JP2009502113A - Compositions and methods for treating breast cancer - Google Patents

Abstract

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

Description

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 to a composition comprising a nucleic acid capable of inhibiting the expression of a gene encoding A2254 or A5623. In some embodiments, the compound is a small interfering RNA (siRNA) corresponding to subsequences from these genes. The invention also relates to methods and kits for identifying compounds useful in the treatment and prevention of cancer. Furthermore, the present invention relates to a method for treating or preventing cancer, particularly breast cancer, and a composition comprising such a binding inhibitor, comprising the step of administering a compound that inhibits the binding between A2254 and A5623.

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

Background Art Breast cancer (BRC) is a complex disease characterized by the accumulation of genetic and epigenetic changes in many genes (Nathanson KL., Et al., Nat Med, 7 (5): 552-6, 2001 (Non-Patent Document 1)). The transformation of normal cells through the stages of breast carcinogenesis, atypical ductal hyperplasia, noninvasive ductal carcinoma (DCIS), and invasive ductal carcinoma (IDC), Although similar to the stage, the exact molecular mechanism that promotes breast cancer remains unclear. Clearly, the molecular factors that lead to the development, progression, and metastasis of primary breast cancer appear to be promising targets for developing better means of prevention and treatment of the disease.

  Gene expression profiles obtained by cDNA microarray analysis can provide a great deal of information to characterize the nature of individual cancers; such information is promising for neoplastic disease through the development of new drugs (Petricoin, EF, 3rd, et al., Nat Genet, 32 Suppl: 474-479, 2002); Bange J,., et al., Nat Med, 7 (5): 548-52, 2001 (non-patent document 3)). For this purpose, we have used a combination of laser microbeam microdissection (LMM) and a cDNA microarray corresponding to 27,648 genes, and 77 breast tumors including 12 DCIS and 69 IDC. (Nishidate T, et al., Int J Oncol. 2004 Oct; 25 (4) 797-819 (non-patent document 4)). The data from these experiments should not only provide important information about breast tumorigenesis, but also help identify candidate genes whose products can be diagnostic markers and / or molecular targets for breast cancer treatment.

Nathanson KL., Et al., Nat Med, 7 (5): 552-6, 2001 Petricoin, E. F., 3rd, et al., Nat Genet, 32 Suppl: 474-479, 2002 Bange J,., Et al., Nat Med, 7 (5): 548-52, 2001 Nishidate T, et al., Int J Oncol. 2004 Oct; 25 (4) 797-819

DISCLOSURE OF THE INVENTION The present invention shows that A2254 and A5623 are significantly overexpressed in breast cancer cells, and inhibition of A2254 or A5623 expression is effective in inhibiting cell proliferation of various cancer cells including those involved in BRC. Based on the discovery that there is. The invention described in this application is based in part on this discovery.

  To isolate a novel molecular target for breast cancer treatment, we used a combination of cDNA microarray and laser microbeam microdissection to accurately represent the entire genome of 77 cases with premenopausal breast cancer I investigated. Of the up-regulated genes, we have up-regulated expression in 44 out of 60 breast cancer cases and 37 out of 58 cases, respectively, for which we were able to obtain expression data A2254 indicating kinesin family member 2C (KIF2C) and A5623 indicating protein control factor 1 of cytokinesis (PRC1) were identified. Subsequent semi-quantitative RT-PCR and Northern blot analysis showed that A2254 and A5623 were significantly overexpressed in clinical breast cancer samples and breast cancer cell lines compared to normal human tissues including duct cells or normal breasts. It was confirmed that Immunocytochemical staining indicates that foreign A2254 and A5623 are localized in the cytoplasm and / or organelles 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 RNA (siRNA) effectively inhibited expression of A2254 and A5623 and suppressed cell / tumor growth of breast cancer cells T47D and HBC5, respectively. In addition, the inventors have found that transient overexpression of both A2254 [and A5623] in HT1080 cells promotes cell migration in a scratch assay, which indicates that these genes are in cell migration. It was suggested to play an important role (data not shown). These findings suggest that overexpression of A2254 and A5623 may be involved in breast tumorigenesis or metastasis and may be a promising strategy for treatment specific to breast cancer patients.

  In addition, we have shown that A5623 can bind to A2254 via its N-terminal part, and that A5623 and A2254 co-localize in the centrosome during late mitosis and cytokinesis. Demonstrated. Immunocytochemical staining indicates that in breast cancer cells, endogenous A2254 is localized at the spindle pole in the early division and then in the entire spindle from the middle stage to the early stage of the division stage. These findings suggest that the A2254 and A5623 complex may be involved in breast tumorigenesis and may be a promising strategy for treatment specific to breast cancer patients.

  A2254, representing kinesin family member 2C, from exons 21 and 20 corresponding to A2254V1 (GenBank accession number: AB264115, SEQ ID NO: 35, 36) and A2254V2 (GenBank accession number; AY026505, SEQ ID NO: 37, 38), respectively. With two different transcription variants (Figure 3A, upper panel). Therefore, in the present invention, A2254 includes A2254V1 and A2254V2. Exon 1 and 2 of the V1 variant are 185 bp and 94 bp, respectively, while the V2 variant does not have exon 1 and 2 of V1 and has a new exon consisting of 346 bp as exon 1. The final exon of the V2 variant (exon 20) was 537 bp shorter than the 3 ′ end of the final exon of the V1 variant (exon 21). The full length cDNA sequences of the A2254V1 and A2254V2 variants contained 2886 and 2401 nucleotides, respectively. The ORF of these mutants starts from within each exon 1. Finally, the V1 and V2 transcripts encode 725 and 671 amino acids, respectively.

  A5623, which shows protein regulator 1 of cytokinesis, is similarly A5623V1 (GenBank accession number; NM_003981; SEQ ID NO: 39, 40), A5623V2 (GenBank accession number; NM_199413; SEQ ID NO: 41, 42), and 5623V3 ( It has three different transcriptional variants consisting of 15, 14, and 14 exons corresponding to GenBank accession number; NM_199414; SEQ ID NO: 43, 44) (FIG. 3B, upper panel). Accordingly, in the present invention, A5623 includes A5623V1, A5623V2, and A5623V3. There were selective changes in exons 13 and 14 of V1, and the other remaining exons were common to all mutants. The V2 variant does not have exon 14 of V1 and a new early stop codon occurs within the final exon. Exon 14 of the V3 variant was completely deleted, exon 13 of V3 was 77 bp shorter than exon 13 of V1 at the 3 'end, as well as a new early stop codon in the final exon . The full-length cDNA sequences of A5623V1, A5623V2, and A5623V3 variants consist of 3128, 3091, and 3011 nucleotides, respectively. The ORF of these mutants starts from within each exon 1. Finally, the V1, V2, and V3 transcripts encode 620, 606, and 566 amino acids, respectively.

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

A further aspect of the invention is a method of screening for compounds useful for the treatment or prevention of cancer, particularly breast cancer, comprising the following steps:
(a) contacting a polypeptide comprising the A2254 binding domain of the A5623 polypeptide with a polypeptide comprising the A5623 binding domain of the A2254 polypeptide in the presence of the test compound;
(b) detecting binding between polypeptides; and
(c) selecting a test compound that inhibits binding between polypeptides.

  One skilled in the art can determine the A2254 binding domain of the A5623 polypeptide or the A5623 binding domain of the A2254 polypeptide by methods known in the art for determining protein interaction domains. For example, interaction domains can be identified by interaction domain mapping. For example, various portions of the A2254 or A5623 polypeptide are expressed, respectively, and screened for interaction, for example, by applying a yeast two-hybrid system. The interactions identified by the yeast two-hybrid system can be verified by applying biochemical assays such as, for example, immunoprecipitation or column interactions. Those skilled in the art can readily apply other methods known in the art to identify and map interaction domains, so the above method for identifying and / or mapping interaction domains is never It is not limited.

  Preferably, a polypeptide comprising an A2254 binding domain applied to a method for screening an inhibitor of binding between A5623 polypeptide and A2254 polypeptide is A5623V1 (SEQ ID NO: 40), A5623V2 (SEQ ID NO: 42), or A5623V3. A5623 polypeptide such as (SEQ ID NO: 44).

  In another preferred aspect, a polypeptide comprising an A5623 binding domain applied to a method for screening an inhibitor of binding between A5623 polypeptide and A2254 polypeptide is A2254V1 (SEQ ID NO: 36) or A2254V1 (SEQ ID NO: 38). A2254 polypeptide such as

A further aspect of the present invention is a kit for screening compounds for treating or preventing breast cancer, including the following:
(a) a polypeptide comprising the A2254 binding domain of the A5623 polypeptide,
(b) a polypeptide comprising the A5623 binding domain of the A2254 polypeptide, and
(c) A reagent for detecting an interaction between polypeptides.

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

  In yet another aspect, the invention relates to a method of treating or preventing breast cancer in a subject comprising administering a pharmaceutically effective amount of a compound that inhibits binding of A5623 polypeptide to A2254 polypeptide. .

  Furthermore, the present invention also provides a composition for treating or preventing breast cancer, comprising a pharmaceutically effective amount of a compound that inhibits the binding between A5623 polypeptide and A2254 polypeptide, and a pharmaceutically acceptable carrier. It is related with the composition containing.

  As used herein, the term “organism” refers to any organism composed of at least one cell. The organism may be as simple as eukaryotic single cells, for example, or as complex as mammals including humans.

  As used herein, the term `` biological sample '' refers to an entire organism or a portion of a tissue, cell, or component thereof (e.g., blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic membrane) Fluid, including but not limited to cord blood, urine, vaginal fluid, and semen). A “biological sample” further includes a homogenate, lysate, extract, cell culture, or tissue culture, or a fraction or portion thereof, prepared from the entire organism, or a portion of its cells, tissues, or components. Point to. Finally, a “biological sample” refers to a medium such as a nutrient broth or gel that contains cellular components such as proteins or nucleic acid molecules in which the organism grows.

  The invention features a method of inhibiting cell proliferation. Cell proliferation is inhibited by contacting the cells with a composition of A2254 or A5623 small interfering RNA (siRNA). The cell is further contacted with a transfection facilitating agent. The cells are provided in vitro, in vivo, or ex vivo. The subject is a mammal, such as a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The cell is a ductal cell. Alternatively, the cell is a tumor cell (ie, a cancer cell) such as a carcinoma cell or an adenocarcinoma cell. For example, the cell is a breast cancer cell. Inhibition of cell growth means that treated cells grow at a slower rate than untreated cells or that viability is reduced. Cell proliferation is measured by proliferation assays known in the art.

  The term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including techniques for transcribing RNA from DNA as a template. The siRNA comprises an A2254 or A5623 sense nucleic acid sequence, an A2254 or A5623 antisense nucleic acid sequence, or both. siRNAs may contain two complementary molecules, or a single transcript may be constructed to have both a sense sequence and a complementary antisense sequence from the target gene, examples of which are In embodiments, there is a hairpin that results in the production of microRNA (miRNA).

  When siRNA binds to the A2254 or A5623 transcript in the target cell, the production of A2254 or A5623 by the cell is reduced. The length of the oligonucleotide is at least about 10 nucleotides and may be as long as the natural 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 A2254 or A5623 siRNA oligonucleotides that inhibit the expression of A2254 or A5623 in mammalian cells include oligonucleotides comprising the target sequence, eg, SEQ ID NO: 21 or 25 or 29 or 33 nucleotides, respectively.

  Methods for designing double stranded RNA having the ability to inhibit gene expression in target cells are known. (See, eg, US Pat. No. 6,506,559, which is incorporated herein by reference in its entirety). For example, a computer program for designing siRNA can be used from the Ambion website (http://www.ambion.co.jp/techlib/tb/tb_506.html). A computer program available from Ambion, Inc. selects nucleotide sequences for siRNA synthesis based on the following procedure.

siRNA location selection
1. Scan downstream for AA dinucleotide sequences starting from the AUG start codon of the transcript. Record the occurrence of each AA and the 3 ′ adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al., Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13 (24): 3191-7 (1999) is a 5 'and 3' untranslated region (UTR) and a region near the start codon (75 It is recommended not to design siRNAs for these because there may be abundant regulatory protein binding sites in the base). UTR binding proteins and / or translation initiation complexes may interfere with the binding of the siRNA endonuclease complex.
2. Compare potential target sites with appropriate genomic databases (human, mouse, rat, etc.) and exclude any target sequences that have significant homology with other coding sequences from consideration. BLAST (Altschul SF, et.al., Nucleic Acids Res. 1997; 25: 3389-402; J Mol Biol. 1990; 215: 403, found on the NCBI server at www.ncbi.nlm.nih.gov/BLAST/ -10.) Is suggested.
3. Select a qualified target sequence for synthesis. To assess, it is typical to select several target sequences along the length of the gene.

  An isolated nucleic acid molecule comprising a nucleic acid sequence of the target sequence, for example, SEQ ID NOs: 21, 25, 29, and 33, or complementary to the nucleic acid sequence of the nucleotides of SEQ ID NOs: 21, 25, 29, and 33 Nucleic acid molecules are also included in the present invention. As used herein, an “isolated nucleic acid” is a nucleic acid that has been removed from its original environment (eg, the natural environment if it is naturally occurring), and thus has been artificially altered from its natural state. It is a nucleic acid. In the present invention, isolated nucleic acids include DNA, RNA, and derivatives thereof. If the isolated nucleic acid is RNA or a derivative thereof, the base “t” must be replaced with “u” in the nucleotide sequence. As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen-type base pairing between nucleotide units of a nucleic acid molecule, and the term “binding” refers to two nucleic acids. Or means a physical or chemical interaction between compounds, or related nucleic acids or compounds, or combinations thereof. Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes with little or no mismatch. For the purposes of the present invention, two sequences with 5 or fewer mismatches are considered to be complementary. Furthermore, the sense and antisense strands of the isolated nucleotides of the present invention can form double stranded nucleotides or hairpin loop structures by hybridization. In preferred embodiments, such duplexes do not contain more than one mismatch for every 10 matches. In particularly preferred embodiments where the duplex strands are completely complementary, such duplexes do not contain mismatches. For A2254 or A5623, respectively, the nucleic acid molecule is less than 2886 or 3128 nucleotides in length. For example, the nucleic acid molecule is less than about 500, about 200, or about 75 nucleotides in length. Also included in the invention are vectors containing one or more of the nucleic acids described herein, and cells containing the vectors. The isolated nucleic acids of the invention are useful for siRAN against A2254 or A5623, or DNA encoding siRNA. When the nucleic acid is used for siRNA or its coding DNA, the sense strand is preferably longer than about 19 nucleotides, more preferably longer than 21 nucleotides.

The present invention is based in part on the discovery that the gene encoding A2254 or A5623 is overexpressed in breast cancer (BRC) compared to non-cancerous breast tissue. The A2254V1 and A2254V2 cDNAs are 2886 and 2401 nucleotides in length, respectively. The A5623V1, A5623V2, and A5623V3 cDNAs are 3128, 3091, and 3011 nucleotides in length, respectively. The nucleic acid and polypeptide sequences of A2254V1 and A2254V2 are shown in SEQ ID NOs: 35 and 36 and SEQ ID NOs: 37 and 38, respectively. The nucleic acid and polypeptide sequences of A5623V1, A5623V2, and A5623V3 are shown in SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, and SEQ ID NOs: 43 and 44, respectively. Sequence data is also available with the following accession numbers.
A2254: AY026505
A5623: NM_003981, NM_199413, NM_199414

Methods for Inhibiting Cell Growth The present invention relates to inhibiting cell growth, ie cancer cell growth, by inhibiting the expression of A2254 or A5623. Alternatively, since it has been found that A5623 and A2254 polypeptides can interact and these interactions are thought to play an important role in carcinogenesis, particularly breast cancer carcinogenesis, the present invention It relates to inhibiting cell growth, ie cancer cell growth, in particular breast cancer growth, by inhibiting the binding between them. Accordingly, the present invention relates to means and methods for treating or preventing breast cancer in a subject by inhibiting the binding of A5623 and A2254 polypeptides. Furthermore, the present invention contemplates inhibitors of binding between A5623 and A2254 polypeptides for use in treating or preventing cancer, particularly as a medicament for treating or preventing breast cancer.

  Expression of A2254 or A5623 is inhibited, for example, by small interfering RNA (siRNA) that specifically targets the A2254 or A5623 gene. A2254 or A5623 targets include, for example, the nucleotides of SEQ ID NOs: 21, 25, 29, and 33.

  In non-mammalian cells, double-stranded RNA (dsRNA) has been shown to have a powerful and specific silencing effect on gene expression, which is called RNA interference (RNAi) (Sharp PA. Genes Dev. 1999; 13 (2): 139-41.). dsRNA is processed into 20-23 nucleotide dsRNA called small interfering RNA (siRNA) by an enzyme containing RNase III motif. siRNAs, together with multicomponent nuclease complexes, specifically target complementary mRNAs (Hammond SM et. al., 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 non-complementary thymidine or uridine dimer induces global changes in gene expression Without having been shown to have a gene-specific knockdown effect (Elbashir SM et al., Nature. 2001; 411 (6836): 494-8.). In addition, plasmids containing nuclear small RNA (snRNA) U6 or polymerase III H1-RNA promoters mobilize type III class RNA polymerase III to efficiently produce such short RNAs, thus their target mRNA Can be constitutively suppressed (Miyagishi M. et al., Nat Biotechnol. 2002; 20 (5): 497-500 .; Brummelkamp TR, et al., Science. 296 (5567): 550-3, 2002.).

  Cell proliferation is inhibited by contacting the cells with a composition comprising an A2254 or A5623 siRNA. The cells are further contacted with a transfection agent. Suitable transfection agents are known in the art. Inhibiting cell growth means that the cells grow at a slower rate or have reduced viability compared to cells not exposed to the composition. Cell proliferation is measured by methods known in the art such as the MTT cell proliferation assay.

A2254 or A5623 siRNAs target a single target of the A2254 or A5623 gene sequence. Alternatively, siRNAs target multiple targets of the A2254 or A5623 gene sequence. For example, the composition comprises an A2254 or A5623 siRNA directed against two, three, four, 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, open reading frame (ORF), or 3 ′ untranslated region of the human A2254 or A5623 gene. Alternatively, the siRNA is a nucleic acid sequence that is complementary to an upstream or downstream modulator of A2254 or A5623 gene expression. Examples of upstream or downstream modulators include transcription factors that bind to the A2254 or A5623 gene promoter, kinases or phosphatases that interact with the A2254 or A5623 polypeptide, A2254 or A5623 promoters or enhancers. The A2254 or A5623 siRNA that hybridizes to the target mRNA usually binds to a single-stranded mRNA transcript, thereby preventing translation and thus preventing expression of the protein, thereby preventing the expression of the A2254 or A5623 gene. Or reduces or inhibits the production of A5623 polypeptide product. Thus, siRNA molecules of the invention can be defined by their ability to specifically hybridize with mRNA or cDNA from the A2254 or A5623 gene under stringent conditions. In the present invention, the terms “hybridize” or “specifically hybridize” are used to refer to 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 with its target sequence, but not detectably with other sequences, typically in complex nucleic acid mixtures. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidance on nucleic acid hybridization can be 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 5-10 ° C. lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength, pH. T _m is the temperature (at a given ionic strength, pH, and nucleic acid concentration) at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (when the target sequence is present in excess). , T _m occupies 50% of the probe 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 twice background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5x SSC, and 1% SDS, 42 ° C incubation, or 5x SSC, 1% SDS, 65 ° C Incubation and washing at 50 ° C. in 0.2 × SSC and 0.1% SDS.

  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 sequences for generating A2254 or A5623 siRNA include the nucleotide sequence of SEQ ID NO: 21 or 25, or 29 or 33, respectively, as the target sequence. Furthermore, in order to enhance the inhibitory activity of siRNA, nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence. The number of “u” added is at least about 2, generally from about 2 to about 10, and preferably from about 2 to about 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA.

  The cell is any cell that expresses or overexpresses A2254 or A5623. The cell is an epithelial cell such as a ductal cell. Alternatively, the cell is a tumor cell such as a carcinoma, adenocarcinoma, blastoma, leukemia, myeloma, or sarcoma. The cell is breast cancer.

  The A2254 or A5623 siRNA is introduced directly into the cell in a form that can bind to the mRNA transcript. Alternatively, DNA encoding A2254 or A5623 siRNA is present in the vector.

  The vector may be, for example, an A2254 or A5623 target sequence into an expression vector having a functionally linked control sequence adjacent to the A2254 or A5623 sequence so that expression of both strands is possible (by transcription of the DNA molecule). It is produced by cloning (Lee, NS, 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 on the 3 ′ side of the cloned DNA), and an RNA molecule that is the sense strand of A2254 or A5623 mRNA is Transcribed by a second promoter (eg, a promoter sequence 5 ′ of the cloned DNA). The sense and antisense strands hybridize in vivo to create a siRNA construct for silencing the A2254 or A5623 gene. Alternatively, two constructs are utilized to create the sense and antisense strands of the siRNA construct. The cloned A2254 or A5623 may encode a construct with a secondary structure, such as a hairpin, where a single transcript has both sense and complementary antisense sequences from the target gene.

In order to form a hairpin loop structure, a loop sequence consisting of any nucleotide sequence can be placed between the sense and antisense sequences. Thus, the present invention also provides a general formula
5 '-[A]-[B]-[A']-3 '
Wherein [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes to mRNA or cDNA from A2254 or A5623. In a preferred embodiment, [A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of the nucleotides of SEQ ID NOs: 21, 25, 29, and 33, and [B] consists of 3 to 23 nucleotides It is a ribonucleotide sequence, and [A ′] is a ribonucleotide sequence consisting of a complementary sequence of [A].

Region [A] hybridizes with [A ′], and then a loop consisting of region [B] is formed. The loop sequence may preferably be about 3 to about 23 nucleotides in length. The loop sequence can be selected, for example, from the group consisting of the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). Furthermore, an active siRNA is also obtained by a loop sequence consisting of 23 nucleotides (Jacque, JM et al., (2002) Nature 418: 435-8.).
CCC, CCACC, or CCACACC: Jacque, JM et al., (2002) Nature, 418: 435-8.
UUCG: Lee, NS et al., (2002) Nature Biotechnology 20: 500-5; Fruscoloni, P. et al., (2003) Proc. Natl. Acad. Sci. USA 100 (4): 1639-44.
UUCAAGAGA: Dykxhoorn, DM et al., (2003) Nature Reviews Molecular Cell Biology 4: 457-67.

As an example, a preferred siRNA having the hairpin loop structure of the present invention is shown below. In the following structure, the loop sequence can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. A preferred loop sequence is UUCAAGAGA (“ttcaagaga” in DNA).
GAGAAGAAGGCCCAGAACU- [B] -AGUUCUGGGCCUUCUUCUCUC (for the target sequence of SEQ ID NO: 21)
CUCUAGGACUUGCAUGAUU- [B] -AAUCAUGCAAGUCCUAGAG (when the target sequence is SEQ ID NO: 25)
GGAAAGACUCAUCAAAAGC- [B] -GCUUUUGAUGAGUCUUUCC (for the target sequence of SEQ ID NO: 29)
GCAUAUCCGUCUGUCAGAA- [B] -UUCUGACAGACGGAUAUGC (for the target sequence of SEQ ID NO: 33)

  The control sequences adjacent to the A2254 or A5623 sequence are the same or different so that their expression can be regulated independently or temporally or spatially. The siRNA is transcribed intracellularly by cloning the A2254 or A5623 gene template into a vector containing, for example, an RNA polymerase III transcription unit derived from a nuclear small RNA (snRNA) U6 or a human H1 RNA promoter. In order to introduce the vector into the cell, a transfection facilitating agent can be used. FuGENE (Roche Diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako Pure Chemical Industries) are useful as transfection promoters.

  Oligonucleotides of various parts of A2254 or A5623 mRNA, and oligonucleotides complementary to those various parts, in tumor cells (e.g., using a breast cell line such as a breast cancer (BRC) cell line) A2254 or A5623 The ability to reduce the production of was tested in vitro according to standard methods. A2254 or A5623 specific antibody or other detection of decreased A2254 or A5623 gene product in cells contacted with the candidate siRNA composition compared to cells cultured in the absence of the candidate composition Detect using method. A sequence that reduces the production of A2254 or A5623 in an in vitro cell assay or cell free assay is then tested for its inhibitory effect on cell proliferation. Sequences that inhibit cell growth in an in vitro cell assay are tested in vivo in rats or mice to confirm a decrease in A2254 or A5623 production and a decrease in tumor cell growth in animals with malignant neoplasms.

How to treat malignant tumors
Patients with tumors characterized by overexpression of A2254 or A5623 are treated by administering A2254 or A5623 siRNA. For example, siRNA therapy is used to inhibit the expression of A2254 or A5623 in patients suffering from breast cancer (BRC) or at risk of developing BRC. Such patients are identified by standard methods for specific tumor types. Breast cancer (BRC) is diagnosed by, for example, CT, MRI, ERCP, MRCP, computed tomography, or ultrasound. A treatment is effective if the treatment results in a clinical benefit such as a decrease in A2254 or A5623 expression in the subject, or a decrease in tumor size, spread, or metastatic potential. “Effective” when the treatment is carried out prophylactically means that the treatment delays or prevents the formation of the tumor or prevents or reduces the clinical symptoms of the tumor. Efficacy is determined by any known method for diagnosing or treating a particular tumor type.

  siRNA therapy is performed by administering siRNA to a patient by standard vectors encoding the siRNA of the invention and / or by gene delivery systems such as by delivery of 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 a backbone and nucleotides that are modified to prevent the action of ribonucleases. Other modifications are possible, for example cholesterol-binding siRNAs have shown improved pharmacological properties (Song et al. Nature Med. 9: 347-351 (2003)). Suitable gene delivery systems can include liposomes, receptor-mediated delivery systems, or viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. The therapeutic nucleic acid composition is formulated in a pharmaceutically acceptable carrier. The therapeutic composition may also include a gene delivery system as described above. A pharmaceutically acceptable carrier is a biologically compatible excipient suitable for administration to animals, such as saline. A therapeutically effective amount of a compound is an amount that can produce a medically desirable result in a treated animal, such as reduced production of the A2254 or A5623 gene product, cell growth, eg, decreased proliferation, or decreased tumor growth.

  Parenteral administration such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes can be used to deliver A2254 or A5623 siRNA compositions. For the treatment of breast tumors, direct injection into the celiac, splenic, or common hepatic artery is useful.

Dosage to any patient is many, including the patient's physique, body surface area, age, the specific nucleic acid to be administered, sex, time and route of administration, general health, and other drugs to be administered simultaneously Depends on the factors. When the nucleic acid is administered intravenously, the dose is about 10 ⁶ to 10 ²² copies of the nucleic acid molecule.

  Polynucleotides are administered by standard methods, such as injection into gaps in tissues such as muscle or skin, introduction into the circulation or body cavities, or inhalation or infusion. The polynucleotide is injected or delivered to an animal with a pharmaceutically acceptable liquid carrier, such as a water-soluble or partially water-soluble liquid carrier. The polynucleotide is associated with liposomes (eg, cationic or anionic liposomes). The polynucleotide contains genetic information necessary for expression by the target cell, such as a promoter.

  Alternatively, patients with tumors, particularly breast tumors, can be treated by administering a compound that inhibits the binding of A5623 and A2254 polypeptides. Without being bound by theory, inhibitors of the interaction between the A5623 polypeptide and the A2254 polypeptide alter the binding of the A5623 polypeptide to the A2254 polypeptide or the binding of the A2254 polypeptide to the A5623 polypeptide, respectively. It is thought to act, regulate, prevent or extinguish. This suggests that both proteins can no longer interact to interact in cancer cells. Therefore, the important role of both proteins in carcinogenesis, especially breast cancer carcinogenesis, as suggested by the findings obtained by the present inventors, can be blocked, thus achieving prevention or treatment of cancer, especially breast cancer. I can think more.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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.

Production and Identification of Compounds that Reduce or Inhibit Binding of A5623 to A2254 In view of the matters provided in the Examples, one aspect of the present invention is to test compounds that reduce or prevent binding of A5623 to A2254. Identifying.

  Methods for determining A5623 / A2254 binding include any method for determining the interaction of two proteins. Further methods are described below. Such assays include, but are not limited to, conventional approaches such as cross-linking, co-immunoprecipitation, and simultaneous purification through gradient or chromatographic columns. In addition, protein-protein interactions are described in the yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature 340: 245-6 (1989); Chien et al., Proc. Natl Acad. Sci. USA 88, 9578-82 (1991)) and monitoring as disclosed in Chevray and Nathans (Proc. Natl. Acad. Sci. USA 89: 5789-93 (1992)). Can do. Many transcriptional activators, such as yeast GALA, consist of two physically distinct modular domains, one serving as a DNA binding domain and the other serving as a transcriptional activation domain. The yeast expression system described in the above-mentioned publication (generally called “two-hybrid system”) uses two hybrid proteins taking advantage of this property, and one of the target proteins is fused to the DNA binding domain of GAL4. The other has a candidate activation protein fused to the activation domain. Expression of the GAL1-lacZ reporter gene under the control of a GALA-activated promoter depends on reconstitution of GAL4 activity via protein-protein interactions. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER ™) is commercially available from Clontech for identifying protein-protein interactions between two specific proteins using two-hybrid technology. The system can be extended to map protein domains involved in specific protein interactions, or to accurately identify amino acid residues that are very important for these interactions.

  Although this application refers to “A5623” or “A2254”, when the interaction between the two is analyzed or manipulated, one or both binding portions of these proteins should be used instead of full-length copies of these proteins. It is understood that A fragment of A5623 that binds to A2254 can be readily identified by identifying fragments that bind to A2254 using standard A5623 deletion analysis and / or mutagenesis. Similar analysis can be used to identify the A5623 binding fragment of A2254.

  As disclosed herein, any test compound, including, for example, proteins (including antibodies), muteins, polynucleotides, nucleic acid aptamers, and peptides and non-peptide organic small molecules can be tested in the present invention. Can be a compound. Test compounds may be isolated from natural sources and may be prepared synthetically or recombinantly or in any combination thereof.

  For example, the peptide is described in "Solid Phase Peptide Synthesis" by G. Barany and RB Merrifield in Peptides, Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York, NY, pp. 100-118 (1980) Can be produced synthetically using solid phase techniques such as those described in. Similarly, nucleic acids can be immobilized as described in Beaucage, SL, & Iyer, RP (1992) Tetrahedron, 48, 2223-311; and Matthes et al., EMBO J., 3: 801-5 (1984). Can be synthesized using phase technology.

  Once an inhibitory peptide is identified, it is particularly useful to modify the peptides of the invention with various amino acid mimetics or unnatural amino acids to increase the stability of the peptide in vivo. Stability can be assayed in a number of ways. For example, stability has been tested using peptidases and various biological media such as human plasma and serum. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokinet. 11: 291-302 (1986). Other useful peptide modifications known in the art include glycosylation and acetylation.

  Both recombinant and chemical synthesis techniques can be used to produce a test compound of the invention. For example, the nucleic acid of a test compound can be produced by inserting it into an appropriate vector, which can be transfected into a competent cell and expressed. Alternatively, the nucleic acid can be amplified using PCR techniques or expression in a suitable host (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA). ).

  Peptides and proteins can also be obtained using recombinant techniques well known in the art, for example, Morrison, J. Bact., 132: 349-51 (1977) and Clark-Curtiss & Curtiss, Methods in Enzymology, 101: 347- It can be expressed by transforming a suitable host cell with a recombinant DNA construct as described in 362 (Wu et al., Eds, 1983).

Anti-A5623 and anti-A2254 antibodies In some aspects of the invention, the test compound is an anti-A5623 or anti-A2254 antibody. In some embodiments, these antibodies are chimeric, including but not limited to humanized antibodies. In some cases, antibody embodiments of the invention bind to either A5623 or A2254 at the interface where one of these proteins associates with the other. In some embodiments, these antibodies are at least about 10 ⁵ mol ⁻¹ , 10 ⁶ mol ⁻¹ or more, 10 ⁷ mol ⁻¹ or more, 10 ⁸ mol ⁻¹ or more, or 10 ⁹ mol ⁻¹ or more under physiological conditions. binding to A5623 or A2254 in more K _a. Such antibodies can be purchased from commercial sources such as Chemicon, Inc. (Temecula Calif.) Or substantially purified A5623 or A2254 proteins, such as human proteins or fragments thereof, It can be prepared using as an immunogen. Methods for 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, eg, PCT / US02 / 07144 (WO / 03/077838), the contents of which are incorporated herein by reference. For example, antibody purification methods using an antibody affinity matrix to form an affinity column are also well known in the art and commercially available (AntibodyShop, Copenhagen, Denmark). The identification of antibodies capable of inhibiting the A5623 / A2254 association is performed using the same test assay as described in detail below for the test compound in general.

Converting enzyme Converting enzyme can be a test compound of the present invention. In the context of the present invention, a conversion enzyme is a molecular catalyst that performs covalent post-translational modifications to A5623 or A2254, or both. The converting enzyme of the present invention causes one or more amino acid residues of A5623 and / or A2254 to cause an allosteric change in the structure of the modified protein or to prevent binding between A5623 and A2254 A5623 / A2254 is modified covalently by changing the chemical nature of the molecular binding site or the structure of the modified protein. The interference with binding between these two molecules, for binding K _a, 30 ° C., 0.1 ionic strength, as compared with coupling Ka between these proteins measured in the absence of a surfactant, at least 25%, 30 Refers to a reduction of%, 40%, 50%, 60%, 70% or more. Exemplary converting enzymes of the present invention include kinases, phosphatases, amidases, acetylases, glycosidases and the like.

Construction of a test compound library Although the construction of a test compound library is well known in the art, in this section the identification of test compounds and the screening of such compounds to screen for effective inhibitors of the A5623 / A2254 interaction Provide further guidance on library construction. Further guidance regarding compound libraries is provided herein below.

The construction of a molecular modeling test compound library is facilitated by knowledge of the molecular structure of the compound known to have the required properties and / or the molecular structure of the target molecules to be inhibited, namely 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 / or A2254 provides insight into the details of the interaction itself and also includes possible molecular inhibitors of the interaction Possible ways to inhibit are suggested.

  Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with that molecule. Three-dimensional constructs typically rely on X-ray crystallographic or NMR imaging data of selected molecules. Molecular dynamics requires force field data. The computer graphic system can predict how new compounds are associated with the target molecule, and can experimentally manipulate the structure of the compound and target molecule to complete the binding specificity. Molecular mechanics in conjunction with a menu-driven interface between a molecular design program and the user, usually useful to the user, to predict what happens to a molecule-compound interaction when a small change occurs in one or both Software and computationally intensive computers are required.

  One example of the above-described molecular modeling system generally described consists of the CHARMm and QUANTA programs (Polygen Corporation, Waltham, Mass). CHARMm performs energy minimization and molecular dynamics functions. QUANTA performs molecular structure construction, graphic modeling, and analysis. QUANTA enables the mutual construction, modification, visualization, and analysis of each other's behavior of molecules.

  Computer modeling of drugs that interact with specific proteins has been outlined in many papers. For example, Rotivinen, et al. Acta Pharmaceutica Fennica 97, 159-166 (1988); Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinlay and Rossmann, Annu. Rev. Pharmacol. Toxiciol. 29, 111- 122 (1989); Perry and Davies, Prog Clin Biol Res. 291: 189-93 (1989); Lewis and Dean, Proc. R. Soc. Lond B Biol Sci. 236, 125-40 and 141-62 (1989) And for model receptors for nucleic acid components, Askew, et al., J. Am. Chem. Soc. 111, 1082-90 (1989).

  Other computer programs for screening and rendering chemicals are available from companies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. Is possible. For example, 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 the A5623 / A2254 interaction is identified, any number of variants can be constructed using combinatorial chemistry techniques based on the chemical structure of the identified putative inhibitor, as detailed below. can do. A library of putative inhibitors or “test compounds” thus obtained can be screened using the methods of the present invention to identify test compounds in the library that inhibit A5623 / A2254 association.

Combinatorial chemical synthesis Combinatorial libraries of test compounds can be produced as part of a rational drug design program that includes knowledge of the core structure present in known inhibitors of the A5623 / A2254 interaction. This approach can keep the library at a reasonable size and facilitates high-throughput screening. Alternatively, a simple, particularly short polymer molecule library can be constructed by simply synthesizing all permutations of the molecular families that make up the library. An example of this latter approach would be a library where all peptides are 6 amino acids long. Such a peptide library may contain a permutation of all 6 amino acid sequences. This type of library is called a linear combinatorial chemical library.

  The preparation of combinatorial chemical libraries is well known to those skilled in the art and can be generated by chemical or biological synthesis. Combinatorial chemical libraries include peptide libraries (eg, US Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37: 487-93 (1991) and Houghten et al., Nature 354: 84-6 ( 1991)), but is not limited thereto. Other chemistries for generating chemical diversity libraries can also be used. Such chemistry includes, but is not limited to: peptides (eg PCT publication WO91 / 19735), encoded peptides (eg PCT publication WO93 / 20242), random biooligomers (eg PCT publication WO92) / 00091), diversomers such as benzodiazepines (eg US Pat. No. 5,288,514), 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 (1992)), non-peptidic peptidomimetics with a glucose backbone (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-8 (1992)), similar organic synthesis of small molecule library (Chen et al., J. Amer. Chem. Soc. 116: 2661 (1994)), oligocarbamate (Cho et al. al., Science 261: 1303 (1993)), and / or Petityl phosphonate (Campbell et al., J. Org. Chem. 59: 658 (1994)), nucleic acid libraries (see Ausubel and Sambrook (all above)), peptide nucleic acid libraries (eg, US Pat. No. 5,539,083) Antibody libraries (see, eg, Vaughan et al., Nature Biotechnology, 14 (3): 309-14 (1996) and PCT / US96 / 10287), carbohydrate libraries (eg, Liang et al., Science, 274: 1520-2 (1996) and US Pat. No. 5,593,853), small organic libraries (eg, benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1; 6 (6): 624-31 Isoprenoids, US Pat. No. 5,569,588; thiazolidinone and metathiazanone, US Pat. No. 5,549,974; pyrrolidines, US Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, US Pat. No. 5,506,337; Zojiazepin, see U.S. Pat. No. 5,288,514). Further guidance on combinatorial chemical synthesis is provided herein below.

Another approach to phage display is to produce libraries using recombinant bacteriophages. This “phage method” (Scott and Smith, Science 249: 386-90, 1990; Cwirla, et al, Proc. Natl. Acad. Sci., 87: 6378-82, 1990; Devlin et al., Science, 249: 404-6, 1990) can be used to construct very large libraries (eg 10 ⁶ to 10 ⁸ chemical units). The second approach is mainly using chemical methods, the Geysen method (Geysen et al., Molecular Immunology 23: 709-15, 1986; Geysen et al. J. Immunologic Method 102: 259-74, 1987). For example, the method of Fodor et al. (Science 251: 767-73, 1991). 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, December) 1986), and Rutter et al. (US Pat. No. 5,010,175, Apr. 23, 1991) describe methods for producing mixtures of peptides that can be tested as agonists or antagonists.

  Combinatorial library preparation equipment is commercially available (eg, 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford , MA). A number of combinatorial libraries are commercially available (see, for example, ComGenex, Princeton, NJ, Tripos, Inc., St. Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.) .

Screening of test compound libraries The screening methods of the present invention provide for efficient and rapid identification of test compounds that are likely to interfere with the A5623 / A2254 association. In general, any method of determining the ability of a test compound to interfere with the A5623 / A2254 association is suitable for use with the present invention. For example, ELISA-style competitive and non-competitive inhibition assays may be utilized. Control experiments should be performed to determine the maximum binding capacity of the system (eg, in the example below, bound A5623 is contacted with A2254 to determine the amount of A2254 bound to A5623). Further guidance regarding screening of test compound libraries is provided herein below.

Competitive assay format Competitive assays can be used to screen test compounds of the invention. As an example, a competitive ELISA format can include A5623 (or A2254) bound to a solid support. This bound A5623 (or A2254) is incubated with A2254 (or A5623) and the test compound. After sufficient time has passed for the test compound and / or A2254 (or A5623) to bind to A5623 (or A2254), the substrate is washed to remove unbound material. Next, the amount of A2254 bound to A5623 is determined. This can be done by any of a variety of methods known in the art, such as using an A2254 (or A5623) species tagged with a detectable label or labeling the washed substrate with anti-A2254 (or A5623). It can be performed in contact with an 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 labels, including but not limited to antibodies, are described in Harlow & Lane, Antibodies, A Laboratory Manual (1988).

  In some variations, A5623 (or A2254) is labeled with an affinity tag. The labeled A5623 (or A2254) is then incubated with the test compound and A2254 (or A5623) and immunoprecipitated. The immunoprecipitate is then subjected to Western blotting using an anti-A2254 (or A5623) antibody. As in the competitive assay format described above, the amount of A2254 (or A5623) found to associate with A5623 (or A2254) is inversely proportional to the ability of the test compound to interfere with the A5623 / A2254 association.

Non-competitive assay formats Non-competitive binding assays may also be useful as initial screens for testing compound libraries constructed in a format not readily applicable to screening using competitive assays, as described herein. There is. An example of such a library is a phage display library (see, eg, Barret, et al. (1992) Anal. Biochem 204, 357-364).

  Phage libraries are useful in that they can quickly produce a working quantity of many different recombinant peptides. Although the phage library is not itself suitable for the competition assay of the present invention, it can be efficiently screened in a non-competitive format to determine which recombinant peptide test compound binds to 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 are well known in the art, 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. USA 87, 6378-82.

  An exemplary non-competitive assay follows a procedure similar to that described for the competitive assay without the addition of one of the components (A5623 or A2254). However, since the non-competitive format determines test compound binding to A5623 or A2254, the ability of the test compound to bind to both A5623 and A2254 must be determined for each candidate. Thus, by way of example, binding of a test compound to immobilized A5623 can be accomplished, for example, by washing away unbound test compound and eluting the bound test compound from the support, eg, mass spectrometry, protein measurement (Bradford or It can be determined by analyzing the eluate by Lowry assay, or absorbance measurement at 280 nm. Alternatively, the binding of the test compound may be determined by omitting the elution step and monitoring changes in the spectroscopic properties of the organic layer on the support surface. Methods for monitoring surface spectroscopic properties include, but are not limited to: absorbance, reflectance, transmittance, birefringence, refractive index, diffraction, surface plasmon resonance, ellipsometry Resonance mirror method, grating coupled waveguide technology, and multipole resonance spectroscopy, all of which are known to those skilled in the art. A labeled test compound can also be used in the assay to eliminate the need for an elution step. In this case, the amount of label associated with the support after washing away unbound material is directly proportional to the binding of the test compound.

  Many well-known robotic systems have been developed for liquid phase chemistry. These systems include automated workstations such as automated synthesizers developed by Takeda Chemical Industries, LTD. (Osaka, Japan), and many robotic systems are robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass. .; Orca, Hewlett Packard, Palo Alto, Calif.) And mimics manual synthesis operations performed by chemists. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications (if necessary) to these devices that allow them to operate as described herein will be apparent to those skilled in the relevant arts. In addition, many combinatorial libraries are commercially available per se (eg, ComGenex, Princeton, NJ, Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D See Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

Screening for Converting Enzymes Test compounds that are converting enzymes can be assayed in a non-competitive format using cofactors and auxiliary substrates specific for the converting enzyme being assayed. Such cofactors and auxiliary substrates are known to those skilled in the art given the type of converting enzyme to be investigated.

One exemplary screening procedure for a converting enzyme first involves the A5623 in the presence of cofactors and auxiliary substrates necessary to effect covalent modification of the protein characteristic of the converting enzyme, preferably under physiological conditions. And / or contacting A2254 with a converting enzyme. The modified protein is then tested for the ability to bind to the binding partner (ie, binding of A5623 to A2254). Next, binding to the binding partner of the modified protein, compared to the binding of unmodified control pairs to determine whether a change is required in the above K _a was achieved.

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

Screening Method The above screening embodiment is suitable for high-throughput 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 the A2254 binding domain of the A5623 polypeptide with a polypeptide comprising the A5623 binding domain of the A2254 polypeptide in the presence of the test compound;
(b) detecting binding between polypeptides; and
(c) selecting a test compound that inhibits binding between polypeptides.

  Alternatively, the test compound under investigation may be added to the growing cells to monitor the growth of the treated cells relative to the growth of a control population not receiving the test compound. Suitable cell lines for screening test compounds will be apparent to those skilled in the art from the teachings provided herein.

  For in vivo testing, the test compound may be administered to an accepted animal model. Expression of a foreign gene by introducing a gene into an animal cell can be performed by any method, for example, electroporation (Chu, et al., Nucleic Acids Res 15: 1311-26 (1987)), calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 ( 1984)), Lipofectin method (Derijard B, et al. Cell 7: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)). A gene can express a protein fused to a tag (eg, HA or Myc).

  The intracellular localization of A5623 and A2254 can be examined by immunohistochemical staining. Cells are transfected with tagged A5623, tagged A2254, or combinations thereof. The localization image can be obtained using a microscope such as a fluorescence microscope.

  In a preferred embodiment, cells expressing both A2254 and A5623 can be obtained by transfecting appropriate cell lines with expression vectors for the A2254 and A5623 genes. For example, HEK293, SW480, or COS7 cells can be used as cell lines. Furthermore, the detection reagent is preferably an antibody that recognizes A2254. Alternatively, when A2254 or A5623 is expressed as a fusion protein with a tag, an antibody that recognizes the tag fused with the protein can also be used as a detection reagent. In the kit of the present invention, the antibody may be labeled with a fluorescent substance (for example, FITC, TAMRA, or GFP).

  In particular, as mentioned herein, the inventors have observed that A5623 and A2254 polypeptides interact with each other. Therefore, the interaction of both polypeptides is thought to play an important role in carcinogenesis, particularly breast cancer carcinogenesis. Thus, it is contemplated to screen for compounds useful in the treatment or prevention of cancer, particularly breast cancer, that inhibit the interaction between A5623 polypeptide and A2254 polypeptide or vice versa. That is, in the presence of a test compound, a polypeptide containing the A2254 binding domain of the A5623 polypeptide is contacted with a polypeptide containing the A5623 binding domain of the A2254 polypeptide, and the binding of the two polypeptides is detected. A test compound that inhibits binding between peptides is selected. Of course, alternatively, in the presence of a test compound, a polypeptide comprising the A5623 binding domain of the A2254 polypeptide can be contacted with a polypeptide comprising the A2254 binding domain of the A5623 polypeptide.

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

  The term “contacting” refers to a polypeptide comprising the A2254 binding domain of an A5623 polypeptide in the presence of the test compound by any means and methods known in the art using the test compound. Contacting a polypeptide comprising a binding domain or contacting a polypeptide comprising an A5623 binding domain of an A2254 polypeptide with a polypeptide comprising an A2254 binding domain of an A5623 polypeptide.

  For example, cell assays or cell-free assays can be applied to screen for inhibitors of binding between A5623 and A2254 polypeptides. In an example of a cellular assay, cells that express a polypeptide comprising an A5623 binding domain and a polypeptide comprising an A2254 binding domain can be used for screening. In an example of a cell-free assay, a partially or fully purified polypeptide comprising an A5623 binding domain and a partially or fully purified polypeptide comprising an A2254 binding domain can be used. Of course, it is also possible to use cell extracts of cells that express a polypeptide comprising an A5623 binding domain and a polypeptide comprising an A2254 binding domain.

  Additional examples of test assays or test methods are described herein above.

  "Test compound" or "compound to be tested" means a molecule or substance or compound or composition or agent to be tested by one or more screening methods of the present invention as a putative inhibitor of the interaction between A5623 and A2254, Or any combination thereof. The test compound can be any chemical, such as an inorganic chemical, organic chemical, protein, peptide, carbohydrate, lipid, or combination thereof, or a compound, composition, or drug described herein. It's okay. The term “test compound” as used in the context of the present invention is the term “test molecule”, “test substance”, “potential candidate”, “candidate”, or interchangeable with the above terms herein. It is understood that

  Thus, it is envisaged that small peptides or peptide-like molecules will be used in methods of screening for inhibitors of interaction. Such a small peptide or peptide-like molecule binds and occupies either the A5623 binding domain or the A2254 binding domain or both interaction domains, whereby the A5623 or A2254 binding domain is an A2254 polypeptide or Prevent access to A5623 polypeptide. Furthermore, any biological or chemical composition or substance can be envisaged as an interaction inhibitor. The inhibitory function of an inhibitor can be measured by methods known in the art. Such methods include interaction assays such as immunoprecipitation assays, ELISA methods, RIA methods. Preferred potential candidate molecules or candidate molecule mixtures for use in screening for inhibitors of binding of A5623 to A2254 or vice versa are, inter alia, of chemical or biological origin, natural and / or synthetically. It may be a recombinantly and / or chemically produced substance, compound or composition. Thus, candidate molecules may be proteins, protein fragments, peptides, amino acids, and / or derivatives thereof, or other compounds such as ions that bind to and / or interact with A5623 and / or A2254 polypeptides. . Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevilet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). Yes. A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in bacterial, fungal, plant, and animal extract forms can be obtained, for example, from Pan Laboratories (Bosell, WA) or MycoSearch (North Carolina) or can be easily generated . Furthermore, natural and synthetically produced libraries and compounds are readily modified by 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 with the assay methods described herein. A combinatorial chemical library is a collection of diverse compounds created by combining several chemical “building unit” reactants by chemical or biological synthesis. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in all possible combinations to obtain a peptide of a given length. Millions of compounds can theoretically be synthesized by such combinatorial mixing of chemical building blocks. For example, it is observed that 100 million tetramer compounds or 10 billion pentamer compounds can be synthesized theoretically by systematically combining and mixing 100 compatible chemical building blocks. It has been. (Gallop A, et al. Journal of Medicinal Chemistry, 37 (9) 1233-51 (1994)). Other chemical libraries known to those skilled in the art can also be used, including natural product libraries. Once generated, the combinatorial library is screened for compounds having the desired biological properties.

  In the context of the present invention, a library of compounds is screened to identify compounds that function as inhibitors of binding between A5623 and A2254 polypeptides. For example, a small molecule library is first created using combinatorial library formation methods well known in the art. US Pat. Nos. 5,463,564 and 5,574,656 are two examples of such teachings. The library compound is then screened to identify compounds having the desired structural and functional properties. US Pat. No. 5,684,711 discusses a method for screening a library. Once the screening process has been demonstrated, the polypeptide containing the A2254 binding domain and the polypeptide containing the A5623 binding domain, as well as the compounds in the library are mixed and interacted with each other. In addition, it is next observed whether the compound inhibits the binding of the two polypeptides. As an example, FRET technology can be applied as long as the interaction of both polypeptides emits a fluorescent signal and the test compound inhibits binding between the polypeptides, as long as the fluorescent signal decreases and / or disappears. . Thus, both polypeptides can include a label suitable for FRET. Such labels are generally known in the art.

  The characteristics of each library compound can be analyzed for compounds that exhibit activity in inhibiting polypeptide binding, and features common to the various compounds identified can be isolated and combined into subsequent iterations of the library. Coded as you can. Once the library of compounds has been screened, the next library is created using the chemical building blocks shown in the first round of screening that have the characteristic of having activity against interactions. Using this method, subsequent iterations of the candidate compound will have more structural and functional characteristics necessary for inhibition of the interaction, and ultimately the inhibition of binding between the two polypeptides. A group of inhibitors with high specificity can be found. These compounds can then be further tested for safety and efficacy as drugs for use in animals such as mammals. It will be readily appreciated that this particular screening method is merely exemplary. Other methods are well known to those skilled in the art.

  For example, candidate agents include many chemical species, but typically they are organic molecules, preferably greater than 50 daltons and less than about 2,500 daltons, preferably less than about 750 daltons, more preferably about A low molecular weight organic compound having a molecular weight of less than 350 Daltons.

  Candidate agents also contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonds, and typically contain at least one amine group, carbonyl group, hydroxyl group, or carboxyl group, preferably at least 2 One chemical functional group may be included. Candidate agents often include carbocyclic or heterocyclic structures and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups.

  Exemplary types of candidate agents can include heterocycles, peptides, saccharides, steroids, and the like. The compounds can be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Drug structure identification can be used to identify, create, or screen additional drugs. For example, when a peptide agent is identified, it is functionalized at the amino terminus or carboxyl terminus, using unnatural amino acids such as D-amino acids, especially D-alanine, eg acylation or alkylation for amino groups, carboxyl The group can be modified by various methods such as esterification or amidation to enhance its stability. Other methods of stabilization can include, for example, encapsulation in liposomes.

  As described above, candidate agents are also found in 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, many 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 can be readily generated. Furthermore, natural or synthetically generated libraries and compounds can be readily modified by conventional chemical, physical, and biochemical means and used to create combinatorial libraries. To make structural analogs, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amide formation. The other test compound may be an aptamer, antibody, affybody, Trinectin, Antiticalin, or similar compound. Of course, an aptamer, antibody, affibody, Trinectin, or Anticalin specific for A5623 or A2254 may already itself be an inhibitor of the binding between A5623 and A2254, and thus cancer, especially breast cancer It is considered that it can be easily used in a method for treating or preventing the disease.

  Compounds identified by the methods described herein for screening for inhibitors of binding between A5623 and A2254 polypeptides may be effective in the prevention or treatment of cancer, particularly breast cancer. Thus, inhibitors have the property of interacting directly or indirectly with the A2254 binding domain and / or the A5623 binding domain, and therefore, as described herein, these domains are no longer cancer cells, particularly breast cancer They affect the interaction of these domains so that they cannot interact as they interact in the cell.

  For example, the inhibitor may be an antibody against the A2256 binding domain or the A5623 binding domain. Another example of such an inhibitor may be an affibody, aptamer, Antiticalin, or Trinectin.

Screening and treatment kits
In one embodiment, the present invention provides a product or kit for screening compounds useful for the treatment or prevention of breast cancer, comprising: (a) the A2254 binding domain of A5623 polypeptide (B) the A5623 binding domain of the A2254 polypeptide, and (c) a reagent that detects the interaction between these two polypeptides. As described above, a polypeptide comprising an A2254 binding domain can comprise a full length A5623 polypeptide or an A2254 binding portion thereof. Similarly, a polypeptide comprising an A5623 binding domain can comprise a full length A2254 polypeptide or an A5623 binding portion thereof.

  In a further aspect of the invention, products and kits are provided that comprise substances useful for treating the pathological conditions described herein. The product can include a container of medication as described herein with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container can be formed from a variety of materials such as glass or plastic. In the context of the present invention, a container holds a composition having an active substance effective to treat a cell proliferative disorder, such as breast cancer. In certain embodiments, the active agent in the composition is a test compound (eg, antibody, small molecule, etc.) identified as being capable of inhibiting A5623 / A2254 association in vivo. The label on the container should indicate that the composition is used to treat one or more conditions characterized by abnormal cell growth. The label may also display instructions regarding administration and monitoring techniques such as those described herein.

  In addition to the containers described above, the kit of the present invention may optionally include a second container containing a pharmaceutically acceptable diluent. In addition, along with instructions for use, other materials desirable from a commercial and user standpoint may be included, including other buffers, diluents, filters, needles, syringes, and package inserts.

  The composition may be provided in a pack or dispenser device containing one or more unit dosage forms containing the active ingredients, as appropriate. The pack may include a metal or plastic foil such as, for example, a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In addition, a composition comprising a compound of the invention formulated in a compatible pharmaceutical carrier can be prepared for treatment of a condition in need of treatment and labeled in an appropriate container.

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

[General method]
Cell lines and clinical materials Human breast cancer cell lines HBC4, HBC5, MDA-MB-231 were provided by Dr. Yamori (Cancer Research Foundation, Tokyo), BT-549, MCF-7, T47D, SKBR3, HCC1937, MDA-MB-435S, YMB1, HBL100, and COS-7 were obtained from ATCC. All cells were cultured in appropriate media; i.e. RPMI-1640 (Sigma, St. Louis, MO) was used against BT549, HBC4, HBC5, SKBR3, T47D, YMB1 and HCC1937 (2 mM L-glutamine Add); using Dulbecco's Modified Eagle Medium (Invitrogen, Carlsbad, CA) for HBL100, COS7; 0.1 mM essential amino acid (Roche), 1 mM sodium pyruvate (Roche) for MCF-7, EMEM (Sigma) supplemented with 0.01 mg / ml insulin (Sigma) was used; L-15 (Roche) was used for MDA-MB-231 and MDA-MB-435S. Each medium was supplemented with 10% fetal calf serum (Cansera) and 1% antibacterial / antifungal solution (Sigma). MDA-MB-231 and MDA-MB-435S cells were maintained at 37 ° C. in a humidified environment without CO ₂ . Other cell lines were maintained at 37 ° C. in a humidified environment containing 5% CO ₂ . Clinical samples (breast cancer and normal ducts) were collected from surgical specimens with informed consent for all patients.

To detect genes that were upregulated in common in the isolation breast novel human gene represented by spot A2254 and A5623 on cDNA microarrays of the present inventors, screening the overall expression patterns of 27,648 genes on the microarray I) all 77 premenopausal breast cancer cases, ii) 69 invasive ductal carcinomas, iii) 31 well differentiated disorders, iv) 14 moderately differentiated disorders, or v) 24 poorly differentiated lesions Genes with an expression ratio> 3.0 at each> 50% were selected. Of all 493 genes up-regulated in tumor cells, the expression ratio exceeded 3.0 in more than 50% of informative breast cancer cases, and the expression profile of normal human tissues indicated that the heart, lung, liver, kidney, and We focused on genes with institutional identification numbers A2254 and A5623 because they showed low expression in normal organs including bone marrow.

Semi-quantitative RT-PCR analysis Total RNA was extracted from each population of laser-captured cells and then subjected to T7-based amplification and reverse transcription as previously described (Ono K, et al., Cancer Res., 60, 5007-11, 2000). Appropriate dilutions of each single-stranded cDNA were prepared for subsequent PCR amplification by monitoring glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a quantitative internal control. The PCR primer sequences are as follows.
Against GAPDH
5'-CGACCACTTTGTCAAGCTCA-3 '(SEQ ID NO: 1) and
5'-GGTTGAGCACAGGGTACTTTATT-3 '(SEQ ID NO: 2),
against β2MG
5'-AACTTAGAGGTGGGAGCAG-3 '(SEQ ID NO: 45) and
5'-CACAACCATGCCTTACTTTATC-3 '(SEQ ID NO: 46),
Against A2254V1
5'-ACTCTAGGACTTGCATGATTGCC-3 '(SEQ ID NO: 3) and
5'-TGGGTGTCAAACCAAACAGA-3 '(SEQ ID NO: 4),
Against A2254V2
5'-GTTAGAACTTGTTTCCTCCTCCG-3 '(SEQ ID NO: 5) and
5'-ATCCTCAATGGTATTTCAGC-3 '(SEQ ID NO: 6),
A5623 common area
5'-GTGGTCCTAGGAGACTTGGTTTT-3 '(SEQ ID NO: 7) and
5'-TACATGCATACCCCCAACAA-3 '(SEQ ID NO: 8),
Against A5623V1
5'-GCTTCAGCGAGAACTTTC-3 '(SEQ ID NO: 9) and
5'-CAACTGTAACACTCATTCACATC-3 '(SEQ ID NO: 10),
Against A5623V2
5'-CTATTCTGAGTTTGCGCGAGAAC-3 '(SEQ ID NO: 11) and
5'-CAACTGTAACACTCATTCACATC-3 '(SEQ ID NO: 10),
Against A5623V3
5'-CATCCTGAGTGCGAGAACTTTC-3 '(SEQ ID NO: 12) and
5'-CAACTGTAACACTCATTCACATC-3 '(SEQ ID NO: 10).

Northern blot analysis Total RNA was extracted from all breast cancer cell lines using the RNeasy kit (QIAGEN) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated using an mRNA purification kit (Amersham Biosciences) according to the manufacturer's instructions. 1% denatured agarose in 1 μg aliquots of each mRNA with poly A (+) RNA isolated from normal adult human breast (Biochain), lung, heart, liver, kidney, bone marrow (BD, Clontech, Palo Alto, Calif.) The gel was separated and transferred to a nylon membrane (breast cancer northern blot). Breast cancer northern blots and human multi-tissue northern blots (Clontech, Palo Alto, Calif.) Were hybridized with [α ³² P] -dCTP labeled PCR products of A2254 and A5623 prepared by RT-PCR (see below) ). Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. Blot autoradiography was performed at −80 ° C. for 14 days using an intensifying screen. Specific probes for A2254 (548 bp) and A5623 (454 bp) were prepared by PCR using the following primer sets:
For A2254 common area
5'-ACTCTAGGACTTGCATGATTGCC-3 '(SEQ ID NO: 3) and
5'-TGGGTGTCAAACCAAACAGA-3 '(SEQ ID NO: 4),
A5623 common area
5'-GTGGTCCTAGGAGACTTGGTTTT-3 '(SEQ ID NO: 7) and
5′-TACATGCATACCCCCAACAA-3 ′ (SEQ ID NO: 8).
In addition, a specific A2254V1 probe (350 bp) was prepared by PCR using the following specific primer set:
5'-CTGGAAGAGCAGGCTAGCAG-3 '(SEQ ID NO: 13) and
5′-GCTGCGGAGAAAGCTCTATG-3 ′ (SEQ ID NO: 14).

Construction of expression vector
In order to construct A5623 and A2254 expression vectors, the entire coding sequence of PRC1 cDNA was amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan). The primer sets were as follows:
A5623-forward,
5'-CCG GAATTC TCCGCCATGAGGAGAAGTGA-3 '(SEQ ID NO: 47)
(Underlined indicates the EcoRI site) and
A5623-Reverse,
5'-TTGCCG CTCGAG GGACTGGATGTTGGTTGAA-3 '(SEQ ID NO: 48)
(Underlined indicates XhoI site),
A2254-forward,
5'-CCG GAATTC ATGGCCATGGACTCGTCG-3 '(SEQ ID NO: 49) and
A2254-Reverse,
5′-GCTCCG CTCGAG CTGGGGCCGTTTCTT-3 ′ (SEQ ID NO: 50).
The PCR product was inserted into the EcoRI and XhoI sites of the pCAGGS-nHA or pCAGGS-Flag expression vector.

Anti-A5623 specific polyclonal antibody and anti-A2254 specific polyclonal antibody
Plasmids designed to express two fragments of A5623 (179-360 and 234-360 aa) or A2254 (86-239 and 124-239 aa) with His-tagged epitopes at the C-terminus were respectively expressed as pET21 Prepared using vector (Novagen, Madison, WI). 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 protocol. Purified recombinant protein was mixed and then rabbits were immunized. The immune serum was purified on an affinity column according to standard methods. Affinity purified anti-A5623 or anti-A2254 antibodies were used for Western blotting, immunoprecipitation, and immune cell staining as described below. Western blot analysis confirmed that these antibodies could specifically recognize endogenous A5623 protein or A2254 protein in MCF7 breast cancer cells, respectively.

To examine the subcellular localization of endogenous A5623 or A2254 protein in immunocytochemical staining breast cancer cell lines, MCF7, HBC4, and HBC5, cells were seeded at 1 × 10 ⁵ cells / well (Lab-Tek II chamber slide, Nalgen Nunc International, Naperville, Illinois). After 24 hours of incubation, cells are fixed with PBS (-) containing 4% paraformaldehyde for 15 minutes and permeabilized with PBS (-) containing 0.1% Triton X-100 for 2.5 minutes at 4 ° C. It was. Subsequently, the cells were coated with 3% BSA in PBS (-) for 12 hours at 4 ° C to block non-specific hybridization, followed by 1/1000 dilution of rabbit anti-A5623 polyclonal antibody or 1/1000 dilution of Incubated with anti-A2254 polyclonal antibody. After washing with PBS (-), cells were stained with 1/1000 dilution of Alexa488-conjugated anti-rabbit secondary antibody (Molecular Probe, Eugene, OR). The nuclei were counterstained with 4 ', 6'-diamidino-2'-phenylindole dihydrochloride (DAPI). Fluorescence images were acquired under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Western blotting analysis Using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions, 1 μg of pCAGGS-A2254-HA, pCAGGS-A5623V1-HA, pCAGGS-A5623V2-HA, or pCAGGS-A5623V3- HA was transiently transfected. Cell lysates were separated on 10% SDS polyacrylamide gels, transferred to nitrocellulose membranes, and then incubated with mouse anti-HA antibody (Roche) as primary antibody at 1/1000 dilution. After incubation with sheep anti-mouse IgG-HRP (Amersham Biosciences) as a secondary antibody, signals were visualized using the ECL kit (Amersham Biosciences).

  In addition, endogenous A5623 protein in breast cancer cell lines (BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3, and T47D) and HMEC (human mammary epithelial cells) Alternatively, to detect A2254 protein, cells were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0 / 150 mM NaCl / 0.5% NP-40) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, CA). ). The amount of total protein was measured with a protein assay kit (Bio-Rad, Hercules, Calif.), Then the protein was mixed with SDS sample buffer, boiled and then added to a 10% SDS-PAGE gel. Following electrophoresis, proteins were blotted onto nitrocellulose membrane (GE Healthcare). The membrane containing the protein was blocked with a blocking solution and incubated with anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody to detect endogenous A5623 protein or A2254 protein. Finally, the membrane was incubated with HRP-conjugated secondary antibody and protein bands were visualized with ECL detection reagent (GE Healthcare). β-actin was also tested and used as a control for addition.

Immunocytochemical staining
To examine the subcellular localization of A2254, or A5623V1, A5623V2, and A5623V3, COS7 cells were seeded at 1 × 10 ⁵ cells / well for all four constructs. After 24 hours, 1 μg each of pCAGGS-A2254-HA, pCAGGS-A5623V1-HA, pCAGGS-A5623V2-HA, or pCAGGS-A5623V3 in COS7 cells using FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions. -Transiently transfected with HA. The cells were then fixed for 15 minutes with PBS containing 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100 for 2.5 minutes at 4 ° C. Subsequently, cells were coated with 3% BSA PBS for 12 hours at 4 ° C. to block non-specific hybridization. Next, each transfected COS7 cell was incubated with a 1/1000 dilution of rat anti-HA antibody (Roche). After washing with PBS, all transfected cells were stained with 1/1000 dilution of Alexa594-conjugated anti-rat secondary antibody (Molecular Probe). The nuclei were counterstained with 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescence images were acquired under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Construction of A2254 and A5623-specific siRNA expression vectors using psiU6X3.0 A vector-based RNAi system was established using a psiU6BX siRNA expression vector according to a previous report (WO2004 / 076623). siRNA expression vectors for A2254 (psiU6BX-A2254) and A5623 (psiU6BX-A5623) were prepared by cloning the double-stranded oligonucleotides of Table 1 into the BbsI site of the psiU6BX3.0 vector. In the BbsI site of the psiU6BX3.0 vector
5'-CACCGAAG CAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3 '(SEQ ID NO: 15), and
5'-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3 '(SEQ ID NO: 16)
A control plasmid, psiU6BX-Mock, was prepared by cloning the double stranded oligonucleotide.

Oligonucleotide sequence for siRNA
The oligonucleotide sequences used for the A2254 and A5623 small interfering RNAs are shown below.

下線は特異的siRNA配列を示す。 Table 1 Oligonucleotide sequences for A2254 and A5623 small interfering RNAs

Underline indicates specific siRNA sequences.

A2254 and A5623 gene silencing human breast cancer cell lines, they were plated T47D and HBC5 in 10cm Petri dish (1x 10 ⁶ cells / dish), using FuGENE6 reagent (Roche) according to the recommendations of the supplier, as a negative control psiU6BX-Mock, psiU6BX-A2254, or psiU6BX-A5623 were transfected. Seven days after transfection of each construct, total RNA was extracted from the cells, and then the siRNA knockdown effect was confirmed by semi-quantitative RT-PCR using specific primers for the common region of A2254 and A5623 described above . Primers for GAPDH as an internal control are as follows:
5'-CGACCACTTTGTCAAGCTCA-3 '(SEQ ID NO: 1) and
5'-GGTTGAGCACAGGGTACTTTATT-3 '(SEQ ID NO: 2).
Furthermore, transfectants expressing siRNA using T47D and HBC5 cell lines were cultured for 28 days in a selective medium containing 0.7 mg / ml neomycin. After fixation with 4% paraformaldehyde, the transfected cells were stained with Giemsa solution to assess colony formation. To quantitate cell viability, an MTT assay was performed. After culturing in neomycin-containing medium for 12 days, MTT solution (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma) was added at a concentration of 0.5 mg / ml. . After incubation for 2.5 hours at 37 ° C, acid-SDS (0.01N HCl / 10% SDS) was added; the suspension was mixed vigorously to dissolve the dark blue crystals and then overnight at 37 ° C. Incubated. Absorbance at 570 nm was measured with a Microplate Reader 550 (BioRad).

Immunoprecipitation and Western blotting cells 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, Calif.)) I let you. Equal amounts of total protein were incubated with 1 mg rat anti-HA antibody (Roche) or mouse anti-Flag antibody (Santa Cruz) for 1 hour at 4 ° C. Immune complexes were incubated with protein G-Sepharose (Zymed Laboratories, South San Francisco, Calif.) For 1 hour and then washed with lysis buffer. Coprecipitated proteins were separated by SDS-PAGE. Proteins separated by SDS-PAGE are transferred to nitrocellulose membrane, then incubated with rat anti-HA antibody or mouse anti-Flag antibody, incubated with HRP-conjugated secondary antibody, and then signaled using ECL kit (Amersham Biosciences) Visualized.

Establishment of NIH3T3 cells stably expressing A5623 or A2254
A5623 or A2254 expression vector or mock vector was transfected into NIH3T3 cells using FUGENE6 as described above. Transfected cells were incubated in medium containing 0.9 mg / ml geneticin (G418) (Invitrogen). Clone NIH3T3 cells were subcloned by limiting dilution. The expression of HA-tagged A5623 or A2254 was evaluated by Western blot analysis using anti-HA monoclonal antibody. Finally several clones were established and named A5623-NIH3T3 or A2254-NIH3T3.

  In order to examine the growth promoting effect of A2254, 5000 cells each of 4 independent A2254-NIH3T3 cells and 3 independent MOCK-NIH3T3 cells were seeded, and the number of cells was counted by MTT assay daily for 6 days. These experiments were performed three times.

Furthermore, the infiltration of A2254-NIH3T3 cells (A2254-NIH3T3-3 and -4) was examined by Matrigel. Briefly, seed 10,000 cells each of two independent A2254-NIH3T3 cells (A2254-NIH3T3-3 and -4) and one independent MOCK-NIH3T3 cell in DMEM containing 10% FCS. Culture to confluent stage. Cells were collected by trypsinization, washed with DMEM without addition of serum and protease inhibitors, and suspended in DMEM at a concentration of 1 × 10 ⁵ cells / mL. Prior to preparing the cell suspension, a dried layer of Matrigel matrix (Becton Dickinson Labware, Bedford, Mass.) Was rehydrated with DMEM for 2 hours at room temperature. DMEM containing 10% FBS was added to each lower chamber of the 24-well Matrigel invasion chamber, and 0.5 mL of cell suspension (5 × 10 ⁴ cells) was added to each insert in the upper chamber. Plates were incubated at 37 ° C. for 22 hours. After incubation, the chambers were processed as directed by the supplier (Bection Dickinson Labware) and cells that had invaded through the Matrigel-coated insert were fixed and stained with Giemsa.

Immunohistochemical staining The expression pattern of A5623 protein in breast cancer tissue and normal tissue was examined using anti-A5623 rabbit polyclonal antibody. Briefly, paraffin-embedded specimens were treated with xylene and ethanol and blocked with a protein blocking reagent (Dako Cytomation, Carpinteria, CA). Polyclonal antibody (1/100) in antibody dilution was added and then stained with substrate-chromogen (DAKO liquid DAB chromogen, DakoCytomation). Finally, tissue specimens were stained with hematoxylin to distinguish between nucleus and cytoplasm.

[result]
Identification of A2254 and A5623 as genes up-regulated in breast cancer cells
Using a cDNA microarray corresponding to 27,648 human genes, we analyzed gene expression profiles of cancer cells from 77 premenopausal breast cancer patients and identified 493 genes that were commonly up-regulated in breast cancer cells . Among them, a gene having institutional code A2254 (SEQ ID NO: 35, 36) representing kinesin family member 2C (KIF2C), and a gene having institutional code A5623 representing protein regulator 1 (PRC1) of cytokinesis ( Attention was paid to Genbank accession number NM_003981; SEQ ID NO: 39, 40). A2254 and A5623 gene expression was elevated in 44 of 60 breast cancer cells and 37 of 58 breast cancer cells, respectively, in the microarray compared to normal breast duct cells. To confirm the expression of these upregulated genes, semiquantitative RT-PCR analysis was performed to compare the expression levels of breast cancer samples with normal human tissues including normal ductal cells. Initially, A2254 expression was observed in 7 of 12 clinical breast cancer samples (poorly differentiated) compared to normal breast duct cells and normal human tissues including breast, lung, heart, liver, kidney, and bone marrow. In the example, it was found to show increased expression (FIG. 1A, upper panel). Furthermore, this gene was similarly overexpressed in all nine breast cancer cell lines (FIG. 1A, lower panel). Next, the expression of A5623 shows increased expression in 7 out of 12 clinical breast cancer samples (poorly differentiated) compared to normal human tissues, especially normal duct cells (Figure 1B, upper panel), It was also found that it was overexpressed in all nine breast cancer cell lines examined (FIG. 1B, lower panel).

  To further investigate the expression patterns of these genes, Northern blot analysis was performed on multiple human tissues and breast cancer cell lines using A2254 and A5623 cDNA fragments as probes (see Materials and Methods). A2254 is not expressed at all in normal human tissues except testis and thymus or is undetectable (FIG. 2A; upper panel), whereas breast cancer cells compared to other normal tissues except bone marrow Surprisingly overexpressed in all of the strains (Figure 2A; lower panel). A5623 is also expressed only in the testis (Figure 2B, upper panel), while significantly overexpressed in all breast cancer cell lines compared to other normal tissues except bone marrow, especially normal human breast (Figure 2B, lower panel). Therefore, the present inventors have focused on transcripts that are specifically expressed in breast cancer.

Genomic structure of A2254 and A5623
In order to obtain the entire cDNA sequences of A2254 and A5623, RT-PCR was performed using a breast cancer cell line, T47D as a template. A2254 consists of 21 exons, represents kinesin family member 2C (KIF2C), and is located on chromosome 1p34.1. The full length mRNA sequence of A2254 contained 2886 nucleotides and encoded 725 amino acids.

  A2254 is two different transcriptional mutations consisting of 21 and 20 exons, corresponding to A2254V1 (GenBank accession number; AB264115, SEQ ID NO: 35, 36) and A2254V2 (GenBank accession number; AY026505, SEQ ID NO: 37, 38), respectively Has a body (Figure 3A, upper panel). Exon 1 and 2 of the V1 variant are 185 bp and 94 bp, respectively, while the V2 variant does not have exon 1 and 2 of V1, but has a new exon consisting of 346 bp as exon 1 . The final exon of the V2 variant (exon 20) was 537 bp shorter than the 3 ′ end of the final exon of the V1 variant (exon 21). The full length cDNA sequences of the A2254V1 and A2254V2 variants contained 2886 and 2401 nucleotides, respectively. The ORF of these mutants starts from within each exon 1. Finally, the V1 and V2 transcripts encode 725 and 671 amino acids, respectively. To further confirm the expression pattern of each mutant in breast cancer samples and normal human tissues, semi-quantitative RT-PCR was performed using a primer set that reacts with each mutant. As a result, the A2254 V1 mutant was significantly expressed in breast cancer cells compared to the expression of the V2 mutant, whereas the V2 mutant was found to be expressed only in the testis (FIG. 3A). ; Lower panel). Therefore, we focused on the A2254 V1 mutant.

  A5623 is similarly A5623V1 (GenBank accession number; NM_003981; SEQ ID NO: 39, 40), A5623V2 (GenBank accession number; NM_199413; SEQ ID NO: 41, 42), and 5623V3 (GenBank accession number; NM_199414; SEQ ID NO: 43, 44) with three different transcription variants consisting of 15, 14, and 14 exons (FIG. 3B, upper panel). There were selective changes in exons 13 and 14 of V1, and the other remaining exons were common to all mutants. The V2 variant does not have exon 14 of V1 and a new early stop codon occurs within the final exon. Exon 14 of the V3 variant was completely deleted, exon 13 of V3 was 77 bp shorter than exon 13 of V1 at the 3 'end, as well as a new early stop codon in the final exon . The full-length cDNA sequences of A5623V1, A5623V2, and A5623V3 variants consist of 3128, 3091, and 3011 nucleotides, respectively. The ORF of these mutants starts from within each exon 1. Finally, the V1, V2, and V3 transcripts encode 620, 606, and 566 amino acids, respectively. To further confirm the expression pattern of each mutant in breast cancer samples and normal human tissues, semi-quantitative RT-PCR was performed using a primer set that reacts with each mutant. As a result, all mutants were found to be highly overexpressed in breast cancer cells compared to normal human tissues (FIG. 3B, lower panel). Therefore, further functional analysis is performed on all variants of A5623.

Intracellular localization of A2254 and A5623
To further characterize A2254 and A5623, the subcellular localization of these gene products in COS7 cells was examined. Initially, a plasmid expressing the A2254 protein (pCAGGS-A2254-HA) was transiently transfected into COS7 cells, and 81 KD-A2254 protein was found as predicted by Western blot analysis ( Figure 4A). In addition, immunocytochemical staining reveals that exogenous A2254 was located under the plasma membrane in the transfected cells (FIG. 4B). Surprisingly, the positive signal in immunocytochemical staining disappears in the cell-cell attachment membrane, suggesting that this gene may play an important role in cell-cell interactions or cell polarity. The

  A plasmid expressing the A5623V1, V2, and V3 proteins (pCAGGS-A5623-HA) was then transiently transfected into COS7 and T47D cells as well, and the foreign A5623 V1, V2, and V3 proteins were The size was estimated as predicted by blot analysis (FIG. 4C). In addition, immunocytochemical staining revealed that all mutant A5623 proteins were localized in organelles as intermediate filaments in transfected cells (FIGS. 4D, E, F), which also allowed A5623 It is suggested that it can play an important role in cell-cell interactions.

  After producing polyclonal antibodies against A5623 or A2254, using HMEC (human mammalian epithelial cells) as an experimental control, breast cancer cell lines, BT-474, BT-549, HBC4, HBC5, HBL-100, MCF-7, Endogenous expression of A5623 or A2254 protein in cell lysates from MDA-MB-231, SKBR3, and T47D was examined by Western blot analysis (FIG. 7A). All 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 lines, HBC4, HBC5, and MCF7 using anti-A5623 polyclonal antibodies shows that endogenous A5623 is mainly localized in the cytoplasm and / or nuclear organ of interphase cells It was done. In particular, endogenous A5623 was found in the intermediate filament network in all breast cancer cell lines. As the cells progressed through mitosis, A5623 remarkably redistributed. A5623 was localized with the spindle pole in the early division, and then with the entire spindle from the middle stage to the early stage of the division. By the middle of mitosis, A5623 concentrated in cells like a series of thin rods in the mitotic spindle midzone (FIG. 7B). Subsequent immunocytochemical analysis of a breast cancer cell line, HBC5, using an anti-A2254 polyclonal antibody showed that exogenous A2254 was located under the plasma membrane in the transfected cells, but endogenous A2254 was mainly the cell size of interphase cells. It was shown to be localized in the organ. As the cells progressed to mitosis, A2254 caused a significant redistribution. Although A2254 was still localized in the cytoplasm in metaphase cells, it concentrated like a series of thin bars in the mid-spindle spindle of the cell (FIG. 7B). Eventually, this protein accumulated in the midbody in end-stage cells. These findings suggest an important role for A5623 and A2254 in cytokinesis in breast cancer cells, similar to previously described HeLa cells (Mollinari C, et al. J Cell Biol, 2002; 157; 1175 -86 .; Mollinari C, et al. Mol Biol Cell. 2005; 16; 1043-55.).

  To further investigate A5623 expression in breast cancer tissue sections and normal tissue sections, immunohistochemical staining was performed using anti-A5623 antibody. Strong staining was identified in the cytoplasm and nucleus of three different histological subtypes of breast cancer, secretory ductal carcinoma, papillary duct carcinoma, and hard carcinoma, but its expression was almost undetectable in normal breast tissue (FIG. 7C, upper panel). Furthermore, in agreement with the results of Northern blot analysis, its expression was detected in the testis, and no expression was observed in any of heart, lung, liver, and kidney (FIG. 7C, lower panel).

Growth inhibitory effect of small interfering RNA (siRNA) designed to reduce the expression of A2254 and A5623
Expression of endogenous A2254 and A5623 in breast cancer cell lines T47D and HBC5 that showed overexpression of A2254 and A5623 by mammalian vector-based RNA interference (RNAi) technique to assess the role of A2254 and A5623 in promoting growth Was knocked down (see above). The expression levels of A2254 and A5623 were examined by semi-quantitative RT-PCR experiments. As shown in FIGS. 5 and 6, the two siRNA constructs for each gene, A2254 (si2 and si5) and A5623 (si1 and si2) specific siRNAs, respectively, compared to the control siRNA construct (psiU6BX-Mock), respectively. The expression of the gene was significantly suppressed (FIGS. 5A and 6A). To confirm cell growth inhibition by A2254 and A5623 specific siRNA, colony formation assay and MTT assay were performed, respectively. Introducing A2254 constructs (si2 and si5) (Figures 5B, C) and A5623 constructs (si1 and si2) (Figures 6B, C) suppressed the growth of T47D and HBC5 cells, consistent with the above expression reduction results It was done. Each result was confirmed by three independent experiments. These findings suggest that A2254 and A5623 have important functions in breast cancer cell proliferation.

Carcinogenic activity of A2254
In order to further confirm the growth promoting effect of A2254, NIH3T3-derived cells (NIH3T3-A2254-1, -2, -3, and 4) stably expressing foreign A2254 were established. Western blot analysis showed high levels of foreign A2254 protein in three derived clones (NIH3T3-A2254-1, -2, and -3) and low levels of A2254 in one clone (A2254-4) ( FIG. 9A). Subsequent MTT assay showed that overexpression of exogenous A2254 did not significantly enhance cell proliferation (FIG. 9B). These findings suggest that the absence of the A2254 gene product has a significant effect on the survival of breast cancer cells, but there is no growth enhancing activity when only this gene is overexpressed.

  Furthermore, since it was first observed that exogenous A2254 was located below the plasma membrane in the transfected cells, a matrigel invasion assay was performed to determine whether A2254 has any role in cell motility. Matrigel-mediated invasion of NIH3T3-derived cells stably expressing foreign A2254 (NIH3T3-A2254-3 and -4) depends on the expression of A2254 protein according to Western blot results, and Mock stable transfected cells (NIH3T3- Mock) was significantly enhanced (FIG. 9C), suggesting that the A2254 gene also has an important role in tumor cell invasion.

Interaction of A5623 with A2254 To further investigate the physiological function of A5623 in breast cancer cells, we attempted to identify its interacting protein. As a possible candidate A5623 interacting protein, the kinesin family member 2C / mitotic centromere-associated kinesin (KIF2c / MCAK) protein (A2254) was found. The reason is that this protein is known to localize in the middle body or near the contractile ring in late or postmitotic cells, and to function in intermediate zone formation and cytokinesis. In addition, A5623 has been 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.).

  First, we examined the expression pattern of A2254 in breast cancer cases by semi-quantitative RT-PCR analysis, and found that A2254 and A5623 were simultaneously up-regulated in breast cancer cases (FIG. 8A). Subsequently, co-immunoprecipitation experiments were performed using Flag tag labeled A5623 and HA tag labeled A2254 cotransfected into COS7 cells. Similarly, using anti-Flag and anti-HA antibodies, Flag tag labeled A5623 coprecipitated with HA tag labeled A2254 (Figure 8B), and conversely, HA tag labeled A2254 coprecipitated with Flag tag labeled A5623. Confirmed (FIG. 8C), thus indicating the interaction of these two proteins.

  Next, NIH3T3-derived cells (NIH3T3-A5623 and NIH3T3-A2254) stably expressing foreign A5623 and A2254 were established. From immunocytochemical analysis, endogenous mouse A5623 in NIH3T3 cells and stably expressed exogenous A2254 co-localize in late metaphase late zone formation (Figure 8D, left panel), and NIH3T3 It was revealed that endogenous A2254 in the cell and stably expressed foreign A5623 co-localize with the intermediate zone formation in the late mitotic phase (FIG. 8D). Although further investigation is needed regarding the colocalization of endogenous A5623 and endogenous A2254 in breast cancer cells, these results indicate that the A5623 and A2254 conformation plays an important role in cancer cell cytokinesis. Strongly suggested.

[Discussion]
With the accurate expression profile of whole-genome breast cancer by cDNA microarray, we isolated A2254 and A5623 as novel genes that are significantly overexpressed in breast cancer cells compared to normal human tissues.

  Furthermore, Northern blot analysis showed that A5623 and A2254 expression was almost undetectable in any normal human tissue examined except for testis and bone marrow. Furthermore, immunohistochemical staining experiments using anti-A5623 polyclonal antibody or anti-A2254 polyclonal antibody clearly showed up-regulation of A5623 or A2254 expression in breast cancer tissue sections. These results indicate that this gene is a useful target for developing anticancer agents against breast cancer.

  According to our immunocytochemical staining experiments, A5623 is localized in the cytoplasm and / or nucleus of breast cancer cells in the interphase, in the intermediate zone in the late mitotic phase, and in the contractile ring at the end of division. It was shown to be localized.

  Furthermore, it was demonstrated that treatment of breast cancer cells with siRNA effectively inhibited expression of all three target genes, A2254 and A5623, and significantly suppressed breast cancer cell / tumor growth. These findings suggest that A2254 and A5623 play an important role in tumor cell growth and may be promising targets for developing anticancer agents.

  The inventors have demonstrated that knocking down endogenous A5623 with siRNA prevents cytokinesis in breast cancer cells, resulting in multinucleated cell accumulation and subsequent cell death. These findings also suggest that A5623 plays a role in cytokinesis of breast cancer cells. A5623 has also been 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.). It has also been reported that A5623 can interact with several molecules such as KIF4 or KIF14 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, according to our breast cancer expression profile, neither KIF4 nor KIF14 was expressed in breast cancer (data not shown). Therefore, we further investigated the biological role of A5623 in breast cancer cells by identifying its interacting proteins and identified the A2254 (kinesin family member 2C / mitotic centromere-binding kinesin) protein as a candidate interacting protein . The reason is that this protein is known to localize in the middle body or near the contractile ring in late or postmitotic cells, and to function in intermediate zone formation and cytokinesis. As shown in FIG. 8, in vivo interaction and colocalization of A2254 and A5623 in the midbody during the cell cycle, especially during cytokinesis of end-stage cells, was demonstrated. Such evidence in breast cancer cases suggested that they interact to play an important role in breast carcinogenesis. In addition, the binding region of A5623 to A2254 was determined (51-70 and 200-490 amino acids) (data not shown). Although further analysis of the function of A5623 is needed, the data provided should contribute to a deeper understanding of breast cancer carcinogenesis and the development of new therapies for breast cancer.

Industrial Applicability The present inventors have shown that cell growth is suppressed by small interfering RNA (siRNA) that specifically targets the A2254 or A5623 gene. Therefore, this novel siRNA is a useful target for the development of anticancer drugs. For example, substances that block the expression of A2254 or A5623 or prevent its activity may be therapeutically useful as anticancer agents, particularly anticancer agents for the treatment of breast cancer (BRC).

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

Tumor cells from breast cancer patients (upper panel) (3T, 31T, 149T, 175T, 431T, 453T, 491T, 554T, 571T, 709T, 772T, and 781T), breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, MCF7 , MDA-MB-231, SKBR3, T47D, YMB1) (bottom panel) and semi-quantitative RT-PCR results for (A) A2254 and (B) A5623 expression in normal human tissues. Shown are photographs of Northern blot analysis of (A) A2254 and (B) A5623 transcripts in various human tissues (upper panel) and breast cancer cell lines and normal human vital organs (lower panel). The genomic structures of (A) A2254 and (B) A5623 are shown. A2254 has two different mutants called V1 and V2, and A5623 has three different mutants (V1, V2, and V3) as well. Upper panel; genome structure, lower panel; results of each mutant-specific semi-quantitative RT-PCR. FIG. 4A shows foreign expression of A2254 protein by Western blot analysis. FIG. 4B shows the intracellular localization of A2254 protein. FIG. 4C shows foreign expression of A5623V1, A5623V2, and A5623V3 proteins by Western blot analysis. 4D-F show the intracellular localization of (D) A5623V1, (E) A5623V2, and (F) A5623V3 proteins. FIG. 6 shows the growth inhibitory effect of small interfering RNA (siRNA) designed to reduce the expression of A2254 in breast cancer cells. FIG. 5A shows semi-quantitative 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. FIG. 5B shows an MTT assay showing a decrease in the number of colonies due to A2254 knockdown in T47D cells (left panel) and HBC5 cells (right panel). FIG. 5C shows a colony formation assay showing a decrease in the number of colonies due to A2254 knockdown in T47D cells (left panel) and HBC5 cells (right panel). FIG. 6 shows the growth inhibitory effect of small interfering RNA (siRNA) designed to reduce the expression of A5623 in breast cancer cells. FIG. 6A shows semi-quantitative RT-PCR showing suppression of endogenous expression of A5623 in breast cancer cell lines T47D cells (left panel) and HBC5 cells (right panel). GAPDH was used as an internal control. FIG. 6B shows an MTT assay showing a decrease in the number of colonies due to A5623 knockdown in T47D cells (left panel) and HBC5 cells (right panel). FIG. 6C shows a colony formation assay showing a decrease in the number of colonies due to A5623 knockdown in T47D cells (left panel) and HBC5 cells (right panel). Expression of A5623 and A2254 in breast cancer cell lines and tissue sections. FIG. 7A, Endogenous A5623 and A2254 protein expression in breast cancer cell lines compared to HMEC cell lines, examined by Western blot analysis using anti-A5623 or anti-A2254 antibodies. FIG. 7B, intracellular localization of endogenous A5623 or A2254 protein in breast cancer cells during the cell cycle. Immunize HBC4, HBC5, and MCF7 cells for A5623 and HBC5 cells for A2254 using affinity-purified anti-A5623 or anti-A2254 polyclonal antibodies (green) and DAPI (blue) to identify nuclei Cytochemical staining (see materials and methods). White arrows indicate the localization of A5623 in the centrosome of end-stage cells. FIG. 7C, results of immunohistochemical staining of breast cancer tissue sections and normal tissue sections (normal breast tissue, lung, heart, liver, kidney, and testis). Endogenous A5623 protein was stained with anti-A5623 antibody. Little expression was detected in normal breast tissue (sample # 10441), but examined including solid ductal carcinoma (sample # 234), papillary ductal carcinoma (sample # 240), and hard cancer (sample # 179) In all cancer tissues, cancer cells were strongly stained. A representative figure is from a microscopic observation at the initial magnification, x200. Interaction between A5623 and A2254. FIG. 8A, breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, MCF7, MDA-MB-231, SKBR3, T47D, YMB1) and normal human tissues (N; normal duct cells, MG, by semi-quantitative RT-PCR Expression of A5623 and A2254 in mammary gland, LUN; lung, LIV; liver, HEA; heart, KID; kidney, and BM; 8B, C, co-immunoprecipitation of A5623 and A2254. Cell lysates of COS7 cells transfected with HA tag-tagged A2254 protein and Flag tag-tagged A5623 protein were immunoprecipitated with anti-HA or anti-Flag. Immunoprecipitates were immunoblotted with monoclonal anti-HA antibody or anti-Flag antibody. FIG. 8D, intracellular localization of endogenous A5623 or A2254 in stably expressing cells. The left panel shows that endogenous A5623 protein (red) co-localized with foreign A2254 protein (green) in stable A2254 expressing cells, right panel shows endogenous A2254 (red) and foreign A5623 in stable A5623 cells. Co-localization of (green) is shown. Endogenous A2254 protein (red) co-localized with foreign A5623 protein (right panel). Growth promotion or invasion effect of exogenous A2254 in NIH3T3 cells. FIG. 9A, Western blot analysis of cells expressing high or medium levels of exogenous A2254, or cells transfected with a mock vector. Exogenous induction of A2254 expression was confirmed by anti-HA tag monoclonal antibody. β-actin was added as a control. FIG. 9B, in vitro growth of NIH3T3-A2254 cells. A2254 (NIH3T3-A2254- # 1,-# 2,-# 3, and-# 4) and mock (NIH3T3-Mock- # 1,-# 2,-# 3) were transfected as measured by MTT assay NIH3T3 cells. FIG. 9C, Matrigel invasion assay showing enhanced invasiveness of NIH3T3-A2254-# 3 and-# 4 and NIH3T3-Mock- # 1. The number of cells migrating through the matrigel-coated filter was counted. The assay was performed 3 times in triplicate wells.

Claims (30)

  Breast cancer in a subject comprising administering to the 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). How to treat or prevent.   2. The method of claim 1, wherein the siRNA comprises a sense nucleic acid sequence and an antisense nucleic acid sequence that specifically hybridizes to a sequence derived from A2254 or A5623.   3. The method of claim 2, wherein the siRNA comprises a ribonucleotide sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 21, 25, 29, and 33 as a target sequence. siRNA is a general formula
5 '-[A]-[B]-[A']-3 '
Where
[A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of the 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],
The method of claim 3.   2. The method of claim 1, wherein the composition comprises a transfection facilitating agent.   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 A double-stranded molecule comprising a sense strand containing a ribonucleotide sequence complementary to the sense strand, the sense strand and the antisense strand hybridizing to each other, and expressing a cell expressing the A2254 or A5623 gene A double-stranded molecule that inhibits the expression of the gene when introduced.   7. The double-stranded molecule of claim 6, wherein the target sequence comprises at least about 10 consecutive nucleotides derived from a nucleotide sequence selected from the group of SEQ ID NOs: 35, 37, 39, 41, and 43.   8. The double-stranded molecule of claim 7, wherein the target sequence comprises about 19 to about 25 contiguous nucleotides derived from a nucleotide sequence selected from the group of SEQ ID NOs: 35, 37, 39, 41, and 43. .   9. The double-stranded molecule of claim 8, which is a single ribonucleotide transcript comprising a sense strand and an antisense strand linked via a single-stranded ribonucleotide sequence.   8. The double-stranded molecule of claim 7, which is an oligonucleotide less than about 100 nucleotides in length.   12. The double-stranded molecule of claim 10, which is an oligonucleotide less than about 75 nucleotides in length.   12. The double-stranded molecule of claim 11, which is an oligonucleotide less than about 50 nucleotides in length.   13. The double-stranded molecule of claim 12, which is an oligonucleotide less than about 25 nucleotides in length.   14. The double stranded polynucleotide of claim 13, wherein the double stranded molecule is an oligonucleotide of about 19 to about 25 nucleotides in length.   A vector encoding the double-stranded molecule according to claim 7.   16. The vector according to claim 15, which encodes a transcript having a secondary structure and comprises a sense strand and an antisense strand.   17. The vector of claim 16, wherein the transcript further comprises a single stranded ribonucleotide sequence linking the sense and antisense strands.   A vector comprising a polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises the nucleotide sequence of SEQ ID NOs: 21, 25, 29, and 33, and the antisense strand nucleic acid is A vector consisting of a sequence complementary to the sense strand. Polynucleotide has the general formula
5 '-[A]-[B]-[A']-3 '
Where
[A] is the nucleotide sequence of SEQ ID NOs: 21, 25, 29, and 33;
[B] is a nucleotide sequence consisting of 3 to 23 nucleotides; and
19. The vector according to claim 18, wherein [A ′] is a nucleotide sequence complementary to [A].   A pharmaceutical composition for treating or preventing breast cancer, comprising a pharmaceutically effective amount of a small interfering RNA (siRNA) that inhibits the 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 a target sequence. siRNA is a general formula
5 '-[A]-[B]-[A']-3 '
Where
[A] is a ribonucleotide sequence corresponding to the nucleotide sequence of SEQ ID NOs: 21, 25, 29, and 33;
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides; and
22. The composition according to claim 21, wherein [A ′] is a nucleotide sequence complementary to [A]. A method of screening for compounds useful for the treatment or prevention of breast cancer comprising the following steps:
(a) contacting a polypeptide comprising the A2254 binding domain of the A5623 polypeptide with a polypeptide comprising the A5623 binding domain of the A2254 polypeptide in the presence of the test compound;
(b) detecting binding between polypeptides; and
(c) selecting a test compound that inhibits binding between polypeptides.   24. The method of claim 23, wherein the polypeptide comprising an A2254 binding domain comprises an A5623 polypeptide.   24. The method of claim 23, wherein the polypeptide comprising an A5623 binding domain comprises an A2254 polypeptide. A kit for screening for compounds for treating or preventing breast cancer, comprising:
(a) a polypeptide comprising the A2254 binding domain of the A5623 polypeptide,
(b) a polypeptide comprising the A5623 binding domain of the A2254 polypeptide, and
(c) A reagent for detecting an interaction between polypeptides.   27. The kit of claim 26, wherein the polypeptide comprising an A2254 binding domain comprises an A5623 polypeptide.   27. The kit of claim 26, wherein the polypeptide comprising an A5623 binding domain comprises an A2254 polypeptide.   A method of treating or preventing breast cancer in a subject comprising administering a pharmaceutically effective amount of a compound that inhibits the binding of A5623 polypeptide to A2254 polypeptide.   A composition for treating or preventing breast cancer, comprising a pharmaceutically effective amount of a compound that inhibits the binding between A5623 polypeptide and A2254 polypeptide, and a pharmaceutically acceptable carrier.

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