in other normal tissues except bone marrow (Figure 2A; the bottom panel). A5623 was also exclusively expressed in testis (Figure 2B, the upper panel), while was significantly overexpressed in all of breast cancer cell lines, compared to in other normal tissues except bone marrow, especially in normal human breast (Figure 2B, the bottom panel). Thus, we focused the breast cancer specifically expressed transcripts.

Genomic structure ofA2254 andA5623

To obtain the entire cDNA sequences of A2254 and A5623, we performed RT-PCR using breast cancer cell line, T47D as template. A2254 consists of 21 exons, designed Kinesin family member 2C (KIF2C), located on the chromosome Ip34.1. The full-length mRNA sequences of A2254 contained 2886 nucleotides, encoding 725 amino acids.

A2254 has two different transcriptional variants consisting of 21 and 20 exons, corresponding to A2254V1 (GenBank Accession NO; AB264115, SEQ ID NO; 35, 36) and A2254V2 (GenBank Accession NO; AY026505, SEQ ID NO; 37, 38), respectively (Figure 3A, upper panel). Exon 1 and 2 of the Vl variant was 185bp and 94bp, respectively, while V2 variant has no exon 1 and 2 of Vl, but a novel exon consisting of 346bp as exon 1. Last exon (exon 20) of V2 variant was 537bp shorter than the 3' end of last exon (exon 21) of Vl variant. The full-length cDNA sequences of A2254V1 and A2254V2 variants contained 2886, and 2401 nucleotides, respectively. The ORF of these variants start at within each exon 1. Eventually, Vl and V2 transcripts encode 725 and 671 amino acids, respectively. To further confirm the expression pattern of each variant in breast cancer specimens and normal human tissues, we performed semi-quantitative RT-PCR using the primer sets recognized to each variant. As a result, we found that A2254 Vl variant was dominantly expressed in breast cancer cells, compared to the expression of V2 variant, while V2 variant was expressed only in testis (Figure 3A; lower panel). Therefore, we focused on A2254 Vl variant.

A5623 has also three different transcriptional variants consisting of 15, 14 and 14 exons, corresponding to A5623V1 (GenBank Accession NO; NM_003981; SEQ ID NO; 39, 40), A5623V2 (GenBank Accession NO; NM_199413; SEQ ID NO; 41, 42) and 5623V3

(GenBank Accession NO; NM_199414; SEQ ID NO; 43, 44) respectively (Figure 3B, upper panel). There were alternative variations in exon 13 and 14 of Vl, and the other remaining exons were common to all variants. V2 variant has no exon 14 of the Vl, generating a novel early stop codon within last exon. Exon 14 of the V3 variant was completely deleted, and exon 13 of V3 was 77bp shorter than that of the Vl at the 3' end, generating a novel early stop codon within last exon as well. The full-length cDNA sequences of A5623V1, A5623V2 and 5623V3 variants consist of 3128, 3091 and 3011 nucleotides, respectively. The ORF of these variants start at within each exon 1. Eventually, Vl, V2 and V3 transcripts encode 620, 606 and 566 amino acids, respectively. To further confirm the expression pattern of each variant in breast cancer specimens and normal human tissues, we performed semi-quantitative RT-PCR using the primer sets recognized to each variant. As a result, we found that all of variants were highly overexpressed in breast cancer cells, compared to normal human tissues (Figure 3B, lower panel). Therefore, we further perform functional analysis for all variants of A5623.

Subcellular localization ofA2254 andA5623

To further examine the characterization of A2254 and A5623, we examined the subcellular localization of these gene products in COS7 cells. Firstly, when we transiently transfected plasmids expressing A2254 protein (pCAGGS-A2254-HA) into COS7 cells, we observed the 81KD-A2254 protein as an expected size by Western blot analysis (Figure 4A). In addition, immunocytochemical staining reveals exogenous A2254 was located under the plasma membrane in transfected-cells (Figure 4B). Surprisingly, positive signal in immunocytochemical staining disappeared in cell-cell attached membrane, suggesting this gene might play a key role of interaction of cell to cell or cell polarity.

Next, when we also transiently transfected a plasmid expressing A5623V1, V2 and V3 proteins (pCAGGS-A5623-HA) into COS7 and T47D cells, exogenous A5623 Vl, V2 and V3 proteins were observed as each expected size by Western blot analysis (Figure 4C). Moreover, immunocytochemical staining reveals that all variant proteins of A5623 localized to the cytoplasmic apparatus as the intermediate filaments in transfected-cells (Figure 4D, E, F), suggesting that A5623 may also play a key role of interaction of cell to cell.

We developed a polyclonal antibody against A5623 or A2254 and then investigated endogenous expression of A5623 protein or A2254 protein in cell lysates from breast cancer cell-lines, BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3, and T47D by Western-blot analysis (Figure 7A), using HMEC (Human Mammalian Epithelial Cell) as a control of the experiments. All of breast cancer cell-lines showed high levels of A5623 or A2254 expression, whereas HMEC cells showed very weak expression. Subsequent immunocytochemical analysis of breast cancer cell-line, HBC4, HBC5 and MCF7, using anti-A5623 polyclonal antibody indicated endogenous A5623 mainly localized in the cytoplasmic and/or nucleus apparatus of interphase cells. In particular, endogenous A5623 was observed intermediate filament network in all breast cancer cell lines. A5623 underwent a remarkable redistribution when cells progressed through mitosis. In prophase, it localized with mitotic spindle poles and then with the entire mitotic spindle through metaphase into the early stage of anaphase. By mid-anaphase, A5623 was concentrated as a series of narrow bars at the anaphase spindle midzone in cells (Figure 7B). Subsequent immunocytochemical analysis of breast cancer cell-line, HBC5 using anti-A2254 polyclonal antibody indicated endogenous A2254 was observed to be mainly localized in the cytoplasmic apparatus of interphase cells although exogenous A2254 was located under the plasma membrane in transfected-cells. A2254 underwent a remarkable redistribution when cells progressed through mitosis. Although A2254 was still localized in cytoplasm in metaphase cells, A2254 was concentrated as a series of narrow bars at the anaphase spindle midzone in cells (Figure 7B). Eventually, this protein accumulated to midbody in telophase cells. These findings suggest an important role of A5623 and A2254 in cytokinesis in breast cancer cells as well as in HeLa cells previously described (Mollinari C, et al. J Cell Biol, 2002;157; 1175-86.; Mollinari C, et al MoI Biol Cell. 2005;16;1043-55.).

To further investigate A5623 expression in breast cancer and normal tissue sections, we performed immunohistochemical staining with anti-A5623 antibody. We identified strong staining in the cytoplasm and nuclei of three different histological subtypes of breast cancer, intraductal carcinoma, papillo-tubular carcinoma, and scirrhous carcinoma, but its expression was hardly detectable in normal breast tissues (Figure 7C, upper panel). Furthermore, in concordance with the results of northern blot analysis, its expression was detected in testis, while no expression was observed in any of heart, lung, liver, and kidney (Figure 7C, lower panel).

Growth-inhibitory effects of small-interfering RNA (siRNA) designed to reduce expression of A2254 andA5623

To assess the growth-promoting role of A2254 and A5623, we knocked down the expression of endogenous A2254 and A5623 in breast cancer lines T47D and HBC5 that have shown the overexpression of A2254 and A5623, by means of the mammalian vector-based RNA interference (RNAi) technique (see above). We examined expression levels of A2254 and A5623 by semi-quantitative RT-PCR experiments. As shown in Figure 5 and 6, the two siRNA constructs of each gene, A2254 (si2 and si5), and A5623 (sil and si2)-specific siRNAs significantly suppressed expression of each gene, compared with control siRNA construct (psiU6BX-Mock) (Figure 5A, 6A). To confirm the cell growth inhibition with A2254 and A5623 -specific siRNAs, we performed colony-formation and MTT assays, respectively. Introduction of A2254 (si2 and si5), (Figure 5B, C) and A5623 (sil and si2) (Figure 6B, C) constructs suppressed growth of T47D and HBC5 cells, consisting with the result of above reduced expression. Each result was verified by three independent experiments. These findings suggest that A2254 and A5623 have a significant function in the cell growth of the breast cancer. Oncogenic activity ofA2254.

To further confirm the growth promoting effect of A2254, we established NTH3T3- derivative cells that stably expressed exogenous A2254 (NIH3T3-A2254-1, -2, -3 and -4) cells. Western-blot analysis indicated high level of exogenous A2254 protein in three derivate clones (NIH3T3-A2254-1, -2 and -3), and low level of A2254 in one clone (A2254- 4) (Figure 9A). Subsequent MTT assays showed that overexpression of exogenous A2254 had no significant enhancement of cell growth (Figure 9B). These findings suggest that the lack of A2254 gene product has a critical effect on the survival of breast cancer cells although overexpression of this gene alone does not have growth enhancing activity. Furthermore, since we firstly observed that exogenous A2254 was located under the plasma membrane in transfected-cells, we performed matrigel-invasion assay to determine whether A2254 might have some role in cellular motility. Invasion through matrigel of MH3T3 -derivative cells that stably expressed exogenous A2254 (NIH3T3-A2254-3 and -4) cells was significantly enhanced as compared with the Mock-stably tranesfected cells (NIH3T3-Mock) (Figure 9C) with A2254 protein expression-dependency through the Western blot results, suggesting A2254 gene might has also a critical role invasion of tumor cells.

Interaction ofA5623 with A2254.

To further investigate physiological function of A5623 in breast cancer cells, we attempted to identify its interacting proteins. We found kinesin family member 2C/mitotic- centromere-associated kinesin (KIF2c/MCAK) protein (A2254) as a possible candidate

A5623 -interacting protein since this protein is known to localize in the midbody or near the contractile ring in late anaphase or telophase cells, and to function in midzone formation and cytokinesis. In addition, A5623 was reported to interact with several kinesin family proteins (Kurasawa Y, et al. EMBO J 2004;23; 3237-48.; Zhu C, et al. Proc Natl Acad Sci USA 2005;102;343-8.; Gruneberg U, et al. 2006; 172; 363-72.).

We first investigated the expression pattern of A2254 in breast cancer cases by semiquantitative RT-PCR analysis, and found co-upregulation of A2254 and A5623 in breast cancer cases (Figure 8A). Subsequently, we performed coimmunoprecipitation experiments using Flag-tagged A5623 and HA-tagged A2254 that were co-transfected into COS7 cells. Using anti-Flag and anti-HA antibodies we identified that Flag-tagged A5623 had co- precipitated with HA-tagged A2254 (Figure 8B) and that HA-tagged A2254 had reversely coprecipitated with Flag-tagged A5623 as well (Figure 8C), indicating the interaction of these two proteins.

Subsequently, we established NIH3T3 -derivative cells that stably expressed exogenous A5623 and A2254 (NIH3T3-A5623 and NIH3T3-A2254) cells. Immunocytochemical analyses revealed co-localization of the endogenous mouse A5623 and stably-expressed exogenous A2254 in NIH3T3 cells at midzone formation in late anaphase (Figure 8D, the left panel), and that co-localization of the endogenous A2254 and stably- expressed exogenous A5623^'in NIH3T3 cells at midzone formation in late anaphase (Figure 8D). Although further investigation of the co-localization of endogenous A5623 and endogenous A2254 in breast cancer cells will be necessary, these results strongly suggest that conformation of A5623 and A2254 might play crucial roles in cytokinesis in cancer cells.

[Discussion]

Through the precise expression profiles of breast cancer by means of genome wide cDNA microarray, we isolated novel genes, A2254 and A5623 that were significantly overexpressed in breast cancer cells, compared to normal human tissues.

Furthermore, northern blot analysis showed that expression of A5623 and A2254 was hardly detectable in any normal human tissues examined except the testis and bonemarrow.

Moreover, immunohistochmical staining experiments using anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody clearly indicated up-regulation of A5623 or A2254 expression in breast cancer tissue sections. These results showed that this gene should serve as a valuable target for development of anti-cancer agents for breast cancers.

Our immunocytochemical staining experiments showed that A5623 was localized in the cytoplasmic and/or nucleus in the breast cancer cell at the interphase, in the midzone in late anaphase, and at the contractile ring in telophase. Furthermore, we demonstrated treatment of breast cancer cells with siRNA effectively inhibited expression of all three target genes, A2254 and A5623 and significantly suppressed cell/tumor growth of breast cancer. These findings suggest that A2254 and A5623 might play key roles in tumor cell growth proliferation, and might be promising targets for development of anti-cancer drugs. We demonstrated that knockdown of endogenous A5623 by siRNAs induced failure of cytokinesis in breast cancer cells and resulted in accumulation of multi-nuclear cells and subsequent cell death. These findings suggest that A5623 play a role in cytokinesis of breast cancer cells as well. A5623 was also reported to interact with several kinesin family proteins (Kurasawa Y, et al EMBO J 2004;23; 3237-^-8.; Zhu C, et al. Proc Natl Acad Sci USA 2005;102;343-8.; Gruneberg U, et al. 2006; 172; 363-72.). It has been reported that A5623 could interact with several molecules, for example KIF4 or KIF 14, which are associated with mitotic events, especially cytokinesis (Kurasawa Y, et al. EMBO J 2004; 23; 3237-48.; Zhu C, et al. Proc Natl Acad Sci USA 2005; 102;343-8.). However, none of KIF4 and KIF14 was expressed in breast cancers through our expression profiles of breast cancers (data not shown). Hence, we further examined biological roles of A5623 in breast cancer cells by identification of its interacting protein(s), and identified A2254 (kinesin family member 2C/ mitotic- centromere-associated kinesin) protein as a candidate interacting protein, since this protein is known to localize in the midbody or near the contractile ring in late anaphase or telophase cells, and to function in midzone formation and cytokinesis. As shown in Figure 8, we demonstrated the in vivo interaction and colocalization of A2254 and A5623 during cell cycle, especially midbody in cytokinesis of telophase cells. Their evidences in breast cancer cases suggested their interaction to play a crucial role in breast carcinogenesis. Furthermore, we determined the binding regions of A5623 (51-70 and 200-490 amino-acids) with A2254 (data not shown). Although further analysis of the function of A5623 will be necessary, the data provided should contribute to more profound understanding of breast cancer carcinogenesis and to development of novel therapies for breast cancers.

Industrial Applicability

The present inventors have shown that the cell growth is suppressed by small interfering RNA (siRNA) that specifically targets the A2254 or A5623 gene. Thus, this novel siRNAs are useful target for the development of anti-cancer pharmaceuticals. For example, agents that block the expression of A2254 or A5623 or prevent its activity may find therapeutic utility as anti-cancer agents, particularly anti-cancer agents for the treatment of breast cancer (BRC).

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims

1. A method for treating or preventing breast cancer in a subject comprising administering to said subject a composition comprising a small interfering RNA (siRNA) that inhibits expression of A2254 (SEQ ID NO: 35 or 37) or A5623 (SEQ ID

NO: 39, 41 or 43).

2. The method of claim 1, wherein said siRNA comprises a sense nucleic acid sequence and an anti-sense nucleic acid sequence that specifically hybridizes to a sequence from A2254 or A5623.

3. The method of claim 2, wherein said siRNA comprises a ribonucleotide sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 21, 25, 29, and 33 as the target sequence.

4. The method of claim 3, wherein said siRNA has the general formula 5'-[A]-[B]-[A']- 3', wherein [A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of nucleotides of SEQ ID NOs: 21, 25, 29, and 33,

[B] is a ribonucleotide loop sequence consisting of 3 to 23 nucleotides, and [A'] is a ribonucleotide sequence consisting of the complementary sequence of [A].

5. The method of claim 1, wherein said composition comprises a transfection-enhancing agent.

6. A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 21, 25, 29, and 33, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said double-stranded molecule, when introduced into a cell expressing the A2254 or A5623 gene, inhibits expression of said gene.

7. The double-stranded molecule of claim 6, wherein said target sequence comprises at least about 10 contiguous nucleotides from the nucleotide sequences selected from the group of SEQ ID NOs: 35, 37, 39, 41 and 43.

8. The double-stranded molecule of claim 7, wherein said target sequence comprises from about 19 to about 25 contiguous nucleotides from the nucleotide sequences selected from the group of SEQ ID NOs: 35, 37, 39, 41 and 43.

9. The double-stranded molecule of claim 8, wherein said double-stranded molecule is a single ribonucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded ribonucleotide sequence.

10. The double-stranded molecule of claim 7, wherein the double-stranded molecule is an oligonucleotide of less than about 100 nucleotides in length.

11. The double-stranded molecule of claim 10, wherein the double-stranded molecule is an oligonucleotide of less than about 75 nucleotides in length.

12. The double-stranded molecule of claim 11, wherein the double-stranded molecule is an oligonucleotide of less than about 50 nucleotides in length.

13. The double-stranded molecule of claim 12, wherein the double- stranded molecule is an oligonucleotide of less than about 25 nucleotides in length.

14. The double-stranded polynucleotide of claim 13, wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length.

15. A vector encoding the double-stranded molecule of claim 7.

16. The vector of claim 15, wherein the vector encodes a transcript having a secondary structure and comprises the sense strand and the antisense strand.

17. The vector of claim 16, wherein the transcript further comprises a single-stranded ribonucleotide sequence linking said sense strand and said antisense strand.

18. A vector comprising a polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence of SEQ ID NOs: 21, 25, 29, and 33, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand.

19. The vector of claim 18, wherein said polynucleotide has the general formula

5'-[A]-[B]-[A']-3' wherein [A] is a nucleotide sequence of SEQ ID NOs: 21, 25, 29, and 33; [B] is a nucleotide sequence consisting of 3 to 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].

20. A pharmaceutical composition for treating or preventing breast cancer comprising a pharmaceutically effective amount of a small interfering RNA (siRNA) that inhibits expression of A2254 or A5623 as an active ingredient, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the siRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21, 25, 29, and 33 as the target sequence.

22. The composition of claim 21, wherein the siRNA has the general formula

5'-[A]-[B]-[A']-3' wherein [A] is a ribonucleotide sequence corresponding to a nucleotide sequence of

SEQ ID NOs: 21, 25, 29, and 33; [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and [A'] is a ribonucleotide sequence complementary to [A].

23. A method of screening for a compound useful in treating or preventing breast cancer, said method comprising the steps of: (a) contacting a polypeptide comprising an A2254-binding domain of an A5623 polypeptide with a polypeptide comprising an A5623-binding domain of an A2254 polypeptide in the presence of a test compound;

(b) detecting binding between the polypeptides; and

(c) selecting a test compound that inhibits binding between the polypeptides.

24. The method of claim 23, wherein the polypeptide comprising the A2254-binding domain comprises an A5623 polypeptide.

25. The method of claim 23, wherein the polypeptide comprising the A5623-binding domain comprises an A2254 polypeptide.

26. A kit for screening for a compound for treating or preventing breast cancer, wherein the kit comprises:

(a) a polypeptide comprising an A2254-binding domain of an A5623 polypeptide;

(b) a polypeptide comprising an A5623-binding domain of an A2254 polypeptide, and

(c) a reagent to detect the interaction between the polypeptides.

27. The kit of claim 26, wherein the polypeptide comprising the A2254-binding domain comprises an A5623 polypeptide.

28. The kit of claim 26, wherein the polypeptide comprising the A5623-binding domain comprises an A2254 polypeptide.

29. A method for treating or preventing breast cancer in a subject, wherein the method comprises the step of administering a pharmaceutically effective amount of a compound that inhibits the binding between an A5623 polypeptide and an A2254 polypeptide.

30. A composition for treating or preventing breast cancer, wherein the composition comprises a pharmaceutically effective amount of a compound that inhibits the binding between an A5623 polypeptide and an A2254 polypeptide, and a pharmaceutically acceptable carrier.