WO2011024428A1

WO2011024428A1 - Breast cancer related gene c12orf32

Info

Publication number: WO2011024428A1
Application number: PCT/JP2010/005169
Authority: WO
Inventors: Yusuke Nakamura; Toyomasa Katagiri; Takuya Tsunoda
Original assignee: Oncotherapy Science, Inc.
Priority date: 2009-08-24
Filing date: 2010-08-23
Publication date: 2011-03-03
Also published as: EP2470650A1; JP2013502202A

Abstract

The present invention provides methods for detecting and diagnosing cancer, such methods involving the determination of the expression level of C12orf32 genes. These genes were discovered to discriminate cancer cells from normal cells. Furthermore, the present invention provides methods of screening for therapeutic substances useful in the treatment of cancer. Moreover, the present invention provides siRNAs targeting C12orf32 genes, all of which are useful in the treatment of cancer.

Description

BREAST CANCER RELATED GENE C12ORF32

Priority
The present application claims the benefit of U.S. Provisional Application No. 61/275,047, filed on August 24, 2009, the entire contents of which are incorporated by reference herein.

Technical Field
The present invention relates to methods for detecting and diagnosing cancer as well as methods for treating and preventing cancer.

Breast cancer is the most common cancer in women, with estimated new cases of 1.15 million worldwide in 2002 (NPL 1). Incidence rates of breast cancer are increasing in most countries, and the increasing rate is much higher in countries where its incidence was previously low (NPL 1). While early detection with mammography as well as development of molecular targeted drugs, such as tamoxifen and trastuzumab, have reduced the mortality rate and made the quality of life of the patients better (NPL 2), there remain very limited treatment options for patients with advanced stage disease, particularly those with a hormone-independent tumor. Hence, the development of novel drugs to provide better management to such patients is still necessary.

Gene-expression profiles obtained by cDNA microarray analysis have yielded detailed characterization of individual cancers and such information may prove useful in the selection of more appropriate clinical strategies for individual patients, both through development of novel drugs and by providing a basis for personalized treatment (NPL 3). Through genome-wide expression analysis, a number of genes have been isolated that function as oncogenes in the process of development and/or progression of breast cancers (NPLs 4-6), synovial sarcomas (NPLs 7-8), and renal cell carcinomas (NPLs 9-10). Such molecules are considered to be candidate targets in the development of new therapeutic modalities.

In an attempt to identify novel molecular targets for breast cancer therapy, detailed gene-expression profiles of breast cancer cells purified by laser microbeam microdissection by means of cDNA microarray have been analyzed (NPL 11, PLs 1-3). Although some breast cancer markers have been identified through these studies, new therapeutic agents targeting them are still under development. Therefore, the identification of novel genes to be targeted for anticancer therapy remains a goal in the art.

[PTL 1] WO2005/029067
[PTL 2] WO2006/016525
[PTL 3] WO2007/013670

[NPL 1] Parkin DM, et al. (2005). CA Cancer J Clin 55:74-108
[NPL 2] Navolanic PM and McCubrey JA. (2005). Int J Oncol 27:1341-1344
[NPL 3] Petricoin EF 3rd, et al. (2002) Nat Genet 32 Suppl:474-479
[NPL 4] Park JH, et al. (2006) Cancer Res 66:9186-9195
[NPL 5] Shimo A, et al. (2007) Cancer Sci 98:174-181
[NPL 6] Lin ML, et al. (2007) Breast Cancer Res 9: R17
[NPL 7] Nagayama S, et al. (2004) Oncogene 23:5551-5557
[NPL 8] Nagayama S, et al. (2005) Oncogene 24:6201-6212
[NPL 9] Togashi A, et al. (2005) Cancer Res 65:4817-4826
[NPL 10] Hirota E, et al. (2006) Int J Oncol 29:799-827
[NPL 11] Nishidate T, et al. (2004) Int J Oncol 25:797-819

The present invention relates to the discovery of a specific expression pattern of C12orf32 gene in cancerous cells.
Through the present invention, C12orf32 gene was revealed to be frequently up-regulated in human tumors, in particular, breast tumors. Moreover, since the suppression of the C12orf32 gene by small interfering RNA (siRNA) resulted in growth inhibition and/or cell death of breast cancer cells, this gene may serve as a novel therapeutic target for human breast cancers.

The C12orf32 gene identified herein, as well as its transcription and translation products, find diagnostic utility as a marker for breast cancer and as an oncogene target, the expression and/or activity of which may be altered to treat or alleviate a symptom of cancer. Similarly, by detecting changes in the expression of the C12orf32 gene that arise from exposure to a test substance, various agents for treating or preventing cancer can be identified.

Accordingly, it is an object of the present invention to provide methods for diagnosing or determining a predisposition to breast cancer in a subject by determining the expression level of the C12orf32 gene in a subject-derived biological sample, such as tissue sample. An increase in the level of expression of the gene as compared to a normal control level indicates that the subject suffers from or is at risk of developing breast cancer.

In the context of the present invention, the phrase "control level" refers to the expression level of the C12orf32 gene detected in a control sample and encompasses both a normal control level and a cancer control level. A control level can be a single expression pattern derived from a single reference population or the average calculated from a plurality of expression patterns. Alternatively, the control level can be a database of expression patterns from previously tested cells. The phrase "normal control level" refers to a level of the C12orf32 gene expression detected in a normal healthy individual or in a population of individuals known not to be suffering from cancer. A normal individual is one with no clinical symptom of breast cancer. A normal control level can be determined using a normal cell obtained from a non-cancerous tissue. A "normal control level" may also be the expression level of the C12orf32 gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from breast cancer. On the other hand, the phrase "cancer control level" refers to an expression level of the C12orf32 gene detected in the cancerous tissue or cell of an individual or population suffering from breast cancer.

An increase in the expression level of the C12orf32 gene detected in a sample as compared to a normal control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing breast cancer.
Alternatively, the expression level of the C12orf32 gene in a sample can be compared to cancer control level of a C12orf32 gene. A similarity between the expression level of a sample and the cancer control level indicates that the subject (from which the sample has been obtained) suffers from or is at risk of developing cancer.

Herein, gene expression levels are deemed to be "altered" when the gene expression increases by, for example, 10%, 25%, or 50% from, or at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more compared to a control level. The expression level of the C12orf32 gene can be determined by the hybridization intensity of nucleic acid probes to gene transcripts in a sample.
In the context of the present invention, subject-derived tissue samples may be any tissues obtained from test subjects, e.g., patients known to have or suspected of having cancer. For example, tissues may include epithelial cells. More particularly, tissues may be cancerous epithelial cells.

It is another object of the present invention to provide methods for identifying substances that inhibit the expression or activity of the C12orf32 protein, by contacting a test cell expressing the C12orf32 protein with test substances and determining the expression level of the C12orf32 gene or the activity of the gene product, the C12orf32 protein. The test cell may be an epithelial cell, such as cancerous epithelial cell. A decrease in the expression level of the gene or the activity of its gene product as compared to a control level in the absence of the test substance indicates that the test substance may be used to reduce symptoms of breast cancer.
The present invention also provides a kit that includes at least one detection reagent that binds to a transcription or translation product of the C12orf32 gene.

It is yet another object of the present invention to provide methods for cancer therapeutic methods targeting C12orf32 gene and its products. Therapeutic methods of the present invention include methods for treating or preventing breast cancer in a subject including the step of administering an antisense composition to the subject. In the context of the present invention, the antisense composition reduces the expression of the C12orf32 gene. For example, the antisense compositions may contain a nucleotide that is complementary to the C12orf32 gene sequence. Alternatively, the present methods may include the step of administering double-stranded molecule (e.g., siRNA) composition to the subject. In the context of the present invention, the double-stranded molecule (e.g., siRNA) composition reduces the expression of the C12orf32 gene. In yet another method, the treatment or prevention of breast cancer in a subject may be carried out by administering a ribozyme composition to the subject. In the context of the present invention, the nucleic acid-specific ribozyme composition reduces the expression of the C12orf32 gene.

To that end, the present inventors confirmed inhibitory effects of siRNAs for the C12orf32 gene. In particular, the inhibition of cell proliferation of cancer cells by siRNAs is demonstrated in the Examples section. The data herein demonstrate the utility of the C12orf32 gene as a preferred therapeutic target for breast cancer. Thus, the present invention also provides double-stranded molecules that serve as siRNAs against the C12orf32 gene as well as vectors expressing the double-stranded molecules.
One advantage of the methods described herein is that the disease is identified prior to detection of overt clinical symptoms of breast cancer. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.

The present invention provides the following inventions.
[1] A method for diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of a C12orf32 gene in a subject-derived biological sample, wherein an increase in the expression level as compared to a normal control level of the gene indicates that the subject suffers from or is at a risk of developing cancer, wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting mRNA of a C12orf32 gene;
(b) detecting a protein encoded by a C12orf32 gene; and
(c) detecting a biological activity of a protein encoded by a C12orf32 gene.
[2] The method of [1], wherein the expression level is at least 10% greater than the normal control level.
[3] The method of [1], wherein the cancer is breast cancer.
[4] A kit for detecting cancer comprising a detection reagent which binds to a transcription or translation product of a C12orf32 gene.
[5] A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting binding between the polypeptide or fragment and the test substance; and
(c) selecting the test substance that binds to the polypeptide or fragment as a candidate substance for treating or preventing cancer.
[6] A method of screening a candidate substance for treating or preventing cancer, wherein the method comprises steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment;
(c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the test substance; and
(d) selecting the test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.
[7] The method of [6], wherein the biological activity is cell proliferative activity.
[8] A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
(a) contacting a test substance with a cell expressing a C12orf32 gene;
(b) detecting expression level of the C12orf32 gene;
(c) comparing the expression level with the expression level detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer.
[9] A method of screening a candidate substance for treating or preventing cancer, wherein the method comprises steps of:
(a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of a C12orf32 gene and a reporter gene expressed under control of the transcriptional regulatory region;
(b) measuring expression level or activity of the reporter gene;
(c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level or activity as a candidate substance for treating or preventing cancer.
[10] A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence consisting of SEQ ID NO: 8, 9 or 14, and wherein the antisense strand comprises a nucleotide sequence which is complementary to the target sequence, wherein the sense molecule, and wherein the double-stranded molecule, when introduced into a cell expressing the C12orf32 gene, inhibits expression of the gene.
[11] The double-stranded molecule of [10], wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
[12] The double-stranded molecule of [10] or [11], wherein the double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
[13] The double-stranded molecule of [12], which has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 8, 9 and 14, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence.
[14] A vector encoding the double-stranded molecule of any one of [10] to [13].
[15] Vectors comprising each of a combination of polynucleotides comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 8, 9 or 14 and the antisense strand nucleic acid comprises a sequence complementary to the sense strand, wherein the transcripts of the sense strand and the antisense strand hybridize to each other to form a double stranded molecule, and wherein the vectors, when introduced into a cell expressing C12orf32 gene, inhibit the cell proliferation.
[16] A method of treating or preventing cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a double-stranded molecule against a C12orf32 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing C12orf32 gene, inhibits the expression of the C12orf32 gene.
[17] The method of [16], wherein the double-stranded molecule is that of any one of [10] to [13], wherein the vector is that of [14] or [15].
[18] The method of [16] or [17], wherein the cancer is breast cancer.
[19] A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against a C12orf32 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing C12orf32 gene, inhibits the expression of the C12orf32 gene, and a pharmaceutically acceptable carrier.
[20] The composition of [19], wherein the double-stranded molecule is that of any one of [10] to [13], wherein the vector is that of [14] or [15].
[21] The composition of [19] or [20], wherein the cancer is breast cancer.
[22] A fragment of C12orf32 protein obtained by following steps of:
(a) transfecting a vector expressing the C12orf32 protein with breast cancer cell line or COS-7 cells, and
(b) recovering the translation products from the cells, wherein the molecular weight of the translation products determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is selected from the group consisting of about 27-kDa, 23-kDa and 16-kDa.

Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments that follows:

Fig. 1 depicts the expression pattern of C12orf32 in the clinical breast cancer cells and normal human organs assayed in Example (Parts (A)-(C)).

Part (D) depicts the results of RT-PCR and mapping analysis of the region corresponding to the ORF region of C12orf32.

Fig. 2 depicts the results of Northern blot analysis of breast cancer cell lines and transfectants thereof (Parts (A)-(B)).

Fig. 3 depicts the results of FACS (Part(A)), western blot(Part(B)) and RT-PCR (Part(C)) analyses of C12orf32 expression using T47D cells after synchronization of the cell cycle by aphidicolin treatment.

Part (D) depicts the results of immunocytostaining of C12orf32 in T47D cells.

Fig. 4 depicts the effect of C12orf32 knockdown by siRNA on the growth of breast cancer cells in Example (Parts (A)-(E)). Parts (A) and (B) depict that two siRNAs (si-#2 and si-#3) significantly suppressed the C12orf32 expression, compared with a control siRNA construct (si-control) (upper panels). In concordance with the knockdown effect, MTT (middle panels) and colony formation assays (lower panels) revealed significant growth-suppressive effects by si-#2 and si-#3 (MTT assays: HBC4). Part (C) depicts that siRNA contained 4-base replacement in si-#2 sequence (si-C12orf32-mismatch (si-mis)), and found no suppressive effect on the expression of C12orf32 or on cell growth of T47D cells. Part (D) depicts fluorescence-activated cell sorting (FACS) analysis using siRNA-oligonucleotides to measure the proportions of apoptotic cell population. Part (E) depicts that knockdown of C12orf32 protein was validated using anti-C12orf32 antibody by western analysis.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Definitions
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated. The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free of at least one substance that may else be included in the natural source. Thus, an isolated or purified antibody refers to antibodies that are substantially free of cellular material such as carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which the polypeptide is isolated or recombinantly produced. Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In a preferred embodiment, antibodies and polypeptides of the present invention are isolated or purified. An "isolated" or "purified" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a preferred embodiment, nucleic acid molecules encoding antibodies of the present invention are isolated or purified.

The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.
Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The polynucleotide, oligonucleotide, nucleotides, nucleic acids, or nucleic acid molecules may be composed of DNA, RNA or a combination thereof.
Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the C12orf32 gene, in particular, breast cancer.

As used herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).

As used herein, the term "target sequence" refers to a nucleotide sequence within mRNA or cDNA sequence of a target gene, which will result in suppression of translation of the whole mRNA of the target gene if a double-stranded nucleic acid molecule targeting the sequence is introduced into a cell expressing the target gene. A nucleotide sequence within mRNA or cDNA sequence of a gene can be determined to be a target sequence when a double-stranded molecule including a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. The double stranded polynucleotide which suppresses the gene expression may consists of the target sequence and 3' overhang having 2 to 5 nucleotides in length (e.g., uu).

When a target sequence is shown by cDNA sequence, a sense strand sequence of a double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.

Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s.

Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand.
A double-stranded molecule may has one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.

As used herein, the term "siRNA" refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a C12orf32 sense nucleic acid sequence (also referred to as "sense strand"), a C12orf32 antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siRNA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA may either be a dsRNA or shRNA.

As used herein, the term "dsRNA" refers to a construct of two RNA molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" RNAs selected from a protein coding sequence of target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.

The term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, wherein the first and second regions are joined by a loop region, wherein the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".

As used herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form the double-stranded molecule, whereas a chimera indicates that one or both of the strands composing the double stranded molecule may contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand") or both. The siD/R-NA may be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA may either be a dsD/R-NA or shD/R-NA.

As used herein, the term "dsD/R-NA" refers to a construct of two molecules including complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands may include not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).

The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, including the first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, wherein the first and second regions are joined by a loop region, wherein the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of a shD/R-NA is a single-stranded region intervening between the sense and antisense strands and may also be referred to as "intervening single-strand".

As used herein, an "isolated nucleic acid" is a nucleic acid removed from its original environment (e.g., the natural environment if naturally occurring) and thus, synthetically altered from its natural state. In the context of the present invention, examples of isolated nucleic acid include DNA, RNA, and derivatives thereof.

Gene and Polypeptide
The present invention is based in part on the discovery of elevated expression of C12orf32 gene in cells from patients of breast cancers. The nucleotide sequence of the human C12orf32 gene is shown in SEQ ID NO: 1. Such nucleotide sequence is also available as GenBank Accession No. NM_031465. Herein, the C12orf32 gene encompasses allelic variants of the human C12orf32 gene as well as those of other animals including, but not limited to, non-human primate, mouse, rat, dog, cat, horse, and cow, and further includes allelic mutants and genes found in other animals as corresponding to the C12orf32 gene.

In the present invention, two transcript variants were confirmed to be overexpressed in breast cancer cell lines. The cDNA sequences of these transcript variants are shown in SEQ ID NO: 18 (GenBank Accession No. NR_027365.1) and SEQ ID NO: 19 (GenBank Accession No. NR_027363.1), respectively. Herein, any transcript variants of the C12orf32 gene, including the above-mentioned variants, are included in mRNA or transcription product of the C12orf32 gene. Also, cDNA sequences of such transcript variants are included in the C12orf32 gene.
The amino acid sequence encoded the human C12orf32 gene is shown in SEQ ID NO: 2 and is also available as GenBank Accession No. NM_031465. In the context of the present invention, the polypeptide or protein encoded by the C12orf32 gene is referred to as "C12orf32", and sometimes as "C12orf32 polypeptide" or "C12orf32 protein".

As demonstrated in Examples, western blot analysis implies that three small-size polypeptides (approximately 27-kDa, 23-kDa and 16-kDa) are produced from approximately 34-kDa polypeptide which corresponds to the protein predicted from the open reading frame of C12orf32 gene. Among those small-size polypeptides, it was confirmed that 23-kDa polypeptide and 16-kDa polypeptide were particularly overexpressed in breast cancer cell lines. Accordingly, those small-size polypeptides are also included in the polypeptide or protein encoded by the C12orf32 gene.

According to an aspect of the present invention, functional equivalents are also included in the C12orf32 protein. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptides that retain the biological ability of the C12orf32 protein may be used as such functional equivalents of each protein in the present invention.
The biological activities of the C12orf32 protein include, for example, cancer cell proliferation activity.

Such functional equivalents include those in which one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the C12orf32 protein. Alternatively, the polypeptide may be one that includes an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the C12orf32 protein, more preferably at least about 90% to 95% homology, even more preferably 96% to 99% homology. In other embodiments, the polypeptide can be encoded by a polynucleotide that hybridizes under stringent conditions to the naturally occurring nucleotide sequence of the C12orf32 gene.

The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times of background, preferably 10 times of background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 degrees C, or, 5x SSC, 1% SDS, incubating at 65 degrees C, with wash in 0.2x SSC, and 0.1% SDS at 50 degrees C.

In the context of the present invention, a condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the human C12orf32 protein can be routinely selected by a person skilled in the art. For example, hybridization may be performed by conducting pre-hybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. An exemplary low stringent condition may include 42 degrees C, 2x SSC, 0.1% SDS, preferably 50 degrees C, 2x SSC, 0.1% SDS. High stringency conditions are often preferably used. An exemplary high stringency condition may include washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors, such as temperature and salt concentration, can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.

In general, modifications of one, two, or more amino acids in a protein will not influence the function of the protein. In fact, mutated or modified proteins (i.e., peptides composed of an amino acid sequence in which one, two, or several amino acid residues have been modified through substitution, deletion, insertion and/or addition) have been known to retain the original biological activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)). Accordingly, one of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence that alter a single amino acid or a small percentage of amino acids or those considered to be a "conservative modification" wherein the alteration of a protein results in a protein with similar functions, are acceptable in the context of the instant invention. Thus, in one embodiment, the peptides of the present invention may have an amino acid sequence wherein one, two or even more amino acids are added, inserted, deleted, and/or substituted in the human C12orf32 sequence.

So long as the activity the protein is maintained, the number of amino acid mutations is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence. Accordingly, in a preferred embodiment, the number of amino acids to be mutated in such a mutant is generally 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 5 or 6 amino acids or less, and even more preferably 3 or 4 amino acids or less.

An amino acid residue to be mutated is preferably mutated into a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (d), Glutamic acid (E);
3) Aspargine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified polypeptides are included in the present C12orf32 protein. However, the present invention is not restricted thereto and the C12orf32 protein includes non-conservative modifications so long as they retain at least one biological activity of the C12orf32 protein. Furthermore, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

Moreover, the C12orf32 gene of the present invention encompasses polynucleotides that encode such functional equivalents of the C12orf32 protein. In addition to hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a polynucleotide encoding a polypeptide functionally equivalent to the C12orf32 protein, using a primer synthesized based on the sequence information of the protein encoding DNA (SEQ ID NO: 1). Polynucleotides and polypeptides that are functionally equivalent to the human C12orf32 gene and protein, respectively, normally have a high homology to the originating nucleotide or amino acid sequence thereof . "High homology" typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 90% to 95% or higher, even more preferably 96%, 97%, 98%, 99% or higher. The homology of a particular polynucleotide or polypeptide can be determined by following the algorithm in "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)".

I. Diagnosing cancer
I-1. Method for diagnosing cancer or a predisposition for developing cancer
The expression of the C12orf32 gene was found to be specifically elevated in patients with cancer, more particularly, breast cancer. Accordingly, the gene identified herein as well as its transcription and translation products find diagnostic utility as a marker for breast cancer, and by measuring the expression of the C12orf32 gene in a cell sample, breast cancer can be diagnosed. More particularly, the present invention provides a method for detecting, diagnosing and/or determining the presence of or a predisposition for developing cancer in a subject by determining the expression level of the C12orf32 gene in a subject. Preferred cancers to be diagnosed by the present method include breast cancer.

Such result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease or is predisposed to developing the disease. Alternatively, the present invention may provide a doctor with useful information to diagnose that the subject suffers from the disease. For example, according to the present invention, when the suspicion or doubt of the presence of cancer cells in the tissue obtained from a subject is indicated, clinical decisions would be made by a doctor with consideration of this observation and another aspect including the pathological finding of the tissue, levels of known tumor marker(s) in blood, or clinical course of the subject, etc. Some blood tumor markers for diagnostic purpose of breast cancer are well known. For example, breast carcinoma-associated antigen 225 (BCA225), carbohydrate antigen 15-3 (CA15-3), or carcinoembryonic antigen (CEA) is preferable blood tumor marker for breast cancer. Namely, in a particular embodiment, according to the present invention, an intermediate result for examining the condition of a subject may also be provided.

In another embodiment, the present invention provides a method for detecting a diagnostic marker of cancer, the method including the step of detecting the expression of the C12orf32 gene in a subject-derived biological sample as a diagnostic marker of cancer. Preferable cancers to be diagnosed by the present method include breast cancer.

In the context of the present invention, the term "diagnosing" is intended to encompass predictions and likelihood analysis. The present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for cancer. According to the present invention, an intermediate result for examining the condition of a subject may also be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to determine that a subject suffers from the disease. That is, the present invention provides a diagnostic marker C12orf32 for examining cancer. Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived breast tissue sample, wherein the method including the step of determining the expression level of the C12orf32 gene in a subject-derived biological sample, wherein an increase in the expression level as compared to a normal control level of the gene indicates the presence or suspicion of cancer cells in the tissue. Such result may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the C12orf32 gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic breast tumor markers in blood are BCA225, CA15-3, CA72-4, CEA, IAP, NSE, SP1, and TPA. Namely, in this particular embodiment of the present invention, the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.

A subject to be diagnosed by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, human, non-human primate, mouse, rat, dog, cat, horse, and cow.
It is preferred to collect a biological sample from the subject to be diagnosed. Any biological material can be used as the biological sample for the determination so long as it includes the objective transcription or translation product of the C12orf32 gene. The biological samples include, but are not limited to, bodily tissues and fluids, such as blood, sputum, and urine. Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a cancerous breast epithelial cell or a breast epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell may be purified from the obtained bodily tissues and fluids, and then used as the biological sample. In a preferred embodiment, the biological sample may contain breast tissue collected from the subject to be diagnosed.

According to the present invention, the expression level of the C12orf32 gene is determined in the subject-derived biological sample. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of the C12orf32 gene may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including the present C12orf32 gene. Those skilled in the art can prepare such probes utilizing the sequence information of the C12orf32 gene. For example, the cDNA of the C12orf32 gene may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.

Furthermore, the transcription product of the C12orf32 gene may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers (SEQ ID NOs: 3, 4, 11, 12, 16 and 17) used in the Example may be employed for the detection by RT-PCR, but the present invention is not restricted thereto.

Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the C12orf32 gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the quantity of the C12orf32 protein may be determined. A method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment retains the binding ability to the C12orf32 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.

As another method to detect the expression level of the C12orf32 gene based on its translation product, the intensity of staining may be observed via immunohistochemical analysis using an antibody against the C12orf32 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the C12orf32 gene.

In the present invention, the translation product of C12orf32 gene includes any fragments thereof, as long as such fragments can be detected in cancer cells or cancer cell culture. For example, some small-size bands were observed in cell lysate of COS-7 transfected with C12orf32 expressing vector (see "Cleaved forms of C12orf32 protein in human breast cancer cells" of Example and Fig. 2) in the western blot analysis using anti-C12orf32 polyclonal antibody. Accordingly, a fragment of C12orf32 protein corresponding to those small-size bands may be detected for diagnosis of breast cancer and/or determination of predisposition for developing cancer. Herein, the fragments, which were detected in cell lysate of COS-7 cells transfected with a vector expressing a C12orf32 protein or breast cancer cell lines in Examples, are also included in the translation product of the C12orf32 gene. Those fragments can be detected by any immunological methods described above using polyclonal antibodies against C12orf32 protein, or monoclonal or polyclonal antibodies that specifically recognizes such fragments. According to the present invention, approximately 16-kDa fragment may be preferably detected for diagnosis of breast cancer, as it has been confirmed that such fragment was more up-regulated than the other fragments in breast cancer cell lines.

As mentioned above, the fragments of the C12orf32 protein also find a use for diagnosis of breast cancer. Accordingly, present invention also provides the fragments of the C12orf32 protein. The fragment of the C12orf32 protein is obtainable from cell lysate of cells transfected with a vector expressing the C12orf32 protein or a breast cancer cell line. The molecular weight of such fragments are approximately 27-kDa, 23-kDa or 16-kDa. These molecular weight may be determined by SDS-PAGE. In the present invention, such fragment of the C12orf32 protein may be purified from the cell lysate through immuno affinity chromatography technique by using antibody against C12orf32 protein or the fragment thereof. Alternatively, gel filtration may also be used to purify the fragment of the present invention from the cell lysate. The cleaved fragments of C12orf32 protein can be detected by antibodies specifically recognize each fragments (27-kDa, 23-kDa and 16-kDa). Once the amino acid sequences of the fragments of C12orf 32 protein are determined, it is possible for ordinary skill in the art to obtain such the fragments by using methods known in the art. For example, the fragment of the present invention can be prepared by introducing a vector expressing the fragments into host cells, and recovering the fragment from the host cells. Host cells into which the vectors are introduced are not particularly limited and may be in any form as long as they can produce recombinant proteins. Alternatively, such fragments can be prepared through in vitro translation from cDNA encoding the fragments.

Furthermore, the translation product may be detected based on its biological activity. Specifically, the C12orf32 protein was demonstrated herein to be involved in the growth of cancer cells. Thus, the cancer cell growth promoting ability (cell proliferative activity) of the C12orf32 protein may be used as an index of the C12orf32 protein existing in a biological sample.

Moreover, in addition to the expression level of the C12orf32 gene, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in breast cancer, may also be determined to improve the accuracy of the diagnosis. Alternatively, the combination of the expression level among the cancer-associated genes may be determined for more accurate diagnosis.
The expression level of cancer marker genes including the C12orf32 gene in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer marker gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.

The control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of the C12orf32 gene in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of the C12orf32 gene in a biological sample may be compared to multiple control levels, which control levels are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample. Moreover, it is preferred, to use the standard value of the expression levels of the C12orf32 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as standard value.

In the context of the present invention, a control level determined from a biological sample that is known to be non-cancerous is called "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it will be called "cancerous control level".
When the expression level of the C12orf32 gene is increased compared to the normal control level or is similar to the cancerous control level, the subject may be diagnosed to be suffering from or at a risk of developing cancer. Furthermore, in case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing cancer.

Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes. Genes whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
Furthermore, the present invention provides the use of the C12orf32 gene as cancerous markers. These genes are particularly useful for breast cancerous markers. For example, it can be determined whether a biological sample contains cancerous cells, especially breast cancerous cells, by detecting the expression level of the C12orf32 gene as cancerous markers. Specifically, increasing the expression level of the C12orf32 gene in a biological sample as compared to a normal control level indicates that the biological sample contains cancerous cells. The expression level can be determined by detecting the transcription or translation products of these marker genes as described above. The translation product may be determined as the biological activity.

I-2. Assessing efficacy of cancer treatment
The C12orf32 gene differentially expressed between normal and cancerous cells also allow for the course of treatment for cancers to be monitored, and the above-described method for diagnosing cancer can be applied for assessing the efficacy of a treatment on cancer. Specifically, the efficacy of a treatment for cancer can be assessed by determining the expression level of the C12orf32 gene in a cell(s) derived from a subject undergoing the treatment. If desired, test cell populations are obtained from the subject at various time points, before, during, and/or after the treatment. The expression level of the C12orf32 gene can be, for example, determined following the method described above under the item of 'I-1. Method for diagnosing cancer or a predisposition for developing cancer'. In the context of the present invention, it is preferable that the control level to which the detected expression level is compared be obtained from the C12orf32 gene expression in a cell(s) not exposed to the treatment of interest.

If the expression level of the C12orf32 gene is compared to a control level that is obtained from a normal cell or a cell population containing no cancer cell, a similarity in the expression level indicates that the treatment of interest is efficacious and an increase in the expression level indicates less favorable clinical outcome or prognosis of that treatment. On the other hand, if the comparison is conducted against a control level that is obtained from a cancer cell or a cell population containing a cancer cell(s), a decrease in the expression level indicates efficacious treatment, while a similarity in the expression level indicates less favorable clinical outcome or prognosis.

Furthermore, the expression levels of the C12orf32 gene before and after a treatment can be compared according to the present method to assess the efficacy of the treatment. Specifically, the expression level detected in a subject-derived biological sample after a treatment (i.e., post-treatment level) is compared to the expression level detected in a biological sample obtained prior to treatment onset from the same subject (i.e., pre-treatment level). A decrease in the post-treatment level compared to the pre-treatment level indicates that the treatment of interest is efficacious while an increase in or similarity of the post-treatment level to the pre-treatment level indicates less favorable clinical outcome or prognosis.

As used herein, the term "efficacious" indicates that the treatment leads to a reduction in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene or a decrease in size, prevalence, or metastatic potential of carcinoma in a subject. When a treatment of interest is applied prophylactically, "efficacious" means that the treatment retards or prevents the forming of tumor or retards, prevents, or alleviates at least one clinical symptom of cancer. Assessment of the state of tumor in a subject can be made using standard clinical protocols.
In addition, efficaciousness of a treatment can be determined in association with any known method for diagnosing cancer. Cancers can be diagnosed, for example, by identifying symptomatic anomalies, e.g., weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and generalized malaise, weakness, and jaundice.

To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels." While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g., reducing the proliferation and metastasis of tumors.

The treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, and the reduction or inhibition of metastasis. Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms that constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.

In the present invention, it is revealed that C12orf32 is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting C12orf32 can be achieved by the present invention. In the present invention, the cancer treatment targeting C12orf32 refers to suppression or inhibition of C12orf32 activity and/or expression in the cancer cells. Any anti-C12orf32 agents may be used for the cancer treatment targeting C12orf32. In the present invention, the anti-C12orf32 agent include following substance or active ingredient:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.

Accordingly, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated with anti-C12orf32 agent, and/or (ii) selecting a subject for cancer treatment targeting C12orf32, which method includes the steps of:
(a) determining the expression level of C12orf32 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
(b) comparing the expression level of C12orf32 with a normal control level;
(c) diagnosing the subject as having the cancer to be treated, if the expression level of C12orf32 is increased as compared to the normal control level; and
(d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).

Alternatively, such a method includes the steps of:
(a) determining the expression level of C12orf32 in in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
(b) comparing the expression level of C12orf32 with a cancerous control level;
(c) diagnosing the subject as having the cancer to be treated, if the expression level of C12orf32 is similar or equivalent to the cancerous control level; and
(d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step (c).

II. Kits
The present invention also provides reagents for detecting cancer, i.e., reagents that can detect the transcription or translation product of the C12orf32 gene. The present invention also provides reagents for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of a cancer treatment. Examples of such reagents include those capable of:
(a) detecting mRNA of the C12orf32 gene;
(b) detecting the C12orf32 protein; and/or
(c) detecting the biological activity of the C12orf32 protein in a subject-derived biological sample.

Suitable reagents include nucleic acids that specifically bind to or identify a transcription product of the C12orf32 gene. For example, a nucleic acid that specifically binds to or identifies a transcription product of the C12orf32 gene includes, for example, oligonucleotides (e.g., probes and primers) having a sequence that is complementary to a portion of the C12orf32 gene transcription product. Such oligonucleotides are exemplified by primers and probes that are specific to the mRNA of the gene of interest and may be prepared based on methods well known in the art. Alternatively, antibodies can be exemplified as reagents for detecting the translation product of the gene. The probes, primers, and antibodies described above under the item of 'I-1. Method for diagnosing cancer or a predisposition for developing cancer' can be mentioned as suitable examples of such reagents. These reagents may be used for the above-described diagnosis of cancer. The assay format for using the reagents may be, for example, Northern hybridization or sandwich ELISA, both of which are well-known in the art.

The detection reagents may be packaged together in the form of a kit. For example, the detection reagents may be packaged in separate containers. Furthermore, the detection reagents may be packaged with other reagents necessary for the detection. For example, a kit may include a nucleic acid or antibody (either bound to a solid matrix or packaged separately with reagents for binding them to the matrix) as the detection reagent, a control reagent (positive and/or negative), and/or a detectable label. A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes. These reagents and such may be retained in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay may also be included in the kit.

Although the present kit is suited for the detection and diagnosis of breast cancer, it may also be useful in assessing the prognosis of cancer and/or monitoring the efficacy of a cancer therapy. Also, it may be useful in determining a subject suffering from cancer that can be treated with anti-C12orf32 agents (e.g., the double-stranded molecule of the present invention or vector encoding thereof).

As an aspect of the present invention, the reagents for detecting cancer may be immobilized on a solid matrix, such as a porous strip, to form at least one site for detecting cancer. The measurement or detection region of the porous strip may include a plurality of sites, each containing a detection reagent (e.g., nucleic acid). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized detection reagents (e.g., nucleic acid), i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test biological sample, the number of sites displaying a detectable signal provides a quantitative indication of the expression level of the C12orf32 gene in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.

The kit of the present invention may further include positive and/or negative controls sample, and/or an C12orf32 standard sample. The positive control sample of the present invention may be prepared by collecting C12orf32 positive samples. Such C12orf32 positive samples may be obtained, for example, from established breast cancer cell lines, including Human breast cancer cell lines, HBC4, HBC5, BT-549, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SK-BR-3, T47D, YMB-1, ZR-75-1 and BSY-1. Alternatively, positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an C12orf32 mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may be include an C12orf32 standard sample containing a cut-off value amount of an C12orf32 mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal breast tissues, or may be prepared by preparing a sample containing an C12orf32 mRNA or protein less than cut-off value.

Alternatively, the present invention provides use of a reagent for preparing a diagnostic reagent for diagnosing cancer. In some embodiments, the reagent can be selected from the group consisting of:
(a) a reagent for detecting mRNA of the C12orf32 gene;
(b) a reagent for detecting the C12orf32 protein; and
(c) a reagent for detecting the biological activity of the C12orf32 protein.
Specifically, such reagent is an oligonucleotide that hybridizes to the C12orf32 polynucleotide, or an antibody that binds to the C12orf32 polypeptide.

III. Screening methods
Using the C12orf32 gene, polypeptides encoded by the gene or fragments thereof, or transcriptional regulatory region of the gene, it is possible to screen substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene. Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, breast cancer. Thus, the present invention provides methods of screening for candidate substances for treating or preventing cancer using the C12orf32 gene, polypeptides encoded by the genes or fragments thereof, or transcriptional regulatory region of the gene.

In the present invention, the fragments of C12orf32 polypeptide can be used to screen substances that alter the biological activity of the fragments of C12orf32 polypeptide. For example, such fragments include the partial peptides of C12orf32 polypeptide whose molecular weight in SDS-PAGE is 27-kDa, 23-kDa or 16-kDa. Such fragments may be isolated from cells transfected with full-length C12orf32 gene or breast cancer cell lines. Full-length of C12orf32 polypeptide expressed in the cells or the breast cancer cell lines is cleaved to form these fragments. Those fragments, especially 23-kDa and 16-kDa fragments, are expressed more than the full length of C12orf32 polypeptide, in breast cancer cells. Furthermore, it has been confirmed that those fragments were decreased in breast cancer cell lines transfected of siRNA against C12orf32 gene (Fig.2A, 4E). Taken together, those fragments of C12orf32 polypeptide may play a crucial roll in breast cancer survival. Thus, substances that binds to those fragments or alter a biological activity of those fragments may serve as candidate drug for cancer therapy. Accordingly, substances that bind to such fragment are potential candidates of antagonists of C12orf32.

A substance isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the C12orf32 gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, breast cancer). Namely, the substances screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.

In the context of the present invention, substances to be identified through the present screening methods may be any compound or composition including several compounds. Furthermore, the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single compound or a combination of compounds. When a combination of compounds is used in the methods, the compounds may be contacted sequentially or simultaneously.

Any test substances, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micromolecular compounds (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention. Test substances useful in the screenings described herein can also be antibodies that specifically bind to a protein of interest or a partial peptide thereof that lacks the biological activity of the original proteins in vivo.

The test substance of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
(1) biological libraries,
(2) spatially addressable parallel solid phase or solution phase libraries,
(3) synthetic library methods requiring deconvolution,
(4) the "one-bead one-compound" library method and
(5) synthetic library methods using affinity chromatography selection.

The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds may be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002103360).

Although the construction of test substance libraries is well known in the art, herein below, additional guidance in identifying test substances and construction libraries of such substances for the present screening methods are provided.

A. Molecular Modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of C12orf32. One approach to preliminary screening of test substances suitable for further evaluation utilizes computer modeling of the interaction between the test substance and its target.

Computer modeling technology allows for the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from X-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.

An example of the molecular modeling system described generally above includes the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles have been published on the subject of computer modeling of drugs interactive with specific proteins, examples of which include Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.

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

Once a putative inhibitor has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified putative inhibitor, as detailed below. The resulting library of putative inhibitors, or "test substances" may be screened using the methods of the present invention to identify test substances suited to the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer, particularly breast cancer.

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

Preparation of combinatorial chemical libraries is well known to those of skill in the art, and may be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemicals for generating chemical diversity libraries can also be used. Such chemicals include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology 1995 supplement; Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

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

C. Other Candidates:
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) disclose SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) can be used for screening.

A compound in which a part of the structure of the compound screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.
Furthermore, when the screened test substance is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein may be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein may be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.

III-1. Protein based screening methods
According to the present invention, the expression of the C12orf32 gene is crucial for the growth and/or survival of cancer cells, in particular breast cancer cells. Accordingly, substances that suppress the function of the polypeptide encoded by the genes would be presumed to inhibit the growth and/or survival of cancer cells, and therefore find use in treating or preventing cancer. Thus, the present invention provides methods of screening a candidate substance for treating or preventing cancer, using the C12orf32 polypeptide. Further, the present invention also provides methods of screening a candidate substance for inhibiting the growth and/or survival of cancer cells, using the C12orf32 polypeptide.

In addition to the C12orf32 polypeptide, fragments of the polypeptides may be used for the present screening, so long as it retains at least one biological activity of the natural occurring C12orf32 polypeptide. As demonstrated in Examples, cleaved fragments of C12ord32 polypeptide are crucial as well as the full length of C12orf32 polypeptide. Those fragments were detected as approximately 27-kDa, 23-kDa and 16-kDa size bands in western blotting using polyclonal antibody against C12orf32. Those approximately 27-kDa, 23-kDa and 16-kDa size fragments may be preferably used as fragments of the C12orf32 polypeptide in the present screening.

The polypeptides or fragments thereof may be further linked to other substances, so long as the polypeptides and fragments retain at least one of their biological activities. Usable substances include: peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc. These kinds of modifications may be performed to confer additional functions or to stabilize the polypeptide and fragments.

The polypeptides or fragments used for the present method may be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
1) Peptide Synthesis, Interscience, New York, 1966;
2) The Proteins, Vol. 2, Academic Press, New York, 1976;
3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; and
7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, the proteins may be obtained through any known genetic engineering methods for producing polypeptides (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector including a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence including a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the C12orf32 polypeptide is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8. A promoter may be used for the expression. Any commonly used promoters may be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF-alpha promoter (Kim et al., Gene 1990, 91:217-23), the CAG promoter (Niwa et al., Gene 1991, 108:193), the RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152:684-704), the SRalpha promoter (Takebe et al., Mol Cell Biol 1988, 8:466), the CMV immediate early promoter (Seed et al., Proc Natl Acad Sci USA 1987, 84:3365-9), the SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982, 1:385-94), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 1989, 9:946), the HSV TK promoter, and such. The introduction of the vector into host cells to express the C12orf32 gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 1987, 15:1311-26), the calcium phosphate method (Chen et al., Mol Cell Biol 1987, 7:2745-52), the DEAE dextran method (Lopata et al., Nucleic Acids Res 1984, 12:5707-17; Sussman et al., Mol Cell Biol 1985, 4:1641-3), the Lipofectin method (Derijard B, Cell 1994, 7:1025-37; Lamb et al., Nature Genetics 1993, 5:22-30; Rabindran et al., Science 1993, 259:230-4), and such.

The C12orf32 protein may also be produced in vitro adopting an in vitro translation system.
The C12orf32 polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.

III-1-1. Identifying substances that bind to the polypeptides
A substance that binds to a protein is likely to alter the expression of the gene coding for the protein or the biological activity of the protein. Thus, as an aspect, the present invention provides a method of screening a candidate substance for treating or preventing cancer, which includes steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting binding (or binding activity) between the polypeptide or fragment and the test substance; and
(c) selecting the test substance that binds to the polypeptide as a candidate substance for treating or preventing cancer.

According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease, using the C12orf32 polypeptide or fragments thereof including the steps as follows:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and
(c) correlating the binding of (b) with the therapeutic effect of the test substance.

In the context of the present invention, the therapeutic effect may be correlated with the binding level to C12orf32 polypeptide or a functional fragment thereof. For example, when the test substance binds to C12orf32 polypeptide or a functional fragment thereof, the test substance may identified or selected as the candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not bind to a C12orf32 polypeptide or a functional fragment thereof, the test substance may identified as the substance having no significant therapeutic effect.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of C12orf32, the method including steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting the binding (or binding activity) between the polypeptide or fragment and the test substance; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance binds to the polypeptide.

In the context of the present invention, the therapeutic effect may be correlated with the binding level of the test substance and C12orf32 protein(s). For example, when the test substance binds to a C12orf32 protein, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not bind to a C12orf32 protein, the test substance may characterized as having no significant therapeutic effect.
In the present invention, it is revealed that suppressing the expression of C12orf32 reduces cancer cell growth. Thus, by screening for candidate substances that binds to C12orf32, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.

The binding of a test substance to the C12orf32 polypeptide may be, for example, detected by immunoprecipitation using an antibody against the polypeptide. Therefore, for the purpose for such detection, it is preferred that the C12orf32 polypeptide or fragments thereof used for the screening contains an antibody recognition site. The antibody used for the screening may be one that recognizes an antigenic region (e.g., epitope) of the present C12orf32 polypeptide of which preparation methods are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.

Alternatively, the C12orf32 polypeptide or a fragment thereof may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 1995, 13:85-90). Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase (GST), green fluorescence protein (GFP), and such by the use of its multiple cloning sites are commercially available and can be used for the present invention. Furthermore, fusion proteins containing much smaller epitopes to be detected by immunoprecipitation with an antibody against the epitopes are also known in the art (Experimental Medicine 1995, 13:85-90). Such epitopes, composed of several to a dozen amino acids so as not to change the property of the C12orf32 polypeptide or fragments thereof, can also be used in the present invention. Examples include polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), and such and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the C12orf32 polypeptide (Experimental Medicine 13: 85-90 (1995)).

Glutathione S-transferase (GST) is also well-known as the counterpart of the fusion protein to be detected by immunoprecipitation. When GST is used as the protein to be fused with the C12orf32 polypeptide or fragment thereof to form a fusion protein, the fusion protein can be detected either with an antibody against GST or a substance specifically binding to GST, i.e., such as glutathione (e.g., glutathione-Sepharose 4B).

In immunoprecipitation, an immune complex is formed by adding an antibody (recognizing the C12orf32 polypeptide or a fragment thereof itself, or an epitope tagged to the polypeptide or fragment) to the reaction mixture of the C12orf32 polypeptide and the test substance. If the test substance has the ability to bind the polypeptide, then the formed immune complex will consists of the C12orf32 polypeptide, the test substance, and the antibody. On the contrary, if the test substance is devoid of such ability, then the formed immune complex only consists of the C12orf32 polypeptide and the antibody. Therefore, the binding ability of a test substance to C12orf32 polypeptide can be examined by, for example, measuring the size of the formed immune complex. Any method for detecting the size of a substance can be used, including chromatography, electrophoresis, and such. For example, when mouse IgG antibody is used for the detection, Protein A or Protein G sepharose can be used for quantitating the formed immune complex.

For more details on immunoprecipitation see, for example, Harlow et al., Antibodies, Cold Spring Harbor Laboratory publications, New York, 1988, 511-52. SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Detection may be achieved using conventional staining method, such as Coomassie staining or silver staining, or, for difficulty to detect protections, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cystein, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.

Furthermore, the C12orf32 polypeptide or a fragment thereof used for the screening of substances that bind thereto may be bound to a carrier. Example of carriers that may be used for binding the polypeptides include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicon; preferably commercially available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials may be used. When using beads, they may be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables to readily isolate polypeptides and substances bound on the beads via magnetism.

The binding of a polypeptide to a carrier may be conducted according to routine methods, such as chemical bonding and physical adsorption. Alternatively, a polypeptide may be bound to a carrier via antibodies specifically recognizing the polypeptide. Moreover, binding of a polypeptide to a carrier can also be conducted by means of interacting molecules, such as the combination of avidin and biotin.

Screening using such carrier-bound C12orf32 polypeptide or fragments thereof include, for example, contacting a test substance to the carrier-bound polypeptide, incubating the mixture, washing the carrier, and detecting and/or measuring the substance bound to the carrier. The binding may be carried out in buffer, for example, but are not limited to, phosphate buffer and Tris buffer, as long as the buffer does not inhibit the binding.

When such carrier-bound C12orf32 polypeptide or fragments thereof and a composition (e.g., cell extracts, cell lysates, etc.) are used as the test substance in a screening method, such method is generally called affinity chromatography. For example, the C12orf32 polypeptide may be immobilized on a carrier of an affinity column, and a test substance, containing a substance capable of binding to the polypeptides, is applied to the column. After loading the test substance, the column is washed, and then the substance bound to the polypeptide is eluted with an appropriate buffer.

A biosensor using the surface plasmon resonance phenomenon may be used as a mean for detecting or quantifying the bound substance in the present invention. When such a biosensor is used, the interaction between the C12orf32 polypeptide and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of the polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide and test substance using a biosensor such as BIAcore.

Methods of screening for molecules that bind to a specific protein among synthetic chemical compounds, or molecules in natural substance banks or a random phage peptide display library by exposing the specific protein immobilized on a carrier to the molecules, and methods of high-throughput screening based on combinatorial chemistry techniques (Wrighton et al., Science 1996, 273:458-64; Verdine, Nature 1996, 384:11-3) to isolate not only proteins but chemical compounds are also well-known to those skilled in the art. These methods can also be used for screening substances (including agonist and antagonist) that bind to the C12orf32 protein or fragments thereof.

When the test substance is a protein, for example, West-Western blotting analysis (Skolnik et al., Cell 1991, 65:83-90) can be used for the present method. Specifically, a protein binding to the C12orf32 polypeptide can be obtained by preparing first a cDNA library from cells, tissues, organs, or cultured cells (e.g., PC cell lines) expected to express at least one protein binding to the C12orf32 polypeptide using a phage vector (e.g., ZAP), expressing the proteins encoded by the vectors of the cDNA library on LB-agarose, fixing the expressed proteins on a filter, reacting the purified and labeled C12orf32 polypeptide with the above filter, and detecting the plaques expressing proteins to which the C12orf32 polypeptide has bound according to the label of the C12orf32 polypeptide.

Labeling substances such as radioisotope (e.g., 3H, 14C, 32P, 33P, 35S, 125I, 131I), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucosidase), fluorescent substances (e.g., fluorescein isothiocyanate (FITC), rhodamine) and biotin/avidin, may be used for the labeling of C12orf32 polypeptide in the present method. When the protein is labeled with radioisotope, the detection or measurement can be carried out by liquid scintillation. Alternatively, when the protein is labeled with an enzyme, it can be detected or measured by adding a substrate of the enzyme to detect the enzymatic change of the substrate, such as generation of color, with absorptiometer. Further, in case where a fluorescent substance is used as the label, the bound protein may be detected or measured using fluorophotometer.

Moreover, the C12orf32 polypeptide bound to the protein can be detected or measured by utilizing an antibody that specifically binds to the C12orf32 polypeptide or a peptide or polypeptide (for example, GST) that is fused to the C12orf32 polypeptide. In case of using an antibody in the present screening, the antibody is preferably labeled with one of the labeling substances mentioned above, and detected or measured based on the labeling substance. Alternatively, the antibody against the C12orf32 polypeptide may be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance. Furthermore, the antibody bound to the C12orf32 polypeptide in the present screening may be detected or measured using protein G or protein A column.

Antibodies to be used in the present screening methods can be prepared using techniques well known in the art. Antigens to prepared antibodies may be derived from any animal species, but preferably is derived from a mammal such as a human, mouse, rabbit, or rat, more preferably from a human. The polypeptide used as the antigen can be recombinantly produced or isolated from natural sources. The polypeptides to be used as an immunization antigen may be a complete protein or a partial peptide derived from the complete protein.

Any mammalian animal may be immunized with the antigen; however, the compatibility with parental cells used for cell fusion is preferably taken into account. In general, animals of the order Rodentia, Lagomorpha or Primate are used. Animals of the Rodentia order include, for example, mice, rats and hamsters. Animals of Lagomorpha order include, for example, hares, pikas, and rabbits. Animals of Primate order include, for example, monkeys of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkeys, sacred baboons and chimpanzees.

Methods for immunizing animals with antigens are well known in the art. Intraperitoneal injection or subcutaneous injection of antigens is a standard method for immunizing mammals. More specifically, antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals. Preferably, it is followed by several administrations of the antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. An appropriate carrier may also be used for immunization. After immunization as above, the serum is examined by a standard method for an increase in the amount of desired antibodies.

Polyclonal antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method. Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies isolated from the serum. Immunoglobulin G or M can be prepared from a fraction which recognizes only the objective polypeptide using, for example, an affinity column coupled with the polypeptide, and further purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion. The immune cells used for cell fusion are preferably obtained from spleen. Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
The above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium). The cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma, to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal is immunized with an antigen for preparing hybridoma, human lymphocytes, such as those infected by the EB virus, may be immunized with an antigen, cells expressing such antigen, or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the antigen (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).

The obtained hybridomas may be subsequently transplanted into the abdominal cavity of a mouse and the ascites may be extracted. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column carrying an objective antigen.

Antibodies against the C12orf32 polypeptide can be used not only in the present screening method, but also for the detection of the polypeptides as cancer markers in biological samples as described in "I. Diagnosing cancer". They may further serve as candidates for agonists and antagonists of the polypeptides of interest. In addition, such antibodies, serving as candidates for antagonists, can be applied to the antibody treatment for diseases related to the C12orf32 polypeptide including breast cancer as described infra.

Monoclonal antibodies thus obtained can be also recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the United Kingdom by MacMillan Publishers LTD (1990)). For example, a DNA encoding an antibody may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant antibody. Such recombinant antibody can also be used in the context of the present screening.

Furthermore, antibodies used in the screening and so on may be fragments of antibodies or modified antibodies, so long as they retain the original binding activity. For instance, the antibody fragment may be an Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). Modified antibodies can be obtained through chemically modification of an antibody. These modification methods are conventional in the field.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by appropriately selected and combined column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)); however, the present invention is not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity, includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.

Alternatively, in another embodiment of the screening method of the present invention, two-hybrid system utilizing cells may be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton et al., Cell 1992, 68:597-612" and "Fields et al., Trends Genet 1994, 10:286-92"). In two-hybrid system, C12orf32 polypeptide or a fragment thereof is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express at least one protein binding to the C12orf32 polypeptide such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the C12orf32 polypeptide is expressed in the yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
The substance isolated by this screening is a candidate for agonists or antagonists of the C12orf32 polypeptide. The term "agonist" refers to molecules that activate the function of the polypeptide by binding thereto. On the other hand, the term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. Moreover, a substance isolated by this screening as an antagonist is a candidate that inhibits the in vivo interaction of the C12orf32 polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).

III-1-2. Identifying substances by detecting biological activity of the polypeptides
The present invention also provides a method for screening a candidate substance for treating or preventing cancer using the C12orf32 polypeptide or fragments thereof including the steps as follows:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof; and
(b) detecting the biological activity of the polypeptide or fragment of the step (a).
(c) selecting the test substance that reduces the biological activity of the polypeptide as compared to the biological activity in the absence of the test substance.

According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease, using the C12orf32 polypeptide or fragments thereof including the steps as follows:
(a) contacting a test substance with a C12orf32 polypeptide or a functional fragment thereof;
(b) detecting the biological activity of the polypeptide or fragment of step (a), and
(c) correlating the biological activity of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of C12orf32, the method including steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a functional fragment thereof;
(b) detecting the biological activity of the polypeptide or fragment of step (a), and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance suppresses the biological activity of the polypeptide encoded by the polynucleotide of C12orf32 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.

In the present invention, the therapeutic effect may be correlated with the biological activity of a C12orf32 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of a C12orf32 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not suppress or inhibit the biological activity of C12orf32 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

In the present invention, it is revealed that suppressing the expression of C12orf32 reduces cancer cell growth. Thus, by screening for candidate substances that suppresses the biological activity of the polypeptide, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance for cancers. For example, when a substance binding to C12orf32 protein inhibits described above activities of the cancer, it may be concluded that such substance has the C12orf32 specific therapeutic effect.

Any polypeptide can be used for the screening so long as it suppresses or reduces a biological activity of the C12orf32 polypeptide. In the context of the instant invention, the phrase "suppress or reduce a biological activity" encompasses at least 10% suppression of the biological activity of C12orf32 in comparison with in the absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression. Such suppression can serve an index in the present screening method.

According to the present invention, the C12orf32 polypeptide has been demonstrated to be required for the growth or viability of breast cancer cells. The biological activities of the C12orf32 polypeptide that can be used as an index for the screening include such cell growth promoting activity of the human C12orf32 polypeptide.

When the biological activity to be detected in the present method is cell proliferation, it can be detected, for example, by preparing cells which express the C12orf32 polypeptide or a fragment thereof, culturing the cells in the presence of a test substance, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony forming activity.

In the preferred embodiments, control cells which do not express the C12orf32 polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease, using the C12orf32 polypeptide or fragments thereof including the steps as follows:
(a) culturing cells which express a C12orf32 polypeptide or a functional fragment thereof, and control cells that do not express a C12orf32 polypeptide or a functional fragment thereof in the presence of the test substance;
(b) detecting the proliferation of the cells which express the protein and control cells; and
(c) selecting the test subtance that inhibits the proliferation in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of the test substance.

In another aspect, the present invention also provides a screening method, comprising the steps of:
(a) contacting a test substance with the C12orf32 polypeptide or a fragment thereof;
(b) detecting the binding between the polypeptide or fragment and the test substance;
(c) selecting the test substance that binds to the polypeptide;
(d) contacting the test substance selected in step (c) with the C12orf32 polypeptide or a fragment thereof;
(e) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the substance; and
(f) selecting the substance that suppresses the biological activity of the polypeptide as a candidate agent for treating or preventing breast cancer.

The substance isolated by the present screening method is a candidate for an antagonist of the C12orf32 polypeptide, and thus, is a candidate that inhibits the in vivo interaction of the polypeptide with molecules (including nucleic acids (RNAs and DNAs) and proteins).

The polypeptides to be used in the present screening methods may be recombinantly produced using standard procedures. For example, a gene encoding a polypeptide of interest or fragment thereof may be expressed in animal cells by inserting the gene into an expression vector for foreign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8. The promoter to be used for the expression may be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF-alpha promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., Gene 108: 193 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) the SR alpha promoter (Takebe et al., Mol Cell Biol 8: 466-72 (1988)), the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946-58 (1989)), the HSV TK promoter and so on. The introduction of the gene into animal cells to express a foreign gene can be performed according to any conventional method, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B, Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)), and so on. The polypeptides may be expressed as a fusion protein including a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. Alternatively, a commercially available epitope-antibody system may be used (Experimental Medicine 13: 85-90 (1995)). Vectors which are capable of expressing a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP), and so on, by the use of its multiple cloning sites are commercially available.

A fusion protein, prepared by introducing only small epitopes composed of several to a dozen amino acids so as not to change the property of the original polypeptide by the fusion, is also provided herein. Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and antibodies recognizing them may be used as the epitope-antibody system for detecting the binding activity between the polypeptides (Experimental Medicine 13: 85-90 (1995)).

III-2. Nucleotide based screening methods
III-2-1. Screening method using C12orf32 gene
As discussed in detail above, by controlling the expression level of the C12orf32 gene, one can control the onset and progression of cancer. Thus, substances that may be used in the treatment or prevention of cancers can be identified through screenings that use the expression levels of the C12orf32 gene as indices. In the context of the present invention, such screening may include, for example, the following steps:
(a) contacting a test substance with a cell expressing a C12orf32 gene;
(b) detecting the expression level of the C12orf32 gene;
(c) comparing the expression level with the expression level detected in the absence of the substance; and
(d) selecting the substance that reduces the expression level as a candidate substance for treating or preventing cancer.

According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing C12orf32 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing C12orf32 associating disease.

In the context of the present invention, such screening may include, for example, the following steps:
(a) contacting a test substance with a cell expressing a C12orf32 gene;
(b) detecting the expression level of the C12orf32 gene; and
(c) correlating the expression level of b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of C12orf32, wherein the method including steps of:
(a) contacting a test substance with a cell expressing a C12orf32 gene;
(b) detecting the expression level of the C12orf32 gene; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression level or activity of said reporter gene.

In the context of the present invention, the therapeutic effect may be correlated with the expression level of the C12orf32 gene. For example, when the test substance reduces the expression level of the C12orf32 gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the C12orf32 gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

Herein, it was revealed that suppressing the expression of C12orf32 reduces cancer cell growth. Thus, by screening for candidate substances that reduces the expression level of C12orf32, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.

A substance that inhibits the expression of the C12orf32 gene or the activity of its gene product can be identified by contacting a cell expressing the C12orf32 gene with a test substance and then determining the expression level of the C12orf32 gene. Naturally, the identification may also be performed using a population of cells that express the gene in place of a single cell. A decreased expression level detected in the presence of a substance as compared to the expression level in the absence of the substance indicates the substance as being an inhibitor of the C12orf32 gene, suggesting the possibility that the substance is useful for inhibiting cancer, thus a candidate substance may be used for the treatment or prevention of cancer.

The expression level of a gene can be estimated by methods well known to one skilled in the art. The expression level of the C12orf32 gene can be, for example, determined following the method described above under the item of 'I-1. Method for diagnosing cancer or a predisposition for developing cancer'.

The cell or the cell population used for such identification may be any cell or any population of cells so long as it expresses the C12orf32 gene. For example, the cell or population may be or contain a breast epithelial cell derived from a breast cancer tissue. Alternatively, the cell or population may be or contain an immortalized cell derived from a carcinoma cell, including breast cancer cell. Cells expressing the C12orf32 gene include, for example, cell lines established from cancers (e.g., breast cancer cell lines such as HCC-1937, BT-549, MCF-7, BSY-1, MDA-MB-435S, SKBR-3, T-47D, MDA-MB-231, YMB-1 etc.). Furthermore, the cell or population may be or contain a cell which has been transfected with the C12orf32 gene.
The present method allows screening of various substances mentioned above and is particularly suited for screening functional nucleic acid molecules including antisense RNA, siRNA, and such.

III-2-2. Screening method using transcriptional regulatory region of C12orf32 gene
According to another aspect, the present invention provides a method which includes the following steps of:
(a) contacting a test substance with a cell into which a vector, including a transcriptional regulatory region of a C12orf32 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) detecting the expression or activity of the reporter gene;
(c) comparing the expression level or activity of (b) with the expression level or activity detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression or activity of the reporter gene as a candidate substance for treating or preventing cancer.

According to another aspect, the present invention provides a method which includes the following steps of:
(a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of a C12orf32 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) detecting the expression or activity of the reporter gene; and
(c) correlating the expression level of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of C12orf32, the method including steps of:
(a) contacting a test substance with a cell into which a vector, composed of a transcriptional regulatory region of a C12orf32 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) detecting the expression or activity of the reporter gene; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduces the expression level or activity of said reporter gene.

In the present invention, the therapeutic effect may be correlated with the expression or activity of the reporter gene. For example, when the test substance reduces the expression or activity of the reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression or activity of the reporter gene as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

Herein, it was revealed that suppressing the expression of C12orf32 reduces cell growth. Thus, by screening for candidate substances that reduces the expression or activity of the reporter gene, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers.

Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared using the transcriptional regulatory region of the C12orf32 gene, which can be obtained as a nucleotide segment containing the transcriptional regulatory region from a genome library based on the nucleotide sequence information of the gene.

The transcriptional regulatory region may be, for example, the promoter sequence of the C12orf32 gene. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of C12orf32 gene. The transcriptional regulatory region of C12orf32 gene herein is the region from start codon to at least 500 bp upstream, preferably 1,000 bp, more preferably 5,000 or 10,000 bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

When a cell(s) transfected with a reporter gene that is operably linked to the regulatory sequence (e.g., promoter sequence) of the C12orf32 gene is used, a substance can be identified as inhibiting or enhancing the expression of the C12orf32 gene through detecting the expression level of the reporter gene product.
Illustrative reporter genes include, but are not limited to, luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa, Ade2 gene, HIS3 gene, and others well-known in the art. Methods for detection of the expression of these genes are well known in the art.

A vector containing a reporter construct may be infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorptiometer, flow cytometer and so on). In the context of the instant invention, the phrase "reduces the expression or activity" encompasses at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, more preferably at least 25%, 50% or 75% reduction and most preferably at 95% reduction.

III-3. Selecting therapeutic substances that are appropriate for a particular individual
Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. A substance that is metabolized in a subject to act as an anti-tumor substance can manifest itself by inducing a change in a gene expression pattern in the subject's cells from that characteristic of a cancerous state to a gene expression pattern characteristic of a non cancerous state. Accordingly, the C12orf32 gene differentially expressed between cancerous and non-cancerous cells disclosed herein allow for a putative therapeutic or prophylactic inhibitor of cancer to be tested in a test cell population from a selected subject in order to determine if the substance is a suitable inhibitor of cancer in the subject.

To identify an inhibitor of cancer that is appropriate for a specific subject, a test cell population from the subject is exposed to a candidate therapeutic substance, and the expression of C12orf32 gene is determined.
In the context of the method of the present invention, test cell populations contain cancer cells expressing the C12orf32 gene. Preferably, the test cell is a breast epithelial cell.
Specifically, a test cell population may be incubated in the presence of a candidate therapeutic substance and the expression of the C12orf32 gene in the test cell population may be measured and compared to one or more reference profiles, e.g., a cancerous reference expression profile or a non-cancerous reference expression profile.

A decrease in the expression of the C12orf32 gene in a test cell population relative to a reference cell population containing cancer indicates that the substance has therapeutic potential. Alternatively, a similarity in the expression of the C12orf32 gene in a test cell population relative to a reference cell population not containing cancer indicates that the substance has therapeutic potential.

IV. Pharmaceutical compositions for treating or preventing cancer
The substances screened by any of the screening methods of the present invention, antisense nucleic acids and double-stranded molecules (e.g., siRNA) of the C12orf32 gene, and antibodies against the C12orf32 polypeptide inhibit or suppress the expression of the C12orf32 gene, or the biological activity of the C12orf32 polypeptide and inhibit or disrupt cancer cell cycle regulation and cancer cell proliferation. Thus, the present invention provides compositions for treating or preventing cancer, which compositions include substances screened by any of the screening methods of the present invention, antisense nucleic acids and double-stranded molecules of the C12orf32 gene, or antibodies against the C12orf32 polypeptide. The present compositions can be used for treating or preventing cancer, in particular, cancer such as breast cancer.

The compositions may be used as pharmaceuticals for humans and other mammals, such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.
In the context of the present invention, suitable pharmaceutical formulations for the active ingredients of the present invention detailed below (including screened substances, antisense nucleic acids, double-stranded molecules, antibodies, etc.) include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Preferably, administration is intravenous. The formulations are optionally packaged in discrete dosage units.

Pharmaceutical formulations suitable for oral administration include capsules, microcapsules, cachets and tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, elixirs, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as a bolus electuary or paste. Alternatively, according to needs, the pharmaceutical composition may be administered non-orally, in the form of injections of sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. For example, the active ingredients of the present invention can be mixed with pharmaceutically acceptable carriers or media, specifically, sterilized water, physiological saline, plant-oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, and such, in a unit dose form required for generally accepted drug implementation. The amount of active ingredient contained in such a preparation makes a suitable dosage within the indicated range acquirable.

Examples of additives that can be admixed into tablets and capsules include, but are not limited to, binders, such as gelatin, corn starch, tragacanth gum and arabic gum; excipients, such as crystalline cellulose; swelling agents, such as corn starch, gelatin and alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin; and flavoring agents, such as peppermint, Gaultheria adenothrix oil and cherry. A tablet may be made by compression or molding, optionally with one or more formulational ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made via molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be coated according to methods well known in the art. The tablets may optionally be formulated so as to provide slow or controlled release of the active ingredient in vivo. A package of tablets may contain one tablet to be taken on each of the month.
Furthermore, when the unit-dosage form is a capsule, a liquid carrier, such as oil, can be further included in addition to the above ingredients.

Oral fluid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle prior to use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Moreover, sterile composites for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection. Physiological saline, glucose, and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection. These can be used in conjunction with suitable solubilizers, such as alcohol, for example, ethanol; polyalcohols, such as propylene glycol and polyethylene glycol; and non-ionic surfactants, such as Polysorbate 80 (TM) and HCO-50.

Sesame oil or soy-bean oil can be used as an oleaginous liquid, which may be used in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and may be formulated with a buffer, such as phosphate buffer and sodium acetate buffer; a pain-killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and/or an anti-oxidant. A prepared injection may be filled into a suitable ampoule.

Formulations for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations for topical administration in the mouth, for example, buccally or sublingually, include lozenges, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and pastilles including the active ingredient in a base such as gelatin, glycerin, sucrose or acacia. For intra-nasal administration of an active ingredient, a liquid spray or dispersible powder or in the form of drops may be used. Drops may be formulated with an aqueous or non-aqueous base also including one or more dispersing agents, solubilizing agents or suspending agents.

For administration by inhalation the compositions are conveniently delivered from an insufflator, nebulizer, pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may include a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder composition, for example, a powder mix of an active ingredient and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflators.

Other formulations include implantable devices and adhesive patches that release a therapeutic agent.
When desired, the above-described formulations, adapted to give sustained release of the active ingredient, may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives.

It should be understood that, in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.
Preferred unit dosage formulations are those containing an effective dose, as recited under the item of 'V. Methods for treating or preventing cancer' (infra), of each of the active ingredients of the present invention or an appropriate fraction thereof.

IV-1. Pharmaceutical compositions containing screened substances
The present invention provides compositions for treating or preventing cancers including any of the substances selected by the above-described screening methods of the present invention.
A substance screened by the method of the present invention can be directly administered or can be formulated into a dosage form according to any conventional pharmaceutical preparation method detailed above.

IV-2. Pharmaceutical compositions including double-stranded molecules
Double-stranded molecules (e.g., siRNA) against the C12orf32 gene can be used to reduce the expression level of the genes. Herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g., double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)) as described in "Definitions". In the context of the present invention, double-stranded molecules include a sense nucleic acid sequence and an antisense nucleic acid sequence against the C12orf32 gene. The double-stranded molecule is constructed so that it includes both portions of the sense and complementary antisense sequences of the target gene (i.e., the C12orf32 gene), and may also be a single construct taking a hairpin structure, wherein the sense and antisense strands are linked via a single-strand.

The double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RNA-induced silencing complex (RISC), when the double-stranded molecule is introduced into cells. The identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the C12orf32 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.

In the context of the present invention, the target sequence of a double-stranded molecule is preferably less than 500, 200, 100, 50, or 25 base pairs in length. More preferably, the target sequence of a double stranded molecule is 19-25 base pairs in length. Exemplary target nucleic acid sequences of double-stranded molecules against the C12orf32 gene include the nucleotide sequences of SEQ ID NO: 8, 9 or 14. The nucleotide "t" in the sequence should be replaced with "u" in RNA or derivatives thereof. Accordingly, for example, the present pharmaceutical composition may include a double-stranded RNA molecule (siRNA) including the nucleotide sequence 5'- AAGCUGACUGCCAUCAGUAAU -3' (for SEQ ID NO: 8), 5'- AACAGUUCAGUUUAGUGUCAU -3' (for SEQ ID NO: 9), and 5'- GCUGACUGCCAUCAGUAAU -3' (for SEQ ID NO: 14)
as the sense strand.

In order to enhance the inhibition activity of the double-stranded molecule, 3' overhangs can be added to the 3'end of the target sequence in the sense and/or antisense strand. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form a single strand at the 3'end of the sense and/or antisense strand of the double-stranded molecule. The nucleotides to be added is preferably "u" or "t", but are not limited to.

A loop sequence consisting of an arbitrary nucleotide sequence can be located between the sense and antisense strands in order to form a hairpin loop structure. Thus, the double-stranded molecule contained in the inventive composition may take the general formula 5'-[A]-[B]-[A']-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to a target sequence. Herein, the polynucleotide strand which includes a sequence corresponding to a target sequence specifically hybridizing to an mRNA or a cDNA of the C12orf32 gene, may be referred to as "sense strand". In preferred embodiments, [A] is the sense strand; [B] is a single stranded polynucleotide consisting of 3 to 23 nucleotides; and [A'] is a polynucleotide strand which includes the antisense strand containing a complementary sequence of a target sequence specifically hybridizing to an mRNA or a cDNA of the C12orf32 gene (i.e., a sequence hybridizing to the target sequence of the sense strand [A]). Herein, the polynucleotide strand which includes a complementary sequence to a target sequence specifically hybridizing to an mRNA or a cDNA of the C12orf32 gene may be referred to as "antisense strand". The region [A] hybridizes to [A'], and then a loop consisting of the region [B] is formed. The loop sequence may be preferably 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from a group consisting of following sequences (www.ambion.com/techlib/tb/tb_506.html):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002, 418: 435-8.
UUCG: Lee NS et al., Nature Biotechnology 2002, 20:500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003, 100(4):1639-44.
UUCAAGAGA: Dykxhoorn DM et al., Nature Reviews Molecular Cell Biology 2003, 4:457-67.
'UUCAAGAGA ("ttcaagaga" in DNA)' is a particularly suitable loop sequence. Furthermore, loop sequence consisting of 23 nucleotides also provides an active siRNA (Jacque JM et al., Nature 2002, 418:435-8).

Exemplary hairpin siRNA suitable for the C12orf32 gene include:
5'- AAGCUGACUGCCAUCAGUAAU -[b]- AUUACUGAUGGCAGUCAGCUU -3'
(target sequence of SEQ ID NO: 8);
5'- AACAGUUCAGUUUAGUGUCAU -[b]- AUGACACUAAACUGAACUGUU -3'
(target sequence of SEQ ID NO: 9); and
5'- GCUGACUGCCAUCAGUAAU -[b]- ATTACTGATGGCAGTCAGC -3'
(target sequence of SEQ ID NO: 14).

Other nucleotide sequences of suitable double-stranded molecules for the present invention can be designed using an siRNA design computer program available from the Ambion website (www.ambion.com/techlib/ misc/siRNA_finder.html). The computer program selects nucleotide sequences for double-stranded molecule synthesis based on the following protocol.

Selection of Target Sites for double-stranded molecules:
1. Beginning with the AUG start codon of the object transcript, scan downstream for AA dinucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential target sites. Tuschl et al. Genes Cev 1999, 13(24):3191-7 don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 nucleotides) as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the human genome database and eliminate from consideration any target sequences with significant homology to other coding sequences. The homology search can be performed using BLAST (Altschul SF et al., Nucleic Acids Res 1997, 25:3389-402; J Mol Biol 1990, 215:403-10.), which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/.
3. Select qualifying target sequences for synthesis. At Ambion, preferably several target sequences can be selected along the length of the gene to evaluate.

The method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that includes the target sequence and antisense thereof is transcribed into the double stranded molecule in vitro. In one embodiment for the annealing, the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.

The regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning C12orf32 gene template into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.

Double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to suppress the growth of cells expressing the target genes. Therefore, the present invention provides double-stranded molecule targeting the sequences of SEQ ID NO: 8, 9 and 14 for C12orf32 gene.
The double-stranded molecule of the present invention may be directed to a single target C12orf32 gene sequence or may be directed to a plurality of target C12orf32 gene sequences.

A double-stranded molecule of the present invention targeting the above-mentioned targeting sequence of C12orf32 gene include isolated polynucleotide that contain the nucleic acid sequences of target sequences and/or complementary sequences to the target sequence. Example of polynucleotide targeting C12orf32 gene includes that containing the sequence of SEQ ID NO: 8, 9 and 14 and/or complementary sequences to these nucleotides. However, the present invention is not limited to this example, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of C12orf32 gene. Herein, the phrase "minor modification" as used in connection with a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.

In an embodiment, a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA.

In an another embodiment, a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand. Generally, the sense strand and the antisense strand are linked by a intervening strand, and hybridize to each other to form a hairpin loop structure. Examples of such double-stranded molecule include shRNA and shD/R-NA.

In other words, a double-stranded molecule of the present invention comprises a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule. In the double-stranded molecule comprising the polynucleotides, a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u".

In one embodiment of the present invention, such a double-stranded molecule of the present invention comprises a stem-loop structure, composed of the sense and antisense strands. The sense and antisense strands may be joined by a loop. Accordingly, the present invention also provides the double-stranded molecule comprising a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.

In the present invention, double-stranded molecules targeting the C12orf32 gene may have a sequence selected from among SEQ ID NOs: 8, 9 and 14 as a target sequence. Accordingly, preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 8, 9 and 14 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 8, 9 and 14 and a complementary sequence thereto.
In the context of the present invention, the term "several" as applies to nucleic acid substitutions, deletions, additions and/or insertions may mean 3-7, preferably 3-5, more preferably 3-4, even more preferably 3 nucleic acid residues.

According to the present invention, a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples. In the Examples herein below, double-stranded molecules composed of sense strands of various portions of mRNA of C12orf32 genes or antisense strands complementary thereto were tested in vitro for their ability to decrease production of C12orf32 gene product in cancer cell lines according to standard methods. Furthermore, for example, reduction in C12orf32 gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for C12orf32 mRNA mentioned under Example item "Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)". Sequences which decrease the production of C12orf32 gene product in in vitro cell-based assays can then be tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in in vitro cell-based assay can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of C12orf32 product and decreased cancer cell growth.

When the isolated polynucleotide is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide, and the term "binding" means the physical or chemical interaction between two polynucleotides. When the polynucleotide includes modified nucleotides and/or non-phosphodiester linkages, these polynucleotides may also bind each other as same manner. Generally, complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. Furthermore, the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization. In a preferred embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In an especially preferred embodiment, where the strands of the duplex are fully complementary, such duplexes contain no mismatches.

The polynucleotide is preferably less than 1872 nucleotides in length for C12orf32. For example, the polynucleotide is less than 500, 200, 100, 75, 50, or 25 nucleotides in length for all of the genes. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against C12orf32 gene or preparing template DNAs encoding the double-stranded molecules. When the polynucleotides are used for forming double-stranded molecules, the polynucleotide may be longer than 19 nucleotides, preferably longer than 21 nucleotides, and more preferably has a length of between about 19 and 25 nucleotides.

Accordingly, the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence. In preferable embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.

Standard techniques are known in the art for introducing a double-stranded molecule into cells. For example, a double-stranded molecule can be directly introduced into the cells in a form that is capable of binding to the mRNA transcripts. In these embodiments, the double-stranded molecules are typically modified as described below for antisense molecules. Other modifications are also available, for example, cholesterol-conjugated double-stranded molecule have shown improved pharmacological properties (Song et al., Nature Med 2003, 9:347-51). These conventionally used techniques may also be applied for the double-stranded molecules contained in the present compositions.

Alternatively, a DNA encoding the double-stranded molecule may be carried in a vector (hereinafter, also referred to as 'siRNA vector') and the double-stranded molecule may be contained in the present composition in the form of vector which enables expression of the double-stranded molecule in vivo. Such vectors may be produced, for example, by cloning a portion of the target sequence sufficient to inhibit the in vivo expression of the target gene into an expression vector having operatively-linked regulatory sequences (e.g., a RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) flanking the sequence in a manner that allows for expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nature Biotechnology 2002, 20: 500-5). For example, an RNA molecule that is antisense to mRNA of the target gene is transcribed by a first promoter (e.g., a promoter sequence 3' of the cloned DNA) and an RNA molecule that is the sense strand for the mRNA of the target gene is transcribed by a second promoter (e.g., a promoter sequence 5' of the cloned DNA). The sense and antisense strands hybridize in vivo to generate the double-stranded molecule construct for silencing the expression of the target gene. Alternatively, the sense and antisense strands may be transcribed together with the help of one promoter. In this case, the sense and antisense strands may be linked via a polynucleotide sequence to form a single-stranded construct having secondary structure, e.g., hairpin.

Thus, the present pharmaceutical composition for treating or preventing cancer may include either the double-stranded molecule (e.g., siRNA) or a vector expressing the double-stranded molecule in vivo. In particular, the present invention provides pharmaceutical compositions for treating or preventing cancer that include a double-stranded molecule that inhibits the expression of the C12orf32 gene, or a vector expressing the double-stranded molecule in vivo.

According to the present invention, the composition may contain plural kinds of the double-stranded molecules, each of the molecules may be directed to the same target sequence, or different target sequences of C12orf32. For example, the composition may contain double-stranded molecules directed to C12orf32. Alternatively, for example, the composition may contain double-stranded molecules directed to one, two or more target sequences C12orf32.

Furthermore, the present composition may contain a vector coding for one or plural double-stranded molecules. For example, the vector may encode one, two or several kinds of the present double-stranded molecules. Alternatively, the present composition may contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
Further, the present invention also provides pharmaceutical compositions for inhibiting cancer cell proliferation, such composition including a double-stranded molecule which inhibits the expression of the C12orf32 gene, or a vector expressing the double-stranded molecule in vivo.

For introducing the double-stranded molecule vector into the cell, transfection-enhancing agent can be used. FuGENE6 (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as the transfection-enhancing agent. Therefore, the present pharmaceutical composition may further include such transfection-enhancing agents.

In another embodiment, the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention or vector encoding thereof in manufacturing a pharmaceutical composition for treating a cancer expressing the C12orf32 gene. For example, the present invention relates to a use of double-stranded nucleic acid molecule that inhibits the expression of C12orf32 gene in a cell that over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule, and targets to a sequence of SEQ ID NOs: 8, 9 or 14 for manufacturing a pharmaceutical composition for treating a cancer expressing the C12orf32 gene.
Alternatively, the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the C12orf32 gene.

Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the C12orf32 gene, wherein the method or process includes step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of C12orf32 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule, and targets to a sequence of SEQ ID NOs: 8, 9 or 14 as active ingredients.

In another embodiment, the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing the C12orf32 gene, wherein the method or process includes step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of C12orf32 gene in a cell, which over-expresses the gene, wherein the molecule includes a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule, and targets to a sequence of SEQ ID NOs: 8, 9 or 14.

IV-3. Pharmaceutical compositions including antisense nucleic acids
Antisense nucleic acids targeting the C12orf32 gene can be used to reduce the expression level of the gene that is up-regulated in cancerous cells including breast cancer cells. Such antisense nucleic acids are useful for the treatment of cancer, in particular breast cancer and thus are also encompassed by the present invention. An antisense nucleic acid acts by binding to the nucleotide sequence of the C12orf32 gene, or mRNAs corresponding thereto, thereby inhibiting the transcription or translation of the gene, promoting the degradation of the mRNAs, and/or inhibiting the expression of the protein encoded by the gene.

Thus, as a result, an antisense nucleic acid inhibits the C12orf32 protein to function in the cancerous cell. Herein, the phrase "antisense nucleic acids" refers to nucleotides that specifically hybridize to a target sequence and includes not only nucleotides that are entirely complementary to the target sequence but also that include mismatches of one or more nucleotides. For example, the antisense nucleic acids of the present invention include polynucleotides that have a homology of at least 70% or higher, preferably of at least 80% or higher, more preferably of at least 90% or higher, even more preferably of at least 95% or higher over a span of at least 15 continuous nucleotides of the C12orf32 gene or the complementary sequence thereof. Algorithms known in the art can be used to determine such homology.

Antisense nucleic acids of the present invention act on cells producing proteins encoded by the C12orf32 gene by binding to the DNA or mRNA of the gene, inhibiting their transcription or translation, promoting the degradation of the mRNA, and inhibiting the expression of the protein, finally inhibiting the protein to function.
Antisense nucleic acids of the present invention can be made into an external preparation, such as a liniment or a poultice, by admixing it with a suitable base material which is inactive against the nucleic acids.

Also, as needed, the antisense nucleic acids of the present invention can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nose-drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, pain-killers, and such. An antisense-mounting medium can also be used to increase durability and membrane-permeability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives of these. These can be prepared by following known methods.

The antisense nucleic acids of the present invention inhibit the expression of the C12orf32 gene and are useful for suppressing the biological activity of the protein. In addition, expression-inhibitors, including antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of the C12orf32 protein.
The antisense nucleic acids of present invention also include modified oligonucleotides. For example, thioated oligonucleotides may be used to confer nuclease resistance to an oligonucleotide.

IV-4. Pharmaceutical compositions including antibodies
The function of a gene product of the C12orf32 gene which is over-expressed in cancers, in particular breast cancer, can be inhibited by administering a compound that binds to or otherwise inhibits the function of the gene products. An antibody against the C12orf32 polypeptide can be mentioned as such a compound and can be used as the active ingredient of a pharmaceutical composition for treating or preventing cancer.

The present invention relates to the use of antibodies against a protein encoded by the C12orf32 gene, or fragments of the antibodies. As used herein, the term "antibody" refers to an immunoglobulin molecule having a specific structure, that interacts (i.e., binds) only with the antigen that was used for synthesizing the antibody (i.e., the gene product of an up-regulated marker) or with an antigen closely related thereto. Molecules including the antigen that was used for synthesizing the antibody and molecules including the epitope of the antigen recognized by the antibody can be mentioned as closely related antigens thereto.

Furthermore, an antibody used in the present pharmaceutical compositions may be a fragment of an antibody or a modified antibody, so long as it binds to the protein encoded by the C12orf32 gene (e.g., an immunologically active fragment of anti-C12orf32 antibody). For instance, the antibody fragment may be Fab, F(ab')2, Fv, or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston JS et al., Proc Natl Acad Sci USA 1988, 85:5879-83). Such antibody fragments may be generated by treating an antibody with an enzyme, such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell (see, for example, Co MS et al., J Immunol 1994, 152:2968-76; Better M et al., Methods Enzymol 1989, 178:476-96; Pluckthun A et al., Methods Enzymol 1989, 178:497-515; Lamoyi E, Methods Enzymol 1986, 121:652-63; Rousseaux J et al., Methods Enzymol 1986, 121:663-9; Bird RE et al., Trends Biotechnol 1991, 9:132-7).

An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG). The present invention includes such modified antibodies. The modified antibody can be obtained by chemically modifying an antibody. Such modification methods are conventional in the field.

Alternatively, the antibody used for the present invention may be a chimeric antibody having a variable region derived from a non-human antibody against the C12orf32 polypeptide and a constant region derived from a human antibody, or a humanized antibody, including a complementarity determining region (CDR) derived from a non-human antibody, a frame work region (FR) and a constant region derived from a human antibody. Such antibodies can be prepared by using known technologies. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see e.g., Verhoeyen et al., Science 1988, 239:1534-6). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

Complete human antibodies including human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom et al., J Mol Biol 1992, 227:381-8). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

When the obtained antibody is to be administered to the human body (antibody treatment), a human antibody or a humanized antibody is preferable for reducing immunogenicity.
Antibodies obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto. A protein A column and protein G column can be used as the affinity column. Exemplary protein A columns to be used include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). The chromatographic procedures can be carried out by liquid-phase chromatography, such as HPLC and FPLC.

V. Methods for treating or preventing cancer
Cancer therapies directed at specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-tumor pharmaceuticals such as trastuzumab (Herceptin) for the treatment of advanced cancers, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Iressa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F et al., Clin Cancer Res 2001, 7:2958-70, Review; Slamon DJ et al., N Engl J Med 2001, 344:783-92; Rehwald U et al., Blood 2003, 101:420-4; Fang G et al., Blood 2000, 96:2246-53). These drugs are clinically effective and better tolerated than traditional anti-tumor agents because they target only transformed cells. Hence, such drugs not only improve survival and quality of life for cancer patients, but also validate the concept of molecularly targeted cancer therapy. Furthermore, targeted drugs can enhance the efficacy of standard chemotherapy when used in combination with it (Gianni L, Oncology 2002, 63 Suppl 1:47-56; Klejman A et al., Oncogene 2002, 21:5868-76). Therefore, future cancer treatments will probably involve combining conventional drugs with target-specific agents aimed at different characteristics of tumor cells such as angiogenesis and invasiveness.

These modulatory methods can be performed ex vivo or in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). The methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract aberrant expression of the differentially expressed genes or aberrant activity of their gene products.

Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) expression levels or biological activities of genes and gene products, respectively, may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity of the over-expressed gene. Therapeutics that antagonize activity can be administered therapeutically or prophylactically.

Accordingly, therapeutics that may be utilized in the context of the present invention include, e.g., (i) a polypeptide of the over-expressed C12orf32 gene or analogs, derivatives, fragments or homologs thereof; (ii) antibodies against the over-expressed gene or gene products; (iii) nucleic acids encoding the over-expressed gene; (iv) antisense nucleic acids or nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the nucleic acids of over-expressed gene); (v) double-stranded molecules (e.g., siRNA); or (vi) modulators (i.e., inhibitors, antagonists that alter the interaction between an over-expressed polypeptide and its binding partner). The dysfunctional antisense molecules are utilized to "knockout" endogenous function of a polypeptide by homologous recombination (see, e.g., Capecchi, Science 1989, 244: 1288 92).

Increased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
Prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

According to the present method to inhibit cell growth and thereby treat cancer, through the administration of plural kinds of the double-stranded molecules (or vectors expressing or compositions containing the same), each of the molecules may have different structures but act on mRNA that matches the same target sequence of C12orf32. Alternatively plural kinds of the double-stranded molecules may act on mRNA that matches a different target sequence of same gene. Alternatively, for example, the method may utilize double-stranded molecules directed to one, two or more target sequence of C12orf32.

Therapeutic methods of the present invention may include the step of contacting a cell with an agent that modulates one or more of the activities of the C12orf32 gene products. Examples of agent that modulates protein activity include, but are not limited to, nucleic acids, proteins, naturally occurring cognate ligands of such proteins, peptides, peptidomimetics, and other small molecule.
Thus, the present invention provides methods for treating or alleviating a symptom of cancer, or preventing cancer in a subject by decreasing the expression of the C12orf32 gene or the activity of the gene product. The present method is particularly suited for treating or preventing breast cancer.

Suitable therapeutics can be administered prophylactically or therapeutically to a subject suffering from or at risk of (or susceptible to) developing cancers. Such subjects can be identified by using standard clinical methods or by detecting an aberrant expression level ("up-regulation" or "over-expression") of the C12orf32 gene or aberrant activity of the gene product.

According to an aspect of the present invention, a substance screened through the present method may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the substances to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration. If the substance is encodable by a DNA, the DNA can be inserted into a vector for gene therapy and the vector administered to a patient to perform the therapy.

The dosage and methods for administration vary according to the body-weight, age, sex, symptom, condition of the patient to be treated and the administration method; however, one skilled in the art can routinely select suitable dosage and administration method.
For example, although the dose of a substance that binds to a C12orf32 polypeptide and regulates the activity of the polypeptide depends on the aforementioned various factors, the dose is generally about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about 50 mg per day and more preferably about 1.0 mg to about 20 mg per day, when administered orally to a normal adult human (60 kg weight).

When administering the substance parenterally, in the form of an injection to a normal adult human (60 kg weight), although there are some differences according to the patient, target organ, symptoms and methods for administration, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 to about 10 mg per day. In the case of other animals, the appropriate dosage amount may be routinely calculated by converting to 60 kg of body-weight.
Similarly, a pharmaceutical composition of the present invention may be used for treating or preventing cancer. Methods well known to those skilled in the art may be used to administer the compositions to patients, for example, as an intraarterial, intravenous, or percutaneous injection or as an intranasal, transbronchial, intramuscular, or oral administration.

For each of the aforementioned conditions, the compositions, e.g., polypeptides and organic compounds, can be administered orally or via injection at a dose ranging from about 0.1 to about 250 mg/kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g/day, preferably about 5 mg to about 10 g/day, and most preferably about 100 mg to about 3 g/day. Tablets or other unit dosage forms of presentation provided in discrete units may conveniently contain an amount which is effective at such dosage or as a multiple of the same, for instance, units containing about 5 mg to about 500 mg, usually from about 100 mg to about 500 mg.

The dose employed will depend upon a number of factors, including the age, body weight and sex of the subject, the precise disorder being treated, and its severity. Also the route of administration may vary depending upon the condition and its severity. In any event, appropriate and optimum dosages may be routinely calculated by those skilled in the art, taking into consideration the above-mentioned factors.
In particular, an antisense nucleic acid against the C12orf32 gene can be given to the patient by direct application onto the ailing site or by injection into a blood vessel so that it will reach the site of ailment.

The dosage of the antisense nucleic acid derivatives of the present invention can be adjusted suitably according to the patient's condition and used in desired amounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.
In the present invention, the inhibitory nucleic acids can be administered to the subject either as a naked nucleic acid, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector that expresses the inhibitory nucleic acids.
Suitable delivery reagents for administration in conjunction with the present inhibitory nucleic acids include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the inhibitory nucleic acids to a particular tissue, such as retinal or tumor tissue, and can also increase the blood half-life of the inhibitory nucleic acids. Liposomes suitable for use in the context of the present invention may be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.

Preferably, the liposomes encapsulating the present inhibitory nucleic acids include a ligand molecule that can deliver the liposome to the cancer site. Ligands which bind to receptors prevalent in tumor cells, such as monoclonal antibodies that bind to tumor antigens, are preferred.
Particularly preferably, the liposomes encapsulating the present inhibitory nucleic acids are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can include both opsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present inhibitory nucleic acids to tumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degree C.

Vectors expressing inhibitory nucleic acids of the present invention are discussed below. Vectors expressing at least one inhibitory nucleic acids of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express inhibitory nucleic acids of the invention, to an area of cancer in a patient are within the skill of the art.
The inhibitory nucleic acids of the invention can be administered to the subject by any means suitable for delivering the inhibitory nucleic acids into cancer sites. For example, the inhibitory nucleic acids can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.

Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravesical and intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the inhibitory nucleic acids or vector be given at or near the site of the cancer.

The inhibitory nucleic acids of the invention can be administered in a single dose or in multiple doses. When the administration of the inhibitory nucleic acids of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent directly into the tissue at or near the site of cancer is preferred. Multiple injections of the agent into the tissue at or near the site of cancer are particularly preferred.

One skilled in the art can also readily determine an appropriate dosage regimen for administering the inhibitory nucleic acids of the invention to a given subject. For example, the inhibitory nucleic acids can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the inhibitory nucleic acids can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the inhibitory nucleic acids are injected at or near the site of cancer once a day for seven days. Where a dosage regimen includes multiple administrations, it is understood that the effective amount of an inhibitory nucleic acids administered to the subject can include the total amount of an inhibitory nucleic acids administered over the entire dosage regimen.

In the present invention, a cancer overexpressing C12orf32 can be treated with at least one active ingredient selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.

The cancer includes, but is not limited to, breast cancer. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of C12orf32 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing C12orf32, which method may include the steps of:
i) determining the expression level of C12orf32 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of C12orf32 with normal control; and
iii) administrating at least one component selected from the group consisting of
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
to a subject with a cancer overexpressing C12orf32 compared with normal control. Alternatively, the present invention also provides a pharmaceutical composition comprising at least one component selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
for use in administrating to a subject having a cancer overexpressing C12orf32. In other words, the present invention further provides a method for identifying a subject to be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
which method may include the step of determining an expression level of C12orf32 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with.

The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.

According to the present invention, the expression level of C12orf32 in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, hybridization methods (e.g., Northern hybridization), a chip or an array, probes, RT-PCR can be used to determine the transcription product level of C12orf32.
Alternatively, the translation product may be detected for the treatment of the present invention. For example, the quantity of observed protein (SEQ ID NO: 2) may be determined.

As another method to detect the expression level of C12orf32 gene based on its translation product, the intensity of staining may be measured via immunohistochemical analysis using an antibody against the C12orf32 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of C12orf32 gene.
Methods for detecting or measuring the C12orf32 polypeptide and/or polynucleotide encoding thereof can be exemplified as described above (I. Daignosing cancer).

VI. Double-stranded molecules and vectors encoding them
Herein, an siRNA including either of the sequences of SEQ ID NOs: 8, 9 or 14 was demonstrated to suppress cell growth or viability of cells expressing the C12orf32 gene. Therefore, double-stranded molecules including any of these sequences and vectors expressing the molecules are considered to serve as preferable pharmaceutics for treating or preventing diseases which involve the proliferation of C12orf32 gene expressing cells, for example, breast cancer. Thus, according to an aspect, the present invention provides double-stranded molecules including the target sequence selected from the group consisting of SEQ ID NOs: 8, 9 and 14 and vectors expressing the molecules. More specifically, the present invention provides a double-stranded molecule, when introduced into a cell expressing the C12orf32 gene, inhibiting expression of the gene, wherein the molecule includes a sense strand and an antisense strand, wherein the sense strand includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 9 and 14 as a target sequence, and the antisense strand includes a nucleotide sequence complementary to the target sequence of the sense strand so that the sense and antisense strands hybridize to each other to form the double-stranded molecule. In preferable embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.

The target sequence for the C12orf32 gene included in the sense strand may consist of a sequence of a portion of SEQ ID NO: 1 that is less than about 500, 400, 300, 200, 100, 75, 50 or 25 contiguous nucleotides. For example, the target sequence may be from about 19 to about 25 contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1. The present invention is not limited thereto, but suitable target sequences include the nucleotide sequences selected from the group consisting of SEQ ID NOs: 8, 9 and 14.

The double-stranded molecule of the present invention may be composed of two polynucleotide constructs, i.e., a polynucleotide including the sense strand and a polynucleotide including the antisense strand. Alternatively, the molecule may be composed of one polynucleotide construct; i.e., a polynucleotide including both the sense strand and the antisense strand, wherein the sense and antisense strands are linked via a single-stranded polynucleotide which enables hybridization of the target sequences within the sense and antisense strands by forming a hairpin structure. Herein, the single-stranded polynucleotide may also be referred to as "loop sequence" or "single-strand". The single-stranded polynucleotide linking the sense and antisense strands may consist of 3 to 23 nucleotides. See under the item of "IV-2. Pharmaceutical compositions including double-stranded molecules" for more details on the double-stranded molecule of the present invention.

The double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which may be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C-methyl nucleotides, and inverted deoxybasic residue incorporation (US20060122137).

In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2'-deoxyribonucleotides (WO2004/029212). In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'-terminal nucleotide overhanging nucleotides may be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. The present invention is not limited to these examples and any known chemical modifications may be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.

Furthermore, the double-stranded molecules of the invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule including both DNA and RNA on any or both of the single strands (polynucleotides), or the like may be formed for enhancing stability of the double-stranded molecule. The hybrid of a DNA strand and an RNA strand may be the hybrid in which either the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may be either the molecule in which both of the sense and antisense strands are composed of DNA and RNA, or the molecule in which any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.

In order to enhance stability of the double-stranded molecule, the molecule preferably contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression. As a preferred example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA. Preferably, the upstream partial region indicates the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in preferable embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention include following combinations.
sense strand:
5'-[---DNA---]-3'
3'-(RNA)-[DNA]-5'
:antisense strand,
sense strand:
5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5'
:antisense strand, and
sense strand:
5'-(RNA)-[DNA]-3'
3'-(---RNA---)-5'
:antisense strand.

The upstream partial region preferably is a domain consisting of 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, preferred examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US20050004064).

In the context of the present invention, the double-stranded molecule may form a hairpin, such as a short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA includes the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.

Alternatively, the present invention provides vectors including each of a combination of polynucleotide having a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid includes nucleotide sequence of SEQ ID NOs: 8, 9 or 14, and the antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of the sense strand and the antisense strand hybridize to each other to form a double-stranded molecule, and wherein the vectors, when introduced into a cell expressing the C12orf32, inhibits expression of the gene. Preferably, the sense strand of the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 1). More preferably, the combination of polynucleotide includes a single nucleotide transcript having the sense strand and the antisense strand linked via a single-stranded nucleotide sequence. More preferably, the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a nucleotide sequence including SEQ ID NO: 8, 9 or 14; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].

Vectors of the present invention can be produced, for example, by cloning C12orf32 sequence into an expression vector so that regulatory sequences are operatively-linked to C12orf32 sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and antisense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.

The vectors of the present invention may also be equipped to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Patent No. 5,922,687).

The vectors of the present invention include, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox (see, e.g., US Patent No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.

Hereinafter, the present invention is described in more detail with reference to the Examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials and Methods
1. Breast cancer cell lines.
Human breast cancer cell lines, BT-549, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SK-BR-3, T47D, YMB-1, ZR-75-1 and BSY-1 were purchased from American Type Culture Collection (ATCC, Rockville, MD), and cultured under their respective depositors' recommendations. HBC4 and HBC5 were kind gifts from Dr. Takao Yamori of Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research. HBC4, HBC5, BT-549, HCC1937, T47D, YBM-1, ZR-75-1 and BSY-1 cell lines were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) (with 2mM L-glutamine). MDA-MB-231 and MDA-MB-435S cell lines were cultured in L-15 (Roche, Basel, Switzerland). SK-BR-3 cell line was cultured in McCoy (Sigma-Aldrich) (with 1.5mM L-glutamine). MCF-7 cell line was cultured in EMEM (Sigma-Aldrich) (with 10 microgram/ml Insulin). Each medium was supplemented with 10% fetal bovine serum (FBS; Cansera International, Ontario, Canada) and 1% antibiotic/antimycotic solution (Sigma-Aldrich). MDA-MB-231 and MDA-MB-435S cell lines were maintained at 37 degrees C in atmosphere of humidified air without CO2, and other cell lines were maintained at 37 degrees C in atmosphere of humidified air with 5% CO2.

2.Semi-quantitative RT-PCR.
Microdissection of breast cancer cells was carried out as described previously (Nishidate et al, Int J oncol, 2004). Total RNA was extracted from each of the microdissected breast cancer clinical samples, microdissected normal breast ductal cells, and breast cancer cell lines using RNeasy Micro Kits (Qiagen, Valencia, CA, USA) and purchased polyA (+) RNAs isolated from mammary gland, heart, lung, liver, kidney and bone marrow from Takara Clontech (Kyoto, Japan). Subsequently, T7-based amplification and RT were carried out as described previously (Nishidate et al, Int J oncol, 2004). Appropriated dilutions of each single-stranded cDNA were prepared for subsequent PCR by monitoring beta-actin as a quantitative control. The sequences of each primer set were as follows; 5'-TTTTAGAGAATCCTGCTTCCATCAG-3' (SEQ ID NO: 3) and 5'-TTTGACTGGGGAAGTCCTTCTG-3' (SEQ ID NO: 4) for C12orf32 (GenBank accession number NM_031465, SEQ ID NO: 1), and 5'-GAACGGTGAAGGTGACAGCA-3' (SEQ ID NO: 5) and 5'-ACCTCCCCTGTGTGGACTTG-3' (SEQ ID NO: 6) for beta-actin.

3.Northern blotting analysis.
Breast cancer northern blot membrane was prepared as described previously (Park et al, Cancer Res, 2006). Human multiple-tissue northern blots (Takara Clontech, Kyoto, Japan) were hybridized with the [alpha32P]-dCTP-labeled PCR products of C12orf32 prepared by RT-PCR (see below). Prehybridization, hybridization, and washing were carried out according to the supplier's recommendations. The blots were autoradiographed with intensifying screens at -80 degrees C for 14 days. Specific probe for C12orf32 (343bp) was prepared by RT-PCR using the following primer set; 5'- TTTTAGAGAATCCTGCTTCCATCAG-3' (SEQ ID NO: 3) and 5'-CAATCCTAAAGAACTCATCTATGTC-3' (SEQ ID NO: 7). It was radioactively labeled with the megaprime DNA labeling system (GE Healthcare, Buckinghamshire, UK).

4. Small interfering RNA (siRNA)-expressing vectors specific to C12orf32.
A vector-based RNAi (RNA interference) expression system was established using psiU6BX3.0 siRNA expression vector as described previously (Shimokawa et al., Cancer Res, 2003). The siRNA expression vectors against C12orf32 (psiU6BX3.0-C12orf32) were prepared by cloning of double-stranded oligonucleotides into the BbsI site in the psiU6BX3.0 vector. The target sequences of synthetic oligonucleotides for siRNAs were as follows; 5'-AAGCTGACTGCCATCAGTAAT-3' (SEQ ID NO: 8) for si-#2, 5'-AACAGTTCAGTTTAGTGTCAT-3' (SEQ ID NO: 9) for si-#3, 5'-AACCTGACTGCGATCTGTAAA-3' (SEQ ID NO: 10) for si-mis (underlined letters indicate mismatched sequence in si-mis). All of the constructs were confirmed by DNA sequencing (ABI3700; PE Applied Biosystems). Human breast cancer cell lines, HBC4 and T47D, were plated onto 10 cm dishes (1 x 106 cells/dish) and transfected with 8 microgram each of psiU6BX3.0-Mock (without insertion) and psiU6BX3.0-C12orf32 (si-#2, si-#3 and si-mis including four-base substitutions in #2) using FuGENE6 transfection reagent (Roche) according to the manufacturer's instructions. Twenty-four hours after transfection, cells were re-seeded for colony formation assay (1 x 106 cells/10 cm dish), RT-PCR (1 x 106 cells/10 cm dish) and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (2 x 105 cells/well). The psiU6BX3.0-introduced HBC4 or T47D cells were selected with culture medium containing 0.4 mg/ml or 0.8 mg/ml of neomycin (Geneticin; Invitrogen), respectively. The culture medium was changed twice a week. Total RNAs were extracted from the cells after 5-day incubation with neomycin, and then the knockdown effect of siRNAs was examined by semi-quantitative RT-PCR using specific primer sets; 5'-CTCATTCACCGGTTGATGCC-3' (SEQ ID NO: 11) and 5'-GCTTTTCACAAGGAATTGGCT-3' (SEQ ID NO: 12) for C12orf32; 5'-GGAACGGTGAAGGTGACAGC-3' (SEQ ID NO: 13) and 5'-ACCTCCCCTGTGTGGACTTG-3' (SEQ ID NO: 6) for beta-actin as an internal control. HBC4 or T47D cells expressing siRNA were grown for 4 weeks in selective media containing 0.4 mg/ml or 0.8 mg/ml of neomycin, and then fixed with 4% paraformaldehyde at 4 degrees C for 30 minutes before staining with Giemsa's solution (Merck, Whitehouse Station, NJ) to assess the colony number. To quantify cell viability, MTT assays were performed with cell counting kit-8 (Wako, Osaka, Japan) according to manufacturer's recommendations. Absorbance at 570 nm wavelengths was measured with a Microplate Reader 550 (Bio-Rad). These experiments were performed in triplicate.

The siRNA oligonucleotides (Sigma Aldrich Japan KK, Tokyo, Japan) were used due to its high transfection efficiency to observe the knockdown-effect of C12orf32. The sequences targeting C12orf32 (si-C12orf32) or EGFP (siEGFP) were as follows: si-C12orf32; 5'-GCUGACUGCCAUCAGUAAU-3' (SEQ ID NO: 14), siEGFP (control); 5'-GCAGCACGACUUCUUCAAG-3' (SEQ ID NO: 15). The sense strand of the above oligonucleotides may be added with several nucleotide sequence such as TT. T47D cells (1X106 cells in 10cm dish for FACS analysis) cells were transfected with those siRNAs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) in OptiMEM (Invitrogen) medium according to the instructions of manufacture.

5. Fluorescence-activated cell sorting (FACS) analysis.
T47D breast cancer cells, which were used for siRNA experiments as indicated above, were harvested at 48 hours after transfection with siRNA-oligonucleotides. Cells were collected and fixed with 70% ethanol, and maintained at 4 degrees C before use. Cells were incubated with 10 mg/ml RNase I in PBS (-) at 37 degrees C for 30 minutes and stained with 50 microgram of propidium iodide (PI) at room temperature for 30 minutes. Cell suspensions were analyzed for DNA content by flow cytometer (FACS calibur; Becton Dickinson, San Diego, CA). The data was analyzed by CELLQuest software (BD Biosciences, Sparks, Md.). Assays were done in duplicate independently.

6. Construction of C12orf32 expression vectors.
To construct C12orf32 expression vector, the entire coding sequence was amplified by PCR using KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan). Primer sets were 5'-CCGGAATTCCTCATTCACCGGTTGATGCC-3' (SEQ ID NO: 16) and 5'-CCGCTCGAGGCTTTTCACAAGGAATTGGCT-3' (SEQ ID NO: 17) (underlines indicate recognition sites of restriction enzymes). The PCR product was inserted into the EcoRI and XhoI sites of pCAGGSnHC expression vector in frame with a hemagglutinin (HA) tag at the C-terminus. DNA sequences of the construct were confirmed by DNA sequencing (ABI3700; PE Applied Biosystems).

7. Generation of anti-C12orf32 specific polyclonal antibody.
A plasmid designed to express a fragment of C12orf32 (codons 1-208) using pET21a (+) vector in frame with a T7 tag at the N-terminus and a histidine (His) tag at the C-terminus (Novagen, Madison, WI). The recombinant peptide was expressed in Escherichia coli, BL21 codon-plus strain (Stratagene, La Jolla, CA), and purified using Ni-NTA resin agarose (QIAGEN) according to the supplier's protocols. The purified recombinant protein was mixed together and then used for immunization of rabbits (Medical and Biological Laboratories, Nagoya, Japan). The immune sera were subsequently purified on antigen affinity columns using Affigel 15 gel (Bio-Rad Laboratories, Hercules, CA) according to supplier's instructions. It was confirmed that this antibody could specifically recognize endogenous C12orf32 protein in breast cancer cell line, T47D and HBC4 using siRNA-oligonucleotides of C12orf32.

8. Western blot analysis.
To examine the expression of endogenous C12orf32 protein in breast cancer cell lines (HBC4, MDA-MB-231, BT-549, T47D, SK-BR-3, ZR-75-1, BSY-1 and MCF-7), cells were lysed with lysis buffer (50mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40) including 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, CA). After homogenization, cell lysates were incubated on ice for 30 minutes and centrifuged at 14,000 rpm for 5 minutes to separate only supernatant from cell debris. The amount of total protein was measured by protein assay kit (Bio-Rad), and then the proteins were mixed with SDS-sample buffer and boiled for 5 minutes before loading at 12% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto nitrocellulose membrane (GE Healthcare). The membranes were blocked by blocking solution for over-night, and incubated with purified anti-C12orf32 polyclonal antibody for another 1 hour to detect endogenous C12orf32 protein. Finally, the membrane was incubated with HRP conjugated secondary antibody for one hour and protein bands were visualized by ECL detection reagents (GE Healthcare).

9. Immunocytochemical staining.
To examine the subcellular localization of endogenous C12orf32 protein in breast cancer cells, T47D cells were seeded at 1x105 cells per well (Lab-Tek II Chamber Slide System; Nalgen Nunc International, Naperville, IL). After 24 hours of incubation, cells were fixed with PBS (-) containing 4% paraformaldehyde at 4 degrees C for 30 minutes and rendered permeable with PBS (-) containing 0.1% Triton X-100 at 4 degrees C for two minutes. Subsequently, the cells were covered with 3% BSA in PBS (-) for 1 hour to block non-specific hybridization followed by incubation with anti-C12orf32 polyclonal antibody diluted at 1:100 for another 1 hour. After washing with PBS (-), cells were stained by Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probe, Eugene, OR) diluted at 1:1,000 for 1 hour. Nuclei were counter-stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescent images were obtained under TCS SP2 AOBS microscope (Leica, Tokyo, Japan).

Results
1. Over-expression of C12orf32 in breast cancer cells.
To elucidate the mechanism of breast carcinogenesis and identify molecules that could be applicable as targets for development of novel therapeutic drugs, genome-wide gene-expression profile analysis of 81 breast cancers was carried out with using cDNA microarray representing 27,648 cDNAs. Among the transactivated genes, chromosome 12 open reading frame 32 (C12orf32) was focused on herein. Its up-regulation was confirmed in 5 of 11 breast cancer specimens compared with normal breast ductal cells and whole mammary gland by semi-quantitative RT-PCR (Fig. 1A). Subsequent northern-blot analysis using a C12orf32 cDNA fragment as a probe confirmed over-expression of an approximately 1.8-kb and 1.5-kb transcripts of C12orf32 in breast cancer cell lines (Fig. 1B). On the other hand, C12orf32 expression was hardly detectable in any of normal human organs except the testis, prostate, ovary, thymus and small intestine as concordant to the results of cDNA microarray analysis (Fig. 1C). Two bands were observed from the results of northern analyses (Fig. 1B). Expected size of C12orf32 mRNA transcript from the NCBI database was 1.8-kb. To investigate whether other transcriptional variants of C12orf32 (approximately 1.5-kb) are existed, the region corresponding to the open reading frame (ORF) region of C12orf32 was amplified by RT-PCR analysis (Fig. 1D). As shown in Fig. 1D, only single band of expected size was observed in the ORF region of C12orf32, suggesting that C12orf32 might have at least two transcripts that share the same open reading frame encoding a 233 amino-acids protein.

2. Cleaved forms of C12orf32 protein in human breast cancer cells.
The C12orf32 expression vector was constructed using the pCAGGSHC to observe the expression of full-length C12orf32 protein (see Material and Methods). It was detected at approximately 34-kDa with another three small-size bands (approximately 27-kDa, 23-kDa and 16-kDa) by western analysis using the lysate of COS-7 cells transfected with pCAGGSHC-C12orf32 (Fig. 2A). Unexpectedly, 16-kDa and 23-kDa bands of exogenously-expressed C12orf32 protein were detected in most of breast cancer cell lines examined, but the predicted size of full-length of C12orf32 protein (approximately 34-kDa) could not be detected. To detect the endogenous C12orf32 protein in breast cancer cells, the polyclonal anti-C12orf32 antibody was generated for western blot analysis using breast cancer cell lines. As a result, a 16-kDa of endogenous C12orf32 was observed (Figs. 2A and 2B) rather than 23-kDa protein. Since any other alternative splicing forms of C12orf32 transcript were found as shown in Fig. 1D, it was speculated that those smaller size proteins might be degradated. To clarify this issue, siRNA experiments using breast cancer cell lines were carried out. The expression level of these two proteins which were detected with anti-C12orf32 antibody were decreased by siRNA-oligonucleotides of C12orf32, suggesting that these two bands are corresponds to endogenous C12orf32 proteins (Figs. 2A and 4E). Furthermore, western analysis using the lysate of COS-7 cells transfected with pCAGGSHC-C12orf32 with anti-C12orf32 antibody or anti-HA antibody showed the different results (Fig. 2B). The C12orf32 protein of predicted full-length (34-kDa) was shown in the both results, but the bands of different small-size were observed, suggesting that C12orf32 protein might have cleaved form.

3. Cell cycle-dependent expression of C12orf32.
FACS and western blot analyses using T47D cells were carried out after synchronization of the cell cycle by aphidicolin treatment. When a large proportion of the cells were considered to be in G2/M phase (at 9-hour after release from cell-cycle arrest) (Figs. 3A-3C), western blot and RT-PCR analyses resulted in detection of the increase of C12orf32 expression, suggesting an important role for C12orf32 in mitosis. Immunocytochemical staining using anti-C12orf32 antibody revealed that C12orf32 protein was localized at the nucleus of T47D cells in interphase (Fig. 3D). From prophase to anaphase, C12orf32 was diffusely localized in the cells. Finally, this protein was concentrated at the midbody of the cells in telophase.

4. Knockdown-effect of C12orf32 using siRNAs on growth of breast cancer cell lines.
To assess a growth-promoting role of C12orf32 in breast cancer cells, the expression of endogenous C12orf32 was knocked down in breast cancer cell lines, HBC4 and T47D, which expressed a high-level of C12orf32, by means of the mammalian vector-based RNA interference (RNAi) technique (see Materials and Methods). The expression level of C12orf32 was examined by semiquantitative RT-PCR analysis. Two siRNAs (si-#2 and si-#3) significantly suppressed the C12orf32 expression, compared with a control siRNA construct, psiU6BX-Mock (si-control) (Figs. 4A and 4B; upper panels). In concordance with the knockdown effect, MTT (Figs. 4A and 4B; middle panels) and colony formation assays (Figs. 4A and 4B; lower panels) revealed significant growth-suppressive effects by si-#2 and si-#3 (MTT assays: HBC4; *, **P<0.0001, T47D; *, **P<0.0001; unpaired t test). The siRNA that contained 4-base replacement in si-#2 sequence (si-C12orf32-mismatch (si-mis), see Materials and Methods) was also generated, and no suppressive effect was found on the expression of C12orf32 or on cell growth of T47D cells (Fig. 4C). These observations suggest that C12orf32 has an important function in the growth of the breast cancer cells. Furthermore, since the depletion of C12orf32 resulted in the significant decrease of the number of colonies and in the cell viability, fluorescence-activated cell sorting (FACS) analysis using siRNA-oligonucleotides was carried out to measure the proportions of apoptotic cell population. The results showed significantly higher percentage in the population of apoptotic cells (Sub-G1) by siRNA-oligonucleotides of C12orf32 (si-C12orf32) compared with si-EGFP (*P=0.0245, **P=0.009; unpaired t test) (Fig. 4D), suggesting that inhibition of C12orf32 expression might induce apoptosis. Knockdown of C12orf32 protein was validated using anti-C12orf32 antibody by western analysis (Fig. 4E).

The gene-expression analysis of cancers described herein using the genome-wide cDNA microarray has identified a specific gene as a target for cancer prevention and therapy. Based on the expression of a differentially expressed gene, C12orf32, the present invention provides molecular diagnostic markers for identifying and detecting cancer, in particular, breast cancer.

The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provide indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.

Claims

A method for diagnosing cancer or a predisposition for developing cancer in a subject, comprising a step of determining an expression level of a C12orf32 gene in a subject-derived biological sample, wherein an increase in the expression level as compared to a normal control level of the gene indicates that the subject suffers from or is at a risk of developing cancer, wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting mRNA of a C12orf32 gene;
(b) detecting a protein encoded by a C12orf32 gene; and
(c) detecting a biological activity of a protein encoded by a C12orf32 gene.
The method of claim 1, wherein the expression level is at least 10% greater than the normal control level.
The method of claim 1, wherein the cancer is breast cancer.
A kit for detecting cancer comprising a detection reagent which binds to a transcription or translation product of a C12orf32 gene.
A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting binding between the polypeptide or fragment and the test substance; and
(c) selecting the test substance that binds to the polypeptide or fragment as a candidate substance for treating or preventing cancer.
A method of screening a candidate substance for treating or preventing cancer, wherein the method comprises steps of:
(a) contacting a test substance with a C12orf32 polypeptide or a fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment;
(c) comparing the biological activity of the polypeptide or fragment with the biological activity detected in the absence of the test substance; and
(d) selecting the test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.
The method of claim 6, wherein the biological activity is cell proliferative activity.
A method of screening a candidate substance for treating or preventing cancer, which comprises steps of:
(a) contacting a test substance with a cell expressing a C12orf32 gene;
(b) detecting expression level of the C12orf32 gene;
(c) comparing the expression level with the expression level detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level as a candidate substance for treating or preventing cancer.
A method of screening a candidate substance for treating or preventing cancer, wherein the method comprises steps of:
(a) contacting a test substance with a cell introduced with a vector that comprises a transcriptional regulatory region of a C12orf32 gene and a reporter gene expressed under control of the transcriptional regulatory region;
(b) measuring expression level or activity of the reporter gene;
(c) comparing the expression level or activity with the expression level or activity detected in the absence of the test substance; and
(d) selecting the test substance that reduces the expression level or activity as a candidate substance for treating or preventing cancer.
A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence consisting of SEQ ID NO: 8, 9 or 14, and wherein the antisense strand comprises a nucleotide sequence which is complementary to the target sequence, wherein the sense molecule, and wherein the double-stranded molecule, when introduced into a cell expressing the C12orf32 gene, inhibits expression of the gene.
The double-stranded molecule of claim 10, wherein the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
The double-stranded molecule of claim 10 or 11, wherein the double-stranded molecule is a single polynucleotide construct comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
The double-stranded molecule of claim 12, which has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 8, 9 and 14, [B] is a single-strand and consists of 3 to 23 nucleotides, and [A'] is an antisense strand comprising a nucleotide sequence complementary to the target sequence.
A vector encoding the double-stranded molecule of any one of claims 10 to 13.
Vectors comprising each of a combination of polynucleotides comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises a nucleotide sequence corresponding to SEQ ID NO: 8, 9 or 14 and the antisense strand nucleic acid comprises a sequence complementary to the sense strand, wherein the transcripts of the sense strand and the antisense strand hybridize to each other to form a double stranded molecule, and wherein the vectors, when introduced into a cell expressing C12orf32 gene, inhibit the cell proliferation.
A method of treating or preventing cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a double-stranded molecule against a C12orf32 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing C12orf32 gene, inhibits the expression of the C12orf32 gene.
The method of claim 16, wherein the double-stranded molecule is that of any one of claims 10 to 13, wherein the vector is that of claim 14 or 15.
The method of claim 16 or 17, wherein the cancer is breast cancer.
A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against a C12orf32 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing C12orf32 gene, inhibits the expression of the C12orf32 gene, and a pharmaceutically acceptable carrier.
The composition of claim 19, wherein the double-stranded molecule is that of any one of claims 10 to 13, wherein the vector is that of claim 14 or 15.
The composition of claim 19 or 20, wherein the cancer is breast cancer.
A fragment of C12orf32 protein obtained by following steps of:
(a) transfecting a vector expressing the C12orf32 protein with breast cancer cell line or COS-7 cells, and
(b) recovering the translation products from the cells, wherein the molecular weight of the translation products determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is selected from the group consisting of about 27-kDa, 23-kDa and 16-kDa.