WO2011161960A1

WO2011161960A1 - C1orf59 for target genes of cancer therapy and diagnosis

Info

Publication number: WO2011161960A1
Application number: PCT/JP2011/003564
Authority: WO
Inventors: Yusuke Nakamura; Yataro Daigo; Takuya Tsunoda
Original assignee: Oncotherapy Science, Inc.
Priority date: 2010-06-23
Filing date: 2011-06-22
Publication date: 2011-12-29

Abstract

Objective methods for diagnosing a predisposition to developing cancer are described herein. In one embodiment, the diagnostic method involves determining an expression level of the C1orf59 gene, the PIWIL4 gene, or piRNA and correlating the determined level with a healthy, diseased or at-risk state. The present invention further provides methods of assessing or determining the prognosis of a patient with esophageal cancer. In one embodiment, the diagnostic method involves determining an expression level of the C1orf59 gene. The present invention further provides methods of screening for therapeutic substances useful in the treatment of a C1orf59-, and/or PIWIL4-associated disease, such as a cancer. The present invention further provides methods of treating or preventing a cancer expressing the C1orf59 gene and/or the PIWIL4 gene. The present invention also features products, including double-stranded molecules and vectors encoding thereof as well as to compositions including them.

Description

C1ORF59 FOR TARGET GENES OF CANCER THERAPY AND DIAGNOSIS

The present invention relates to the field of biological science, more specifically to the field of cancer research, cancer diagnosis and cancer therapy. In particular, the present invention relates to methods for detecting and diagnosing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer as well as methods for treating and preventing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Moreover, the present invention relates to methods of screening for a substance for treating and/or preventing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.
Priority
The present application claims the benefit of U.S. Provisional Application No. 61/357,732, filed on June 23, 2010, the entire contents of which are incorporated by reference herein.

Esophageal squamous-cell carcinoma (ESCC) is one of the most lethal malignancies of the digestive tract, and most diagnoses occur at advanced stages (NPL 1). Despite the use of current surgical techniques combined with various treatment modalities, such as radiotherapy and chemotherapy, the overall 5-year survival rate of ESCC still remains at 40% to 60% (NPL 2). To isolate potential molecular targets for diagnosis, treatment, and/or prevention of esophageal carcinomas, genome-wide analysis of gene expression profiles of cancer cells from 19 ESCC patients was performed using a cDNA microarray consisting of 27,648 genes (NPL 3-9). To verify the biological and clinicopathological significance of the respective gene products, high-throughput screening of loss-of-function effects were performed using the RNAi technique and clinical esophageal-cancer materials were analyzed using tumor-tissue microarray techniques (NPL 10-29). This systematic approach revealed that chromosome 1 open reading frame 59 (C1orf59) was overexpressed in the majority of primary esophageal cancers.
C1orf59 has been postulated to be a homolog of methyltransferase for small RNA, HEN1. HEN1 is a well-studied methyltransferase protein for miRNA in plants (NPL 30). Methylation of miRNA may contribute to the stability of miRNA (NPL 31). Some homologs of HEN1 have been reported to act as methyltransferases for Piwi interacting RNA (piRNA) in mouse, Drosophila and so on (NPL 32-35).

[NPL 1] Shimada H et al. Surgery 2003;133:486-94.
[NPL 2] Tamoto E et al. Clin Cancer Res 2004;10:3629-38.
[NPL 3] Daigo Y and Nakamura Y. Gen Thorac Cardiovasc Surg 2008;56:43-53.
[NPL 4] Kikuchi T et al. Oncogene 2003;22:2192-205.
[NPL 5] Kakiuchi S et al. Mol Cancer Res 2003;1:485-99.
[NPL 6] Kakiuchi S et al. Hum Mol Genet 2004;13:3029-43.
[NPL 7] Kikuchi T et al. Int J Oncol 2006; 28:799-805.
[NPL 8] Taniwaki M et al. Int J Oncol 2006;29:567-75.
[NPL 9] Yamabuki T et al. Int J Oncol 2006;28:1375-84.
[NPL 10] Suzuki C et al. Cancer Res 2003;63:7038-41.
[NPL 11] Ishikawa N et al. Clin Cancer Res 2004;10:8363-70.
[NPL 12] Kato T et al. Cancer Res 2005;65:5638-46.
[NPL 13] Furukawa C et al. Cancer Res 2005;65:7102-10.
[NPL 14] Ishikawa N et al. Cancer Res 2005; 65:9176-84.
[NPL 15] Suzuki C et al. Cancer Res 2005;65:11314-25.
[NPL 16] Ishikawa N et al. Cancer Sci 2006;97:737-45.
[NPL 17] Takahashi K et al. Cancer Res 2006;66:9408-19.
[NPL 18] Hayama S et al. Cancer Res 2006;66:10339-48.
[NPL 19] Kato T et al. Clin Cancer Res 2007;13:434-42.
[NPL 20] Suzuki C et al. Mol Cancer Ther 2007;6:542-51.
[NPL 21] Yamabuki T et al. Cancer Res 2007;67:2517-25.
[NPL 22] Hayama S et al. Cancer Res 2007;67:4113-22.
[NPL 23] Kato T et al. Cancer Res 2007;67:8544-53.
[NPL 24] Taniwaki M et al. Clin Cancer Res 2007;13:6624-31.
[NPL 25] Ishikawa N et al. Cancer Res 2007;67:11601-11.
[NPL 26] Mano Y et al. Cancer Sci 2007;98:1902-13.
[NPL 27] Suda T et al. Cancer Sci 2007;98:1803-8.
[NPL 28] Kato T et al. Clin Cancer Res 2008;14:2363-70.
[NPL 29] Mizukami Y et al. Cancer Sci 2008;99:1448-54.
[NPL 30] Park W et al. Curr Biol 2002; 12:1484-1495
[NPL 31] Ramachandran V and Chen X. Science 2008; 321:1490-1492
[NPL 32] Horwich MD et al. Curr Biol 2007; 17:1265-1272
[NPL 33] Kirino Y and Mourelatos Z. Nucleic Acids Symp Ser (Oxf) 2007; 51:417-418
[NPL 34] Kirino Y and Mourelatos Z. RNA 2007; 13:1397-1401
[NPL 35] Saito K et al. Genes Dev 2007; 21:1603-1608

In the course of the present invention, the methyltransferase activity of C1orf59 for piRNA was discovered, as was the existence of piRNA in cancer cells. The present invention is based, at least in part, on the presumption that oncogenic activity of C1orf59 is brought out through the stabilization of piRNAs, and that this interaction plays an important role in cancer cells. Further analysis revealed that only piwi-like 4 (PIWIL4) is highly expressed in esophageal cancer cells among PIWIL family genes, and further that C1orf59 and PIWIL4 have significant interaction. PIWI in Drosophila melanogaster and PIWIL4 in human have been reported to be associated with methylation on of H3K9 (Yin H and Lin H. Nature 2007; 450:304-308, Sugimoto K et al. Biochem Biophys Res Commun 2007; 359:497-502). The present invention confirms that knock down of C1orf59 results in the reduction of trimethylation of H3K9.

Central to the present invention is the discovery that the overexpression of C1orf59 contributes to the malignant nature of cancer cells. Thus, targeting the C1orf59 molecule may hold promise for the development of a new diagnostic and therapeutic strategy in the clinical management of lung and esophageal cancers. Accordingly, the present invention focuses on C1orf59 as a candidate for the target of cancer/tumor immunotherapy, more particularly the discovery that double-stranded molecules composed of specific sequences (in particular, SEQ ID NOs: 5, 6, 7 and 8) are effective for inhibiting cellular growth of cancer, such as esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Accordingly, it an object of the present invention to provide small interfering RNAs (siRNAs) that target the C1orf59 or PIWIL4 gene. These double-stranded molecules may be utilized in an isolated state or encoded in vectors and expressed from the vectors. Thus, the present invention encompasses such double stranded molecules as well as vectors and host cells expressing them.
It is a further object of the present invention to provide methods for inhibiting cell growth and treating esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer by administering the double-stranded molecules or vectors of the present invention to a subject in need thereof. Such methods encompass administering to a subject in need thereof a composition composed of one or more of the double-stranded molecules or vectors of the present invention.
It is another object of the present invention to provide pharmaceutical compositions formulated for the treatment and/or prophylaxis of cancer and/or a post-operative recurrence thereof, such compositions containing at least one of the double-stranded molecules or vectors of the present invention.

It is yet another object of the present invention to provide a method of diagnosing the presence of or determining a predisposition for the development of cancer, more particularly esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer in a subject by determining an expression level of C1orf59, PIWIL4 and/or piRNA in a patient derived biological sample. An increase in the expression level of C1orf59, PIWIL4 and/or piRNA as compared to a normal control level indicates that the subject suffers from or is at risk of developing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.

The present invention also relates to the discovery that a high expression level of C1orf59 correlates to poor survival rate. Therefore, it is an object of the present invention to provide a method for assessing or determining the prognosis of a patient with esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer, such a method including the steps of detecting the expression level of the C1orf59 gene, comparing it to a pre-determined reference expression level and determining the prognosis of the patient from the difference there between.

It is yet a further object of the present invention to provide a method of screening for a substance for treating and/or preventing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Suitable substances will bind with the C1orf59 polypeptide or PIWIL4 polypeptide or reduce the biological activity of the C1orf59 polypeptide or PIWIL4 polypeptide or reduce the expression of the C1orf59 gene and/or the PIWIL4 gene or a reporter gene surrogating the C1orf59 gene or the PIWIL4 gene or inhibit the binding between the C1orf59 polypeptide and the PIWIL4 polypeptide or inhibit the methyltransferase activity of the C1orf59 polypeptide.

More specifically, the present invention provides the following [1] to [36]:
[1] A method for diagnosing cancer, said method comprising the steps of:
(a) determining the expression level of the C1orf59 gene, the PIWIL4 gene or piRNA in a subject-derived biological sample by a method selected from the group consisting of:
(i) detecting an mRNA of the C1orf59 gene and/or the PIWIL4 gene,
(ii) detecting a protein encoded by the C1orf59 gene and/or the PIWIL4 gene,
(iii) detecting a biological activity of the protein encoded by the C1orf59 gene and/or the PIWIL4 gene, and
(iv) detecting a piR1 and/or piR2;
(b) correlating an increase in the expression level determined in step (a) as compared to a normal control level of the C1orf59 gene, the PIWIL4 gene or piRNA to the presence of cancer;
[2] The method of [1], wherein the expression level determined in step (a) is determined by detecting the binding of an antibody against the protein encoded by the C1orf59 gene or PIWIL4 gene.
[3] The method of [1], wherein the subject-derived biological sample comprises a biopsy specimen;
[4] A method for assessing or determining the prognosis of a patient with cancer, which method comprises the steps of:
(a) detecting an expression level of the C1orf59 gene in a patient-derived biological sample;
(b) comparing the expression level detected in step (a) to a control level; and
(c) assessing or determining the prognosis of the patient based on the comparison of step (b);
[5] The method of [4], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level is determined as poor prognosis;
[6] The method of [4], wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting an mRNA of the C1orf59 gene;
(b) detecting a protein encoded by the C1orf59 gene; and
(c) detecting a biological activity of a protein encoded by the C1orf59 gene;
[7] The method of [4], wherein the patient derived biological sample comprises a biopsy specimen;
[8] A kit for diagnosing cancer or assessing or determining the prognosis of a patient with cancer, which comprises a reagent selected from the group consisting of:
(a) a reagent for detecting an mRNA of the C1orf59 gene and/or the PIWIL4 gene;
(b) a reagent for detecting a protein encoded by the C1orf59 gene and/or the PIWIL4 gene;
(c) a reagent for detecting a biological activity of a protein encoded by the C1orf59 gene and/or the PIWIL4 gene; and
(d) a reagent for detecting a piR1 and/or piR2;
[9] The kit of claim 8, wherein the reagent is a probe that binds to the mRNA of the C1orf59 gene or gene PIWIL4 gene, piR1 or piR2.
[10] The kit of [8], wherein the reagent is an antibody against and binding to a protein encoded by the C1orf59 gene or the PIWIL4gene.
[11] An isolated double-stranded molecule that, when introduced into a cell, inhibits expression of the C1orf59 gene or the PIWIL4 gene as well as cell proliferation, said molecule comprising a sense strand and an antisense strand complementary thereto, said strands hybridized to each other to form the double-stranded molecule.
[12] The double-stranded molecule of [11], wherein the sense strand comprises a sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.
[13] The double-stranded molecule of [11] or [12], wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length.
[14] The double-stranded molecule of any one of [11] to [13], which consists of a single polynucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[15] The double-stranded molecule of [14], which has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A], wherein [A] is the sense strand comprising a sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is the antisense strand comprising a complementary sequence to [A].
[16] A vector encoding the double-stranded molecule of any one of claims 11 to 15.
[17] A method for treating or preventing a cancer expressing at least one gene selected from the group consisting of the C1orf59 gene and the PIWIL4 gene, wherein the method comprises the step of administering at least one isolated double-stranded molecule of any one of [11] to [15] or a vector of [16].
[18] A composition for treating or preventing a cancer expressing at least one gene selected from the group consisting of the C1orf59 gene and the PIWIL4 gene, wherein composition comprised at least one isolated double-stranded molecule of any one of [11] to [15] or a vector of [16].
[19] A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene , or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene and/or the PIWIL4 gene;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) selecting a substance that binds to the polypeptide.
[20] A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene or the PIWIL4 gene , or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene or the PIWIL4 gene;
(b) detecting a biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide as compared to the biological activity of said polypeptide detected in the absence of the test substance.
[21] The method of [20], wherein the biological activity is the facilitation of the cell proliferation.
[22] A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing the C1orf59 gene and/or the PIWIL4 gene and
(b) selecting the test substance that reduces the expression level of the C1orf59 gene and/or the PIWIL4 gene in comparison with the expression level detected in the absence of the test substance.
[23] A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell into which a vector, comprising the transcriptional regulatory region of the C1orf59 gene or the PIWIL4 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the test substance that reduces the expression or activity level of said reporter gene as compared to a control.
[24] A method of screening for a candidate substance for treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene with a substrate to be methylated in the presence of a test substance under a condition capable of methylation of the substrate;
(b) detecting the methylation level of the substrate; and
(c) selecting the test substance that decreases the methylation level of the substrate compared to a control level.
[25] The method of [24], wherein the substrate is piRNA.
[26] The method of [25], wherein the piRNA is piR1 or piR2.
[27] A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising a PIWIL4-binding domain of a C1orf59 polypeptide with a polypeptide comprising a C1orf59-binding domain of a PIWIL4 polypeptide in the presence of a test substance;
(b) detecting binding between the polypeptides; and
(c) selecting the test substance that inhibits binding between the polypeptides.
[28] The method of [27], wherein the polypeptide comprising the PIWIL4-binding domain comprises a C1orf59 polypeptide.
[29] The method of [27], wherein the polypeptide comprising the C1orf59-binding domain comprises a PIWIL4 polypeptide.
[30] A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising an S-adenosylmethionine (SAM)-binding domain of a C1orf59 polypeptide with SAM in the presence of a test substance;
(b) detecting binding between the polypeptide and SAM; and
(c) selecting the test substance that inhibits the binding.
[31] The method of [30], wherein the polypeptide comprising the SAM-binding domain comprises a C1orf59 polypeptide.
[32] A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing the C1orf59 gene and
(b) selecting the test substance that reduces the expression level of piRNA in comparison with the expression level detected in the absence of the test substance.
[33] The method of [32], wherein the piRNA is piR1 or piR2.
[34] A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising a CBX5, SUV39H1 or SUV39H2-binding domain of a PIWIL4 polypeptide with a polypeptide comprising a PIWIL4 -binding domain of a CBX5, SUV39H1 or SUV39H2 polypeptide in the presence of a test substance;
(b) detecting a binding between the polypeptides; and
(c) selecting the test substance that inhibits the binding between the polypeptides;
[35] The method of [34], wherein the polypeptide comprising the CBX5, SUV39H1 or SUV39H2 -binding domain comprises a PIWIL4 polypeptide; and
[36] The method of [34], wherein the polypeptide comprising the PIWIL4-binding domain comprises a CBX5, SUV39H1 or SUV39H2 polypeptide.

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.

It will also be understood that both the foregoing summary of the present invention and the following detailed description are of exemplified embodiments, and not restrictive of the present invention or other alternate embodiments of the present invention. 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. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims. Likewise, other objects, features, benefits and advantages of the present invention will be apparent from this summary and certain embodiments described below, and will be readily apparent to those skilled in the art. Such objects, features, benefits and advantages will be apparent from the above in conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.

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:
Figure 1 depicts C1orf59 expression in cancers and normal tissues. Part A depicts the expression of C1orf59 in a normal esophagus and 10 clinical ESCC tissue samples (top panels) and 11 ESCC cell lines detected by semiquantitative RT-PCR analysis (bottom panels). Part B depicts the expression of C1orf59 in cervical, colon, bile duct, lung squamous cell cancers tissue samples. Part C depicts the expression of C1orf59 in cervical, colon, bile duct, lung squamous cell cancers cell lines.

Part D depicts the expression of C1orf59 in esophageal cancer cell lines, examined by Western blot analyses. Expression of ACTB served as a quantity control. Part E depicts the subcellular localization of endogenous C1orf59 protein in TE1 cells. C1orf59 was stained in the nucleus and cytoplasm. DAPI, 4',6-diamidino-2-phenylindole.

Figure 2 depicts C1orf59 protein expression in normal tissues esophageal cancers, and its association with poorer clinical outcomes for ESCC patients. Part A depicts the results of Northern blot analysis of the C1orf59 transcript in 23 normal human tissues. A strong signal was observed in testis. Part B depicts the expression of C1orf59 in six normal human tissues as well as lung and esophageal cancers, detected by immunohistochemical staining. Magnification, X100. Positive staining appeared predominantly in the nucleus and cytoplasm of primary spermatocytes in the testis and esophageal cancer cells.

Part C depicts the representative examples of expression of C1orf59 in esophageal cancer (squamous cell carcinomas, X100) and normal esophagus (X100), and magnified view (X200). Part D depicts the results of Kaplan-Meier analysis of tumor-specific survival in ESCC patients according to C1orf59 expression level (P = 0.0029; log-rank test).

Figure 3 depicts effect of C1orf59 on growth promotion of cells. Part A depicts the expression of C1orf59 in response to si-C1orf59s (si-1 and -2) or control siRNAs (LUC and EGFP) in TE1 (left) and TE5 (right) cells, analyzed by semiquantitative RT-PCR. Part B depicts the viability of TE1 (left) or TE5 (right) cells evaluated by MTT assay in response to si-1, si-2, si-LUC, or si-EGFP. Part C depicts the results of a colony -formation assays of TE1 (left) and TE5 (right) cells transfected with specific siRNAs or control siRNAs. All experiments were carried out in triplicate assays.

Part D depicts the expression of C1orf59 in COS-7 and HEK293 cells examined by western-blot analysis. Part E depicts the results of an MTT assay on cell viability. Cells transfected with pCAGGSn3Fc-C1orf59 or mock vector were each cultured in triplicate and evaluated.

Figure 4 depicts detection of piRNAs in cancer cells and confirmation of enzyme activity of C1orf59 protein. Part A depicts the methyltransferase activity of C1orf59. Enzyme activity of C1orf59 could be confirmed by in vitro methyltransferase assay. Pre-methylated piRNA on 2'-OH of 3' terminal nucleotide was not methylated. Part B depicts piRNAs in esophageal clinical cancers. Real time PCR was done with the sample, which was reverse transcribed by miScript kit (see above). piR1,2 could be amplified especially in cancers. Part C depicts the results of northern blot analysis, wherein piRNA in TE1 was detected.

Parts D and E depict the impact of C1orf59 on piRNA expression. Knock down of C1orf59 in TE1 and TE5 led to a decrease in expression of piRNA (Part D). Overexpression of C1orf59 in HEK293T led to an increase in piRNA expression (Part E).

Figure 5 depicts the generated mutant recombinant protein, and expression vector. In Part A, a partial mutation was inserted. In Part B, a mutant recombinant C1orf59 was deactivated. In Part C, expression of mutant recombinant C1orf59 was examined by western-blot analysis. In Part D, mutant recombinant C1orf59 was shown to lose the growth promotive effect.

Figure 6 depicts detection of functional downstream of C1orf59 and methylated piRNAs in cancer cells. In Part A, C1orf59 was interacted with PIWIL4. In Parts B, C, and D, PIWIL4 was interacted with H3K9 methylate associating proteins, CBX5 (HP1 subfamily), SUV39H1, and SUV39H2.

In Part E, knock down of C1orf59 was shown to induce the reduction of trimethylated H3K9. Part F proposes a downstream process after methylation of piRNA by C1orf59.

In Part G, knock down of C1orf59 was shown to induce the activation of p16 and p21.

Figure 7 depicts expression of C1orf59 in normal tissues and cancers. Part A depicts the results of a semi-quantitative RT-PCR assay, wherein C1orf59 was strongly expressed in esophageal tumors in spite of no expression in normal esophagus. Part B depicts the results of a immunohistochemical staining, wherein C1orf59 was strongly expressed in tumor in spite of no expression in normal esophagus. Part C depicts the results of a semi-quantitative RT-PCR assay, wherein C1orf59 was strongly expressed in several kinds of tumors in spite of no expression in normal tissues.

Figure 8 depicts identification of over expression of PIWIL4 in several cancers. Parts A and B depict the expression of PIWIL1, PIWIL2, PIWIL3 and PIWIL4. Only PIWIL4 was expressed in esophageal cancers. Part C depicts the expression of PIWIL4 in response to si-PIWIL4s (si-1 and -2) or control siRNAs (LUC and EGFP) in TE1 cells, analyzed by semiquantitative RT-PCR . Part D depicts the viability of TE1 cells evaluated by MTT assay in response to si-1, si-2, si-LUC, or si-EGFP. Part E depicts the results of colony -formation assays of TE1cells transfected with specific siRNAs or control siRNAs. All experiments were carried out in triplicate assays.

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 the present invention belongs. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definition
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
As used herein, the phrase "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). The phrase "biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, the phrase "biological sample" refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or polynucleotides.

The terms "gene", "polynucleotide", "oligonucleotide" "nucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleic acid residues and, unless otherwise specifically indicated are referred to by their commonly accepted single-letter codes. The terms apply to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester bonding. The nucleic acid polymers may be composed of DNA, RNA or a combination thereof and encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.

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 includes DNA, RNA, and derivatives thereof.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms refer to naturally occurring and synthetic amino acids, as well as amino acids analogs and amino acids mimetics 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. 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 substances 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 substances 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 term "amino acid" as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly function to the naturally occurring amino acids. Amino acid may be either L-amino acids or D-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 one or more modified R group(s) 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 term "piR1" refers to a piRNA having a ribonucleotide sequence shown in SEQ ID NO: 9.
The term "piR2" refers to a piRNA having a ribonucleotide sequence shown in SEQ ID NO: 10.
Piwi-interacting RNAs (piRNAs) are a novel class of small RNAs isolated from the mammalian germline cells. piRNAs interact with the Piwi subfamily of proteins and form a ribonucleoprotein complex called Piwi-interacting RNA complex. It is predicted that piRNAs play a role in epigenetic regulation , prevention of retrotransposon transposition, positive regulation of translation and mRNA stability. However, the functions of piRNAs remain unclear.
The term "SAM" refers to S-adenosylmethionine.
Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the C1orf59 gene and/or the PIWIL4 gene. Examples of cancers over-expressing C1orf59 and/or PIWIL4 include, but are not limited to, esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer.

In the context of the present invention, the term "composition" is used to refer to a product including that include the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such terms, when used in relation to the modifier "pharmaceutical" (as in "pharmaceutical composition"), are intended to encompass products including a product that includes the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, in the context of the present invention, the term "pharmaceutical composition" refers to any product made by admixing a molecule or compound of the present invention and a pharmaceutically or physiologically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" or "physiologically acceptable carrier", as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject scaffolded polypharmacophores from one organ, or portion of the body, to another organ, or portion of the body.

The term "active ingredient" herein refers to a substance in composition that is biologically or physiologically active. Particularly, in the context of pharmaceutical composition, the term "active ingredient" refers to a substance that shows an objective pharmacological effect. For example, in case of pharmaceutical compositions for use in the treatment or prevention of cancer, active ingredients in the agents or compositions may lead to at least one biological or physiologically action on cancer cells and/or tissues directly or indirectly. Preferably, such action may include reducing or inhibiting cancer cell growth, damaging or killing cancer cells and/or tissues, and so on. Typically, indirect effect of active ingredients is inductions of CTLs recognizing or killing cancer cells. Before being formulated, the "active ingredient" may also be referred to as "bulk", "drug substance" or "technical product".

As used herein, the term "isolated double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene and includes, 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 use 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 C1orf59 or a PIWIL4 sense nucleic acid sequence (also referred to as "sense strand"), a C1orf59 or a PIWIL4 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 composed of 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, composed of first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, 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 use 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 C1orf59 or a PIWIL4 sense nucleic acid sequence (also referred to as "sense strand"), a C1orf59 or a PIWIL4 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 composed of 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, composed of a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions are sufficient such that base pairing occurs between the regions, the first and second regions are joined by a loop region, the loop results from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an 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".

Genes or Proteins
The present invention makes reference to nucleic acid and polypeptide sequences of genes of interest, examples of which include, but are not limited to, those shown in the following numbers:
C1orf59: SEQ ID NO: 1 and 2;
PIWIL4: SEQ ID NO: 3 and 4;
CBX5: SEQ ID NO: 24 and 25;
SUV39H1: SEQ ID NO: 26 and 27; and
SUV39H2: SEQ ID NO: 28 and 29.

Additional sequence data is available via following accession numbers:
C1orf59: NM_001102592.1 and NM_144584.2;
PIWIL4: NM_152431.2 ;
CBX5: NM_001127321.1, NM_001127322.1, NM_012117.2;
SUV39H1: NM_003173; and
SUV39H2: NM_024670.3.

The present invention also contemplates "functional equivalents" and deems such to be "polypeptides" in context. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to that of the original reference protein. Namely, any polypeptide that retains the biological ability of the original reference peptide may be used as such a functional equivalent in the present invention. Such functional equivalents include those wherein one or more amino acids are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the protein. Alternatively, the polypeptide may be composed an amino acid sequence having at least about 80% homology (also referred to as sequence identity) to the sequence of the respective 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 gene.

A polypeptide of the present invention may have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has a function equivalent to that of a human protein of the present invention, it is within the scope of the present invention.

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 substances 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 include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42degrees 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, the optimal condition of hybridization for isolating a DNA encoding a polypeptide functionally equivalent to the above human 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.

It is generally known that a modification of one, two or more amino acid in a protein will not significantly influence the function of the protein; in some cases, it may even enhance the desired function of the original 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 (i.e., less than 5%, more preferably less than 3%, even more preferably less than 1%) or those considered to be a "conservative modifications", wherein the alteration of a protein results in a protein with similar functions, are acceptable in the context of the instant invention. Thus, 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 an originally disclosed reference sequence.

So long as the activity the protein is maintained, the number of amino acid mutations or modifications is not particularly limited. However, it is generally preferred to alter 5% or less of the amino acid sequence, more preferably less than 3%, even more preferably less than 1%. 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) Asparagine (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 protein. However, the present invention is not restricted thereto and includes non-conservative modifications, so long as the resulting modified peptide retains at least one biological activity of the original protein. In the context of the present invention, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

Moreover, the gene of the present invention encompasses polynucleotides that encode such functional equivalents of the 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 protein, using a primer synthesized based on the sequence above information. Polynucleotides and polypeptides that are functionally equivalent to the human gene and protein, respectively, normally have a high homology to the originating nucleotide or amino acid sequence of. The phrase "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% to 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)".

Double-Stranded Molecules
Double-stranded molecules (e.g., siRNA and the like) against target gene(s) can be used to reduce the expression level of said gene(s). 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, a double-stranded molecule against C1orf59 or PIWIL4 that hybridizes to target mRNA may be used to decrease or inhibit production of proteins encoded by C1orf59 or PIWIL4 gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the protein. As demonstrated herein, the expression of C1orf59 or PIWIL4 in cancer cell lines is inhibited by dsRNA (Fig. 3A, Fig. 8C). Accordingly, the present invention provides isolated double-stranded molecules that are capable of inhibiting the expression of a C1orf59 or a PIWIL4 gene when introduced into a cell expressing the gene. The target sequence of double-stranded molecules may be designed by an siRNA design algorithm such as that mentioned below.

Examples of C1orf59 target sequences include, for example, nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, and examples of PIWIL4 target sequences include, for example, nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8. Therefore, the present invention also provides a double-stranded molecule having the nucleotide sequence of SEQ ID NO: 5, 6, 7, or 8 as the target sequence.

Double stranded molecules of particular interest in the context of the present invention are set forth below:
[1]An isolated double-stranded molecule that, when introduced into a cell, inhibits expression of the C1orf59 gene or the PIWIL4 gene and cell proliferation, such molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule;
[2]The double-stranded molecule of [1], wherein said double-stranded molecule acts on mRNA, matching a target sequence selected from among SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
[3]The double-stranded molecule of [1], wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8;
[4]The double-stranded molecule of any one of [1] to [3], having a length of less than about 100 nucleotides;
[5]The double-stranded molecule of [4], having a length of less than about 75 nucleotides;
[6]The double-stranded molecule of [5], having a length of less than about 50 nucleotides;
[7]The double-stranded molecule of [6] having a length of less than about 25 nucleotides;
[8]The double-stranded molecule of [7], having a length of between about 19 and about 25 nucleotides;
[9]The double-stranded molecule of any one of [1] to [8], composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand;
[10]The double-stranded molecule of [9], having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
[11]The double-stranded molecule of any one of [1] to [10], composed of RNA;
[12]The double-stranded molecule of any one of [1] to [10], composed of both DNA and RNA;
[13]The double-stranded molecule of [12], wherein the molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[14]The double-stranded molecule of [13] wherein the sense and the antisense strands are composed of DNA and RNA, respectively;
[15]The double-stranded molecule of [12], wherein the molecule is a chimera of DNA and RNA;
[16]The double-stranded molecule of [15], wherein 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 are RNA;
[17]The double-stranded molecule of [16], wherein the flanking region is composed of 9 to 13 nucleotides; and
[18]The double-stranded molecule of any one of [1] to [17], wherein the molecule contains one or two 3' overhang(s).

The double-stranded molecule of the present invention is described in more detail below.
Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Patent No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). Such a computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.

Selection of Target Sites:
1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. don't recommend designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these may be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. Basically, BLAST, which can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/, is used (See Altschul SF et al., Nucleic Acids Res 1997 Sep 1, 25(17): 3389-402).
3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
Using the above protocol, the target sequences of the isolated double-stranded molecules of the present invention were designed as:
SEQ ID NO: 5 and 6 for C1orf59 gene, and
SEQ ID NO: 7 and 8 for PIWIL4 gene.

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. Accordingly, the present invention provides double-stranded molecules targeting any of the sequences selected from among:
SEQ ID NOs: 5 (at the position 314-332nt of SEQ ID NO: 1) and 6 (at the position 1073-1091nt of SEQ ID NO: 1) for C1orf59, and
SEQ ID NOs: 7 (at the position 1002-1020nt of SEQ ID NO: 3) and 8 (at the position 2679-2697nt of SEQ ID NO: 3) for PIWIL4 gene.
A double-stranded molecule of the present invention may be directed to a single target C1orf59 or PIWIL4 gene sequence or may be directed to a plurality of target C1orf59 and/or PIWIL4 gene sequences.

A double-stranded molecule of the present invention targeting an above-mentioned targeting sequence of the C1orf59 or PIWIL4 gene may include isolated polynucleotides that contain any of the nucleic acid sequences of target sequences and/or complementary sequences to the target sequences. Examples of polynucleotides targeting the C1orf59 or PIWIL4 gene include those containing the sequence of SEQ ID NO: 5, 6, 7 or 8 and/or complementary sequences to these nucleotides; However, the present invention is not limited to these examples, 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 the C1orf59 or PIWIL4 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 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 suppression ability using the methods utilized in the Examples. In the Examples herein below, double-stranded molecules composed of sense strands of various portions of C1orf59 or PIWIL4 mRNA or antisense strands complementary thereto were tested in vitro for their ability to decrease production of a C1orf59 or a PIWIL4 gene product in esophageal, cervical, colon, bile duct and/or lung cancer cell lines according to standard methods. For example, reduction in a C1orf59 or a PIWIL4 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 C1orf59 or PIWIL4 mRNA mentioned under Example 1 item "Semi-quantitative RT-PCR". Sequences that decrease the production of a C1orf59 or a PIWIL4 gene product in vitro cell-based assays can then be tested for their inhibitory effects on cell growth. Sequences that inhibit cell growth in an in vitro cell-based assay can then be tested for their in vivo suppression ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of a C1orf59 or a PIWIL4 gene 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. However, the present invention extends to complementary sequences that include mismatches of one or more nucleotides. In addition, 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 complementary or antisense polynucleotide is preferably less than 1249 nucleotides in length for C1orf59 or a PIWIL4. Preferably, 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 a C1orf59 or a PIWIL4 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 composed of a sense strand and an antisense strand, wherein the sense strand has 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 composed of between 19 and 25 nucleotide pairs.

The double-stranded molecules of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. It is well known in the art to introduce chemical modifications to increase stability, availability, and/or cell uptake of the double-stranded molecule. A person skilled in the art will readily contemplate the wide array of chemical modifications that may be incorporated into the present molecules (See WO03/070744; WO2005/045037). For example, in one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include, but are not limited to, 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 (See US20060122137).

In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Examples of such modifications include, but are not limited to, 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'-deoxy ribonucleotides (See 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 (See 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 (See Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents such as US20060234970 are available. However, the present invention should not be construed as limited to these examples; any of a number of conventional 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.

The double-stranded molecules of the present invention may include both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. For example, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability and are thus contemplated herein. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule composed of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule containing 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 either have a DNA sense strand coupled to an RNA antisense strand, or vice versa, so long as the resulting double stranded molecule can inhibit expression of the target gene when introduced into a cell expressing the gene. In a preferred embodiment, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may either have either or both sense and antisense strands composed of DNA and RNA, so long as the resulting double stranded molecule 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.

A preferred chimera type double-stranded molecule contains an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of 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. Alternatively, regions flanking to 5'-end of sense strand and/or 3'-end of antisense strand may be referred to as the upstream partial region. That is, in preferred 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 are composed of RNA. For instance, a chimera or hybrid type double-stranded molecule of the present invention may 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 composed 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 (See 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 that match the target sequence of the dsRNA or dsD/R-NA.

A loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Such loop sequence may be joined to 5' or 3' end of a sense strands to form the hairpin loop structure. Thus, the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 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]. The target sequence may be selected from among, for example, nucleotide sequences of SEQ ID NOs: 5 and 6 for C1orf59 and SEQ ID NOs: 7 and 8 for PIWIL4.

The present invention is not limited to these examples, and the target sequence in [A] may be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted C1orf59 or PIWIL4 gene. The region [A] hybridizes to [A'] to form a loop composed of the region [B]. The intervening single-stranded portion [B], i.e., loop sequence may be preferably 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from among the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26;
UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and
UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.

Examples of preferred double-stranded molecules of the present invention having hairpin loop structure are shown below. In the following structures, the loop sequence can be selected from among AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:
CAGUUUAAACCUCCACUAU -[B]- AUAGUGGAGGUUUAAACUG (for target sequence SEQ ID NO: 5);
AUAGUGGAGGUUUAAACUG -[B]- CAGUUUAAACCUCCACUAU (for target sequence SEQ ID NO: 5);
GUGGAAAGCUUAAGAGUGA -[B]- UCACUCUUAAGCUUUCCAC (for target sequence SEQ ID NO: 6);
UCACUCUUAAGCUUUCCAC -[B]- GUGGAAAGCUUAAGAGUGA (for target sequence SEQ ID NO: 6);
GUUACAAAGUCCUCCGGAA -[B]- UUCCGGAGGACUUUGUAAC (for target sequence SEQ ID NO: 7);
UUCCGGAGGACUUUGUAAC -[B]- GUUACAAAGUCCUCCGGAA (for target sequence SEQ ID NO: 7)
GUCAGUAUGCUCACAAGCU -[B]- AGCUUGUGAGCAUACUGAC (for target sequence SEQ ID NO: 8);
AGCUUGUGAGCAUACUGAC -[B]- GUCAGUAUGCUCACAAGCU (for target sequence SEQ ID NO: 8);
Additionally, several nucleotides can be added to 3'end of the sense strand and/or the antisense strand of the target sequence, as 3' overhangs, so as to enhance the inhibition activity of the double-stranded molecules. The number of nucleotides to be added is at least 2, generally 2 to 10, preferably 2 to 5. The added nucleotides form single strand at the 3'end of the sense strand and/or antisense strand of the double-stranded molecule. The preferred examples of nucleotides to be added include "t" and "u", but are not limited to. In cases where double-stranded molecules consists of a single polynucleotide to form a hairpin loop structure, a 3' overhang sequence may be added to the 3' end of the single polynucleotide.

The method for preparing the double-stranded molecule is not particularly limited, though it is preferable to use one of the standard chemical synthetic methods known in the art. According to one 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. In one specific annealing embodiment, the synthesized single-stranded polynucleotides are mixed in a molar ratio of preferably at least about 3:7, more preferably about 4:6, and most preferably 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 C1orf59 or PIWIL4 sequences may 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 C1orf59 or PIWIL4 gene templates 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.

Vectors Containing A Double-Stranded Molecule Of The Present Invention:
Also included in the present invention are vectors containing one or more of the double-stranded molecules described herein, and a cell containing such a vector.
Of particular interest to the present invention are the vectors of [1] to [10] set forth below:
[1] A vector, encoding a double-stranded molecule that, when introduced into a cell, inhibits expression of C1orf59 or PIWIL4 and cell proliferation, such molecules composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule.
[2] The vector of [1], encoding the double-stranded molecule acts on mRNA, matching a target sequence selected from among SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
[3] The vector of [1], wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
[4] The vector of any one of [1] to [3], encoding the double-stranded molecule having a length of less than about 100 nucleotides;
[5] The vector of [4], encoding the double-stranded molecule having a length of less than about 75 nucleotides;
[6] The vector of [5], encoding the double-stranded molecule having a length of less than about 50 nucleotides;
[7] The vector of [6] encoding the double-stranded molecule having a length of less than about 25 nucleotides;
[8] The vector of [7], encoding the double-stranded molecule having a length of between about 19 and about 25 nucleotides;
[9] The vector of any one of [1] to [8], wherein the double-stranded molecule is composed of a single polynucleotide having both the sense and antisense strands linked by an intervening single-strand;
[10] The vector of [9], encoding the double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
A vector of the present invention preferably encodes a double-stranded molecule of the present invention in an expressible form. Herein, the phrase "in an expressible form" indicates that the vector, when introduced into a cell, will express the molecule carried, contained or encoded therein. In a preferred embodiment, the vector includes one or more regulatory elements necessary for expression of the double-stranded molecule. Such vectors of the present invention may be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.

Vectors of the present invention can be produced, for example, by cloning the C1orf59 or PIWIL4 sequences into an expression vector so that regulatory sequences are operatively-linked to the C1orf59 or PIWIL4 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 anti-sense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct having a secondary structure (e.g., hairpin); accordingly, a single transcript of a vector may contain both the sense and complementary antisense sequences of the target gene.

The present invention contemplates a vector that includes each or both of a combination of polynucleotides, including a sense strand nucleic acid and an antisense strand nucleic acid, wherein the antisense strand includes a nucleotide sequence which is complementary to said sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said vector, when introduced into a cell expressing the C1orf59 or PIWIL4 gene, inhibits expression of said gene.

The vectors of the present invention may also be equipped so 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 also, 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.

Methods Of Inhibiting Or Reducing Growth Of A Cancer Cell And Treating Cancer Using A Double-Stranded Molecule Of The Present Invention:
The ability of certain siRNA to inhibit NSCLC has been previously described in WO 2005/89735, the entire contents of which are incorporated by reference herein. In the present invention, two different dsRNA for C1orf59 or PIWIL4 were tested for their ability to inhibit cell growth. The two dsRNA for C1orf59 (Fig. 3) and the two dsRNA for PIWIL4 (Fig. 8) that effectively knocked down the expression of the gene in lung and esophageal cancer cell lines coincided with suppression of cell proliferation.

Accordingly, the present invention provides methods for inhibiting cancer cell growth, for example, esophageal, cervical, colon, bile duct or lung cancer cell growth, by inducing dysfunction of the C1orf59 or PIWIL4 gene via inhibiting the expression of C1orf59 or PIWIL4. The C1orf59 or PIWIL4 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention that specifically target the C1orf59 or PIWIL4 gene or the vectors of the present invention that can express any of the double-stranded molecules.

Such ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cell indicates that they can be used for methods for treating cancer, as well as treating or preventing a post-operative or secondary recurrence thereof. Thus, the present invention provides methods to treat patients with cancer by administering a double-stranded molecule against a C1orf59 or a PIWIL4 gene or a vector expressing the molecules. The treating methods of the present invention are expected to be carried out without adverse effect because those genes were hardly detected in normal organs (Fig. 1, Fig. 2, Fig.7 and Fig.8). The treating method of the present invention may be suitable for treatment of esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.

Of particular interest to the present invention are the methods of [1] to [36] set forth below:
[1] A method for inhibiting growth of a cancer cell and treating a cancer, wherein the cancer cell or the cancer expresses a C1orf59 or a PIWIL4 gene, such method including the step of administering at least one isolated double-stranded molecule inhibiting the expression of the C1orf59 or PIWIL4 genes in a cell over-expressing the gene and the cell proliferation or vector encoding the double-stranded molecule, wherein the double-stranded molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule, wherein the sense strand has a nucleotide sequence corresponding to a contiguous sequence from SEQ ID NO: 1 or 3.
[2] The method of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[3] The method of [1], wherein the sense strand contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[4] The method of any one of [1] to [3], wherein the cancer to be treated is esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer;
[5] The method of [4], wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) and esophageal cancer is esophageal squamous cell cancer (ESCC);
[6] The method of [1], wherein plural kinds of the double-stranded molecules are administered;
[7] The method of any one of [1] to [6], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[8] The method of [7], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[9] The method of [8], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[10] The method of [9], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[11] The method of [10], wherein the double-stranded molecule has a length of between about 19 and about 25 nucleotides in length;
[12] The method of any one of [1] to [11], wherein the double-stranded molecule is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[13] The method of [12], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A];
[14] The method of any one of [1] to [13], wherein the double-stranded molecule is an RNA;
[15] The method of any one of [1] to [13], wherein the double-stranded molecule contains both DNA and RNA;
[16] The method of [15], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[17] The method of [16] wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[18] The method of [15], wherein the double-stranded molecule is a chimera of DNA and RNA;
[19] The method of [18], wherein 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 are composed of RNA;
[20] The method of [19], wherein the flanking region is composed of 9 to 13 nucleotides;
[21] The method of any one of [1] to [20], wherein the double-stranded molecule contains one or two 3' overhang(s);
[22] The method of any one of [1] to [21], wherein the double-stranded molecule is contained in a composition which includes, in addition to the molecule, a transfection-enhancing substance and pharmaceutically acceptable carrier.
[23] The method of [1], wherein the double-stranded molecule is encoded by a vector;
[24] The method of [23], wherein the double-stranded molecule encoded by the vector acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[25] The method of [23], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[26] The method of any one of [23] to [25], wherein the cancer to be treated is esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer;
[27] The method of [26], wherein the lung cancer is NSCLC or SCLC and esophageal cancer is ESCC;
[28] The method of [23], wherein plural kinds of the double-stranded molecules are administered;
[29] The method of any one of [23] to [28], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides;
[30] The method of [29], wherein the double-stranded molecule encoded by the vector has a length of less than about 75 nucleotides;
[31] The method of [30], wherein the double-stranded molecule encoded by the vector has a length of less than about 50 nucleotides;
[32] The method of [31], wherein the double-stranded molecule encoded by the vector has a length of less than about 25 nucleotides;
[33] The method of [32], wherein the double-stranded molecule encoded by the vector has a length of between about 19 and about 25 nucleotides in length;
[34] The method of any one of [23] to [33], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[35] The method of [34], wherein the double-stranded molecule encoded by the vector has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is a intervening single-strand is composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[36] The method of any one of [23] to [35], wherein the double-stranded molecule encoded by the vector is contained in a composition which includes, in addition to the molecule, a transfection-enhancing agent and pharmaceutically acceptable carrier.

Therapeutic methods of the present invention are described in more detail below.
The growth of cells expressing a C1orf59 or a PIWIL4 gene may be inhibited by contacting the cells with a double-stranded molecule against a C1orf59 or a PIWIL4 gene, a vector expressing the molecule or a composition containing the same. The cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability as compared to a cell not exposed to the molecule. Cell growth may be measured by any of a number of methods known in the art, e.g., using the MTT cell proliferation assay.

The growth of any kind of cell may be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include esophageal, cervical, colon, bile duct and lung cancer cells. Lung cancer may be NSCLC or SCLC. Preferably, cancer is esophageal cancer, and more be preferably ESCC.

Thus, patients suffering from or at risk of developing a disease related to the over-expression of theC1orf59 or PIWIL4 genes may be treated with the administration of at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition containing at least one of the molecules. For example, patients suffering from esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer may be treated according to the present methods. The type of cancer may be identified by standard methods according to the particular type of tumor to be diagnosed. Esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer may be diagnosed, for example, with known tumor markers such as Carcinoembryonic antigen (CEA), CYFRA, pro-GRP and so on, or with Chest X-Ray and/or Sputum Cytology. More preferably, patients treated by the methods of the present invention are selected by detecting the expression of C1orf59 or PIWIL4 in a biopsy specimen from the patient by RT-PCR or immunoassay. Preferably, before the treatment of the present invention, the biopsy specimen from the subject is confirmed for C1orf59 or PIWIL4 gene over-expression by methods known in the art, for example, immunohistochemical analysis or RT-PCR.

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 an mRNA that matches the same target sequence of C1orf59 or PIWIL4. Alternatively, plural kinds of double-stranded molecules may act on an mRNA that matches a different target sequence of same gene. For example, the method may utilize double-stranded molecules directed to C1orf59 or PIWIL4.
For inhibiting cell growth, a double-stranded molecule of present invention may be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts. Alternatively, as described above, a DNA encoding the double-stranded molecule may be introduced into cells by means of a vector. For introducing the double-stranded molecules and vectors into the cells, transfection-enhancing agent, such as FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), may be employed.

A treatment is deemed "efficacious" if it leads to clinical benefit such as, reduction in expression of C1orf59 or PIWIL4 gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.

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, the tumor regression, 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 constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.

Those of skill in the art understand that a double-stranded molecule of the invention degrades C1orf59 or PIWIL4 mRNA in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, as compared to standard cancer therapies, the present invention requires the delivery of significantly less double-stranded molecule at or near the site of cancer in order to exert therapeutic effect.

One skilled in the art can readily determine the optimal effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors such as body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of the double-stranded molecule of the invention is an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50 nM, more preferably from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered. The precise dosage required for a particular circumstance may be readily and routinely determined by one of skill in the art.

The present methods can be used to inhibit the growth or metastasis of cancer expressing C1orf59 and/or PIWIL4; for example, esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer. Lung cancer may be NSCLC or SCLC. Preferably, cancer is esophageal cancer, and more preferably ESCC. In particular, a double-stranded molecule containing a target sequence against the C1orf59 or PIWIL4 gene (i.e., SEQ ID NOs: 5, 6, 7 and 8) is particularly preferred for the treatment of esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.

For treating cancer, the double-stranded molecule of the invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule. Alternatively, the double-stranded molecule of the invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecule of the invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).

In the context of present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector that expresses the double-stranded molecule.

Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule 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 double-stranded molecule to a particular tissue, such as esophageal, cervical, colon, bile duct and/or lung tumor tissue, and can also increase the blood half-life of the double-stranded molecule. 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 double-stranded molecule include a ligand molecule that can deliver the liposome to the cancer site. Ligands that bind to receptors prevalent in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, are preferred.
Particularly preferably, the liposomes encapsulating the present double-stranded molecule 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 double-stranded molecule 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 GM.sub.1. 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)BH. sub. 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.

Vectors expressing a double-stranded molecule of the present invention are discussed above. Such vectors expressing at least one double-stranded molecule 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 a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.

The double-stranded molecule of the invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule 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 or 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); 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 suppository or an implant including a porous, non-porous, or gelatinous material); and inhalation. It is preferred that injections or infusions of the double-stranded molecule or vector be given at or near the site of the cancer.

The double-stranded molecule of the invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the substance directly into the tissue is at or near the site of cancer preferred. Multiple injections of the substance 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 double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule 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 double-stranded molecule is 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 a double-stranded molecule administered to the subject can include the total amount of a double-stranded molecule administered over the entire dosage regimen.

Compositions Containing A Double-Stranded Molecule Of The Present Invention:
In addition to the above, the present invention also provides pharmaceutical compositions that include at least one of the present double-stranded molecules or the vectors coding for the molecules. Of particular interest to the present invention are the following compositions [1] to [36]:
[1] A composition for inhibiting a growth of a cancer cell and treating a cancer, wherein the cancer and the cancer cell express at least one C1orf59 or PIWIL4 gene, including at least one isolated double-stranded molecule that inhibits the expression of C1orf59 or PIWIL4 and the cell proliferation, or vector encoding the morelucle, wherein molecule is composed of a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded molecule.
[2] The composition of [1], wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[3] The composition of [1], wherein the double-stranded molecule, wherein the sense strand contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[4] The composition of any one of [1] to [3], wherein the cancer to be treated is esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer;
[5] The composition of [4], wherein the lung cancer is NSCLC or SCLC and esophageal cancer is ESCC;
[6] The composition of [1], wherein the composition contains plural kinds of the double-stranded molecules;
[7] The composition of any one of [1] to [6], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[8] The composition of [7], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[9] The composition of [8], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[10] The composition of [9], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[11] The composition of [10], wherein the double-stranded molecule has a length of between about 19 and about 25 nucleotides;
[12] The composition of any one of [1] to [11], wherein the double-stranded molecule is composed of a single polynucleotide containing the sense strand and the antisense strand linked by an intervening single-strand;
[13] The composition of [12], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand sequence contains a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is the antisense strand contains a sequence complementary to [A];
[14] The composition of any one of [1] to [13], wherein the double-stranded molecule is an RNA;
[15] The composition of any one of [1] to [13], wherein the double-stranded molecule is DNA and/or RNA;
[16] The composition of [15], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[17] The composition of [16], wherein the sense and antisense strand polynucleotides are composed of DNA and RNA, respectively;
[18] The composition of [15], wherein the double-stranded molecule is a chimera of DNA and RNA;
[19] The composition of [18], wherein 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 are composed of RNA;
[20] The composition of [19], wherein the flanking region is composed of 9 to 13 nucleotides;
[21] The composition of any one of [1] to [20], wherein the double-stranded molecule contains one or two 3' overhang(s);
[22] The composition of any one of [1] to [21], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier.

[23] The composition of [1], wherein the double-stranded molecule is encoded by a vector and contained in the composition;
[24] The composition of [23], wherein the double-stranded molecule encoded by the vector acts at mRNA which matches a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[25] The composition of [23], wherein the sense strand of the double-stranded molecule encoded by the vector contains the sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8.
[26] The composition of any one of [23] to [25], wherein the cancer to be treated is esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer;
[27] The composition of [26], wherein the lung cancer is NSCLC or SCLC and esophageal cancer is ESCC;
[28] The composition of [23], wherein plural kinds of the double-stranded molecules are administered;
[29] The composition of any one of [23] to [28], wherein the double-stranded molecule encoded by the vector has a length of less than about 100 nucleotides;
[30] The composition of [29], wherein the double-stranded molecule encoded by the vector has a length of less than about 75 nucleotides;
[31] The composition of [30], wherein the double-stranded molecule encoded by the vector has a length of less than about 50 nucleotides;
[32] The composition of [31], wherein the double-stranded molecule encoded by the vector has a length of less than about 25 nucleotides;
[33] The composition of [32], wherein the double-stranded molecule encoded by the vector has a length of between about 19 and about 25 nucleotides in length;
[34] The composition of any one of [23] to [33], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide containing both the sense strand and the antisense strand linked by an intervening single-strand;
[35] The composition of [34], wherein the double-stranded molecule has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence selected from among SEQ ID NOs: 5, 6, 7 and 8, [B] is a intervening single-strand composed of 3 to 23 nucleotides, and [A'] is the antisense strand containing a sequence complementary to [A]; and
[36] The composition of any one of [23] to [35], wherein the composition includes a transfection-enhancing agent and pharmaceutically acceptable carrier.

Suitable compositions of the present invention are described in additional detail below.
The double-stranded molecules of the invention are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Thus, 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 of the present invention include those suitable for oral, rectal, nasal, topical (including buccal, sub-lingual, and transdermal), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Other formulations include implantable devices and adhesive patches that release a therapeutic agent. When desired, the above-described formulations may be adapted to give sustained release of the active ingredient. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.

The present pharmaceutical formulations contain at least one of the double-stranded molecules or vectors encoding them of the present invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt of the molecule, mixed with a physiologically acceptable carrier medium. Preferred physiologically acceptable carrier media are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

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 C1orf59 or PIWIL4. For example, the composition may contain double-stranded molecules directed to the C1orf59 or PIWIL4 gene. Alternatively, for example, the composition may contain double-stranded molecules directed to one, two or more target sequences of C1orf59 or PIWIL4.

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.
Moreover, the present double-stranded molecules may be contained as liposomes in the present composition. See under the item of "Methods Of Inhibiting OR Reducing Growth Of A Cancer Cell And Treating Cancer Using A Double-Stranded Molecule Of The Present Invention " for details of liposomes.

Pharmaceutical compositions of the invention can also include conventional pharmaceutical excipients and/or additives. Examples of suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administration can include any of the carriers and excipients listed above and 10-95%, preferably 25-75%, of one or more double-stranded molecule of the invention. A pharmaceutical composition for aerosol (inhalational) administration can include 0.01-20% by weight, preferably 1-10% by weight, of one or more double-stranded molecule of the invention encapsulated in a liposome as described above, and propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.

In addition to the above, the present composition may contain other pharmaceutically active ingredients, so long as they do not inhibit the in vivo function of the double-stranded molecules of the present invention. For example, the composition may contain chemotherapeutic agents conventionally used for treating cancers. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressants or preservatives. Furthermore, 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.

In another embodiment, the present invention provides for the use of the double-stranded nucleic acid molecules of the present invention in manufacturing a pharmaceutical composition for use in treating an esophageal, cervical, colon, bile duct and/or lung cancer characterized by the expression of C1orf59 or PIWIL4. For example, the present invention relates to a use of double-stranded nucleic acid molecule inhibiting the expression of a C1orf59 or a PIWIL4 gene in a cell, which 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 selected from among SEQ ID NOs: 5, 6, 7 and 8, for manufacturing a pharmaceutical composition for use in treating esophageal, cervical, colon, bile duct and/or lung cancer expressing C1orf59 or PIWIL4.

The present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a esophageal, cervical, colon, bile duct and/or lung cancer characterized by the expression of C1orf59 or PIWIL4, wherein the method or process includes a step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the expression of C1orf59 or PIWIL4 in a cell, which over-expresses the gene, which 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 selected from among SEQ ID NOs: 5, 6, 7 and 8 as active ingredients.

In another embodiment, the present invention provides a method or process for manufacturing a pharmaceutical composition for treating a esophageal, cervical, colon, bile duct and/or lung cancer characterized by the expression of C1orf59 or PIWIL4, wherein the method or process includes a 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 C1orf59 or PIWIL4 in a cell, which over-expresses the gene, which 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 selected from among SEQ ID NOs: 5, 6, 7 and 8.

Method Of Detecting Or Diagnosing Cancer:
The expression of C1orf59 and PIWIL4 was found to be specifically elevated in esophageal, cervical, colon, bile duct and lung cancer cells (Fig. 1, 2, 7 and 8), and also the elevated expression levels of the piR1 and piR2 was detected in esophageal cancer cells (Fig. 4) Accordingly, the genes identified herein as well as their transcription and translation products find diagnostic utility as markers for esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer. Moreover, by measuring the expression level of C1orf59, PIWIL4, piR1 and/or piR2 in a cell sample, esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer can be diagnosed. Thus, the present invention provides a method for detecting, diagnosing and/or determining the presence of or a predisposition for developing cancer by determining the expression level of C1orf59, PIWIL4, piR1 and/or piR2 in the subject. Preferred examples of cancers to be diagnosed or detected by the present methods include esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer. In the context of the present invention, "lung cancer" includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Likewise, "NSCLC" includes adenocarcinoma, squamous cell carcinoma (SCC) and large-cell carcinoma. The present invention relates to the discovery that C1orf59, PIWIL4, piR1 and/or piR2 can serve as a diagnostic marker of cancer, finding utility in the detection, monitoring, and prognosis of cancers related thereto. 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 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. Alternatively, the present invention may be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.

The present invention also provides a method for detecting or identifying cancer cells in a subject-derived esophageal tissue sample, cervical tissue sample, colon tissue sample, bile duct tissue sample or lung tissue sample, said method including the step of determining the expression level of the C1orf59, PIWIL4, piR1 and/or piR2 in the subject-derived tissue sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the subject-derived tissue sample.

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 C1orf59, PIWIL4, piR1 and/or piR2, 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 lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA. Alternatively, diagnostic esophageal tumor markers in blood such as CEA, DUPAN-2, IAP, NSE, SCC, SLX and Span-1 are also well known. 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. Particularly preferred embodiments of the present invention are set forth below as items [1] to [12]:
[1] A method for diagnosing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer, said method including the steps of:
(a) detecting the expression level of the of the C1orf59 gene, the PIWIL4 gene, piR1 and/or piR2 in a subject-derived biological sample; and
(b) correlating an increase in the expression level detected of step (a) as compared to a normal control level of the gene and/or piRNA to the presence of disease;
[2] The method of [1], wherein the expression level is at least 10% greater than the normal control level;
[3] The method of [1] or [2], wherein the expression level is detected by a method selected from among:
(a) detecting an mRNA of the C1orf59 gene and/or mRNA of the PIWIL4 gene,
(b) detecting a protein encoded by the C1orf59 gene and/or a protein encoded by the PIWIL4 gene,
(c) detecting a biological activity of a protein encoded by the C1orf59 gene and/or a protein encoded by the PIWIL4 gene and
(d) detecting piR1 and/or piR2;
[4] The method of [1], wherein the lung cancer is NSCLC or SCLC, and the esophageal cancer is ESCC;
[5] The method of [3] or [4], wherein the expression level is determined by detecting hybridization of a probe to a gene transcript of the gene;
[6] The method of [5], wherein the expression level is determined by detecting the hybridization of a probe having a complementary sequence to a part of the mRNA of the C1orf59 gene or the PIWIL4 gene, piR1 or piR2 to the mRNA of the C1orf59 gene or the PIWIL4 gene, piR1 or piR2;
[7] The method of [3] or [4], wherein the expression level is determined by detecting the binding of an antibody against the protein encoded by the gene as the expression level of the gene;
[8] The method of c[7], wherein the expression level is determined by detecting the binding of an antibody against the protein encoded by the C1orf59 or PIWIL4 gene and the protein encoded by the C1orf59 gene or the PIWIL4 gene;
[9] The method of any one of [1] to [8], wherein the biological sample includes a biopsy specimen, sputum or blood.
[10] The method of any one of [1] to [9], wherein the subject-derived biological sample includes an epithelial cell.
[11] The method of [9], wherein the subject-derived biological sample includes a cancer cell.
[12] The method of [10], wherein the subject-derived biological sample includes a cancerous epithelial cell.

The method of diagnosing esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer is described in more detail below.
A subject to be diagnosed 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.

It is preferred to collect a biological sample from the subject to be diagnosed to perform the diagnosis. Any biological material can be used as a biological sample for the determination so long as it includes the objective transcription or translation products of the C1orf59 gene and/or the PIWIL4 gene, and/or piR1 and/or piR2. The biological samples include, but are not limited to, bodily tissues which are desired for diagnosing or are suspicion of suffering from cancer, and fluids, such as biopsy, blood, sputum, pleural effusion and urine. Preferably, the biological sample contains a cell population including an epithelial cell, more preferably a cancerous epithelial cell or an 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.

According to the present invention, the expression level of C1orf59, PIWIL4, piR1 and/or piR2 in a subject-derived biological sample is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, C1orf59 mRNA, PIWIL4 mRNA, piR1 and/or piR2 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 or piRNAs (e.g., various cancer specific genes) including C1orf59, PIWIL4, piR1 and/or piR2. Those skilled in the art can prepare such probes utilizing the sequence information of the C1orf59 (SEQ ID NO 1), PIWIL4 (SEQ ID NO 3), piR1 (SEQ ID NO: 9) and/or piR2 (SEQ ID NO 10). For example, the cDNA of C1orf59, PIWIL4, piR1 or piR2 may be used as the probes. If necessary, the probe may be labeled with a suitable label, such as dyes, fluorescent 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 C1orf59 gene and/or the PIWIL4 gene or piR1 and/or piR2 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 or piRNA. For example, the primer pairs (SEQ ID NOs : 16 and 17, or 20 and 21) used in the Example may be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.

A probe or primer suitable for use in the context of the present method will hybridize under stringent, moderately stringent, or low stringent conditions to the C1orf59 mRNA and/or PIWIL4 mRNA or piR1 and/or piR2. 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, diagnosis may involve detection of a translation product. For example, the quantity of C1orf59 protein and/or PIWIL4 protein may be determined and correlated to a disease or normal state. The quantity of translation products/proteins may be determined using, for example, 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 C1orf59 protein or PIWIL4 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.

Alternatively, the intensity of staining may be observed via immunohistochemical analysis using an antibody against C1orf59 protein or PIWIL4 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of the gene encoding such protein.

To improve the accuracy of the diagnosis, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer may also be determined, in addition to the expression level of C1orf59, PIWIL4, piR1 and/or piR2.
The expression level of cancer marker gene including C1orf59, PIWIL4, piR1 and/or piR2 in a biological sample can be considered to be increased if it increases from a 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 C1orf59, PIWIL4, piR1 and/or piR2 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 C1orf59, PIWIL4, piR1 and/or piR2 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 C1orf59, PIWIL4, piR1 and/or piR2 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, the phrase "control level" refers to the expression level of a test gene detected in a control sample and encompasses both a normal control level and a cancer control level. The phrase "normal control level" refers to a level of 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 esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung 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 a test gene detected in a normal healthy tissue or cell of an individual or population known not to be suffering from esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. On the other hand, the phrase "cancer control level" refers to an expression level of a test gene detected in the cancerous tissue or cell of an individual or population suffering from esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.

An increase in the expression level of C1orf59, PIWIL4, piR1 and/or piR2 detected in a subject-derived 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 esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. In the context of the present invention, the subject-derived sample 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.

Alternatively, the expression level of C1orf59, PIWIL4, piR1 and/or piR2 in a subject-derived biological sample can be compared to (a) cancer control levels of C1orf59, PIWIL4, piR1 and/or piR2 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. When the expression levels of other cancer-related genes are also measured and 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.

Herein, gene expression levels are deemed to be "altered" or "increased" when the gene expression changes or 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. Accordingly, the expression level of cancer marker genes including C1orf59, PIWIL4, piR1 and/or piR2 in a biological sample can be considered to be increased if it increases from the normal control level of the corresponding cancer marker gene by, for example, 10% or more, 25% or more, or 50% or more; 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 expression level of the target gene can be determined by detecting, e.g., determined by the hybridization intensity of nucleic acid probes to gene transcripts in a sample.

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, 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.

Method For Assessing The Prognosis Of Cancer:
The present invention further relates to the novel discovery that C1orf59 expression is significantly associated with poorer prognosis of patients. Thus, the present invention provides a method for determining, monitoring or assessing the prognosis of a patient with cancer, in particular esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer, by detecting the expression level of the C1orf59 gene in a biological sample of the patient; comparing the detected expression level to a control level; and determining a increased expression level to the control level as indicative of poor prognosis (poor survival). In particular, the present method may be preferably used to assessing the prognosis of a subject with esophageal caner.

In addition, the expression level of the C1orf59 gene before and after a treatment can be compared according to the present method to assess the efficacy of the treatment and/or monitor disease status (e.g., progression, regression, or remission). Specifically, the expression level detected in a subject-derived biological sample after a treatment (i.e., post-treatment level) may be 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 formation of tumor or retards, prevents, or alleviates at least one clinical symptom of the disease. 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.
Herein, the term "prognosis" refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative, poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.

The terms "assessing the prognosis" refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the patient (e.g., malignancy, likelihood of curing cancer, survival, and the like). For example, a determination of the expression level of C1orf59 over time enables a predicting of an outcome for the patient (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).

In the context of the present invention, the phrase "assessing (or determining) the prognosis" is intended to encompass predictions and likelihood analysis of cancer, progression, particularly cancer recurrence, metastatic spread and disease relapse. The present method for assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.

The patient-derived biological sample used for the method may be any sample derived from the subject to be assessed so long as the C1orf59 gene can be detected in the sample. Preferably, the biological sample is an esophageal, cervical, colon, bile duct and/or lung cell (a cell obtained from the esophageal, cervical, colon, bile duct and/or lung). Furthermore, the biological sample may include bodily fluids such as sputum, blood, serum, or plasma. Moreover, the sample may be cells purified from a tissue. The biological samples may be obtained from a patient at various time points, including before, during, and/or after a treatment. For example, a esophageal, cervical, colon, bile duct and lung cancer cell(s) obtained from a subject to be assessed is a preferable biological sample.

According to the present invention, the higher expression level of the C1orf59 gene measured in the patient-derived biological sample, the poorer prognosis for post-treatment remission, recovery, and/or survival and the higher likelihood of poor clinical outcome. Thus, according to the present method, the "control level" used for comparison may be, for example, the expression level of the C1orf59 gene detected before any kind of treatment in an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment, which herein is referred to as "good prognosis control level". Alternatively, the "control level" may be the expression level of the C1orf59 gene detected before any kind of treatment in an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment, which herein will be referred to as "poor prognosis control level". The "control level" may be a single expression pattern derived from a single reference population or from a plurality of expression patterns. Thus, the control level may be determined based on the expression level of the C1orf59 gene detected before any kind of treatment in a patient of cancer, or a population of the patients whose disease state (good or poor prognosis) is known. Preferably, cancer is esophageal, cervical, colon, bile duct and/or lung cancer. It is preferred, to use the standard value of the expression levels of the C1orf59 gene in a patient group 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.

The control level may be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer patient(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing the expression level of the C1orf59 gene in samples previously collected and stored from a control group. 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 C1orf59 gene in a biological sample may be compared to multiple control levels, such as control levels are determined from multiple reference samples. Nevertheless, 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.

According to the present invention, a similarity between a measured expression level of the C1orf59 gene and a level corresponding to a good prognosis control indicates a more favorable patient prognosis. Likewise, an increase in the measured expression level as compared to the good prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. In the present invention, a good prognosis refers to a positive prognosis or favorable prognosis. On the other hand, a decrease in the measured expression level of the C1orf59 gene as compared to a poor prognosis control level indicates a more favorable prognosis of the patient, with a similarity between the two indicating less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. In the present invention, a poor prognosis refers to a negative prognosis or less favorable prognosis. In the context of the present invention, an esophageal, cervical, colon, bile duct and lung cancer cell(s) obtained from a subject who showed good, or poor prognosis of cancer after treatment is a preferable biological sample for good, or poor prognosis control level, respectively.

The expression level of the C1orf59 gene in a biological sample can be considered altered when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.
The difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., housekeeping gene. For example, polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells, including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1, may be used to normalize the expression level of the C1orf59 gene.

The expression level may be determined by detecting the gene transcript in the patient-derived biological sample using techniques well known in the art. The gene transcripts detected by the present method include both the transcription and translation products, such as mRNA and protein.
For instance, the transcription product of the C1orf59 gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a C1orf59 gene probe to the gene transcript. 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 including the C1orf59 gene. As another example, amplification-based detection methods, such as reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the C1orf59 gene may be employed for the detection (see Example). The C1orf59 gene-specific probe or primers may be designed and prepared using conventional techniques by referring to the whole sequence of the C1orf59 gene (SEQ ID NO: 1). For example, the primers (SEQ ID NOs: 16, 17, 20 and 21) 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 C1orf59 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 assessment of the present invention. For example, the quantity of the C1orf59 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 C1orf59 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 C1orf59 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 C1orf59 gene based on its translation product, the intensity of staining may be observed via immunohistochemical analysis using an antibody against C1orf59 protein. Namely, the observation of strong staining indicates increased presence of the C1orf59 protein and at the same time high expression level of the C1orf59 gene.

Furthermore, the C1orf59 protein is known to have a cell proliferating activity. Therefore, the expression level of the C1orf59 gene can be determined using such cell proliferating activity as an index. For example, cells that express C1orf59 are prepared and cultured in the presence of a biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
Moreover, in addition to the expression level of the C1orf59 gene, the expression level of other esophageal, cervical, colon, bile duct and/or lung cancer-associated genes, for example, genes known to be differentially expressed in esophageal, cervical, colon, bile duct and/or lung cancer may also be determined to improve the accuracy of the assessment. Examples of such other esophageal, cervical, colon, bile duct and/or lung cell-associated genes include those described in WO 2004/031413 and WO 2005/090603, the contents of which are incorporated by reference herein.
Alternatively, according to the present invention, an intermediate result may also be provided in addition to other test results for assessing the prognosis of a subject. Such intermediate result may assist a doctor, nurse, or other practitioner to assess, determine, monitor or estimate the progress and/or prognosis of a subject. Additional information that may be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.

In other words, the expression level of the C1orf59 gene is useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from esophageal cancer. Therefore, the present invention also provides a method for detecting prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from esophageal cancer, which includes steps of:
a) detecting or determining an expression level of a C1orf59 gene in a subject-derived biological sample, and
b) correlating the expression level detected or determined in step a) with the prognosis of the subject.

In particular, according to the present invention, an increased expression level as compared to the control level is indicative of potential or suspicion of poor prognosis (poor survival).
The patient to be assessed for the prognosis of cancer according to the method is preferably a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.

A Kit For Diagnosing Cancer, Assessing The Prognosis Of Cancer And/Or Monitoring The Efficacy Of A Cancer Therapy:
The present invention provides a kit for diagnosing cancer, assessing the prognosis of cancer, and/or monitoring the efficacy of a cancer therapy. Preferably, the cancer is esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Specifically, the kit includes at least one reagent for detecting the expression of the C1orf59, PIWIL4, piR1 and/or piR2 in a patient-derived biological sample, which reagent may be selected from the group of:
(a) a reagent for detecting mRNA of the C1orf59 or PIWIL4 gene;
(b) a reagent for detecting the C1orf59 or PIWIL4 protein;
(c) a reagent for detecting the biological activity of the C1orf59 or PIWIL4 protein; and
(d) a reagent for detecting the piR1 and/or piR2.

Suitable reagents for detecting mRNA of the C1orf59 or PIWIL4 gene include nucleic acids that specifically bind to or identify the C1orf59 or PIWIL4 mRNA, such as oligonucleotides that have a complementary sequence to a part of the C1orf59 or PIWIL4 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the C1orf59 or PIWIL4 mRNA. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the C1orf59 or PIWIL4 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the C1orf59 or PIWIL4 mRNA may be included in the kit.

A probe or primer of the present invention is typically a substantially purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under stringent conditions to at least about 2000, 1000, 500, 400, 350, 300, 250, 200, 150, 100, 50, or 25, consecutive sense strand nucleotide sequence of a nucleic acid having a C1orf59 or PIWIL4sequence, or an anti sense strand nucleotide sequence of a nucleic acid having a C1orf59 or PIWIL4 sequence, or of a naturally occurring mutant of these sequences. In particular, for example, in a preferred embodiment, an oligonucleotide having 5-50bp in length can be used as a primer for amplifying the genes, to be detected. More preferably, mRNA or cDNA of a C1orf59 or PIWIL4 gene can be detected with oligonucleotide probe or primer having 15- 30bp in length. In preferred embodiments, length of the oligonucleotide probe or primer can be selected from 15-25bp. Assay procedures, devices, or reagents for the detection of gene by using such oligonucleotide probe or primer are well known (e.g. oligonucleotide microarray or PCR). In these assays, probes or primers can also contain tag or linker sequences. Further, probes or primers can be modified with detectable label or affinity ligand to be captured. Alternatively, in hybridization based detection procedures, a polynucleotide having a few hundreds (e.g., about 100-200) bases to a few kilo (e.g., about 1000-2000) bases in length can also be used for a probe (e.g., northern blotting assay or cDNA microarray analysis).

On the other hand, suitable reagents for detecting the C1orf59 or PIWIL4 protein include antibodies to the C1orf59 or PIWIL4 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 as the reagent, so long as the fragment retains the binding ability to the C1orf59 or PIWIL4 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. Furthermore, the antibody may be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods may be employed for the present invention. Moreover, more than one reagent for detecting the C1orf59 or PIWIL4 protein may be included in the kit.

Furthermore, the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed C1orf59 or PIWIL4 protein in the biological sample. For example, the cell may be cultured in the presence of a patient-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined. If needed, the reagent for detecting the C1orf59 or PIWIL4 mRNA may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the biological activity of the C1orf59 or PIWIL4 protein may be included in the kit.

Suitable reagents for detecting piR1 and/or piR2 include nucleic acids that specifically bind to or identify the small RNAs, such as oligonucleotides that have a complementary sequence to a part of the piR1 and/or piR2. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the piR1 and/or piR2. These kinds of oligonucleotides may be prepared based on methods well known in the art. If needed, the reagent for detecting the piR1 and/or piR2 may be immobilized on a solid matrix. Moreover, more than one reagent for detecting the piR1 and/or piR2 may be included in the kit.

The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix and reagents for binding probes against the C1orf59, PIWIL4, piR1 and/or piR2 or antibodies against the C1orf59 and/or PIWIL4 protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the C1orf59 or PIWIL4. For example, tissue samples obtained from patient with good prognosis or poor prognosis may serve as useful control reagents. A kit of the present invention may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use. 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.

As an embodiment of the present invention, when the reagent is a probe against the C1orf59 and/or PIWIL4 mRNA, or piR1 and/or piR2, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site. The measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe). A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a strip separated from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of C1orf59 and/or PIWIL4 mRNA, or piR1 and/or piR2 present 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 a positive control sample or C1orf59, PIWIL4, piR1 and/or piR2 standard sample. The positive control sample of the present invention may be prepared by collecting C1orf59, PIWIL4, piR1 and/or piR2 positive cancer tissue samples and then those C1orf59, PIWIL4, piR1 and/or piR2 level are assayed.

Screening For An Anti-Cancer Substance:
In the context of the present invention, substances to be identified through the present screening methods include any substance or composition including several substances. Furthermore, the test substance exposed to a cell or protein according to the screening methods of the present invention may be a single substance or a combination of substances. When a combination of substances is used in the methods, the substances may be contacted sequentially or simultaneously.

Any test substance, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide substances, synthetic micromolecular substances (including nucleic acid constructs, such as antisense RNA, siRNA, Ribozymes, and aptamer etc.) and natural substances can be used in the screening methods of the present invention. 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-substance" 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 substances (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 substances 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).
A substance in which a part of the structure of the substance screened by any one 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 is confirmed it's usefulness in preparing the test substance which is a candidate for treating or preventing cancer.
Test substances useful in the screenings described herein can also be antibodies that specifically bind to C1orf59 and/or PIWIL4 protein or partial peptides thereof that lack the biological activity of the original proteins in vivo.
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.

(i) Molecular modeling:
Construction of test substance libraries is facilitated by knowledge of the molecular structure of substances known to have the properties sought, and/or the molecular structure of C1orf59 or PIWIL4. 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 substances 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 substance will link to the target molecule and allow experimental manipulation of the structures of the substance and target molecule to perfect binding specificity. Prediction of what the molecule-substance 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 wherein the esophageal, cervical, colon, bile duct and/or lung cancer.

(ii) 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 chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., 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 substance 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 substances, 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.).

(iii) 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)) discloses 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.

Screening For A C1orf59 Or A PIWIL4 Binding Substance:
In context of the present invention, over-expression of C1orf59 or PIWIL4 was detected in esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer, in spite of no expression in normal organs (Fig. 1, 2, 7 and 8). Accordingly, using the C1orf59 or PIWIL4 genes, proteins encoded by the genes, the present invention provides a method of screening for a substance that binds to C1orf59 or PIWIL4. Due to the expression of C1orf59 or PIWIL4 in esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer, a substance binds to C1orf59 or PIWIL4 is expected to suppress the proliferation of cancer cells, and thus be useful for treating or preventing cancer, wherein the cancer is esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer. Therefore, the present invention also provides a method of screening for a substance that suppresses the proliferation of cancer cells, and a method of screening for a substance for treating or preventing cancer using the C1orf59 or PIWIL4 polypeptide, particularly wherein the cancer is esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer. One particular embodiment of this screening method includes the steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of (corresponding to) the C1orf59 gene or the PIWIL4 gene (i.e., C1orf59 polypeptide or PIWIL4 polypeptide);
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) selecting the test substance that binds to the polypeptide.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance or compound 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 C1orf59 or PIWIL4, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene or the PIWIL4 gene;
(b) detecting the binding activity between the polypeptide 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 C1orf59 or PIWIL4 protein(s). For example, when the test substance binds to a C1orf59 or PIWIL4 protein, the test substance may identified or selected as a candidate substance having the requisite therapeutic effect. Alternatively, when the test substance does not binds to C1orf59 or PIWIL4 proteins, the test substance may identified as the substance having no significant therapeutic effect.

The method of the present invention is described in more detail below.
The C1orf59 or PIWIL4 polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof. The polypeptide to be contacted with a test substance can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.

As a method of screening for proteins, for example, that bind to the C1orf59 or PIWIL4 polypeptide using the C1orf59 or PIWIL4 polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted using, for example, the immunoprecipitation method, specifically, in the following manner. The gene encoding the C1orf59 or PIWIL4 polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to 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 (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 (1989)), the HSV TK promoter and so on.

The introduction of the gene into host cells to express a foreign gene can be performed according to any methods, 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 polypeptide encoded by the C1orf59 or PIWIL4 gene can 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 13: 85-90 (1995)). Vectors that can express 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 comprised of several to a dozen amino acids so as not to change the property of the C1orf59 or PIWIL4 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 monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the C1orf59 or PIWIL4 polypeptide (Experimental Medicine 13: 85-90 (1995)).

In the context of immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex includes the C1orf59 or PIWIL4 polypeptide, a polypeptide including the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the C1orf59 or PIWIL4 polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above. An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody. If the polypeptide encoded by C1orf59 or PIWIL4 gene is prepared as a fusion protein with an epitope, such as GST, an immune complex can be formed in the same manner as in the use of the antibody against the C1orf59 or PIWIL4 polypeptide, using a substance specifically binding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
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. Since the protein bound to the C1orf59 or PIWIL4 polypeptide is difficult to detect by a common staining method, such as Coomassie staining or silver staining, 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.

West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used as a method of screening for proteins binding to the C1orf59 or PIWIL4 polypeptide using the polypeptide. In particular, a protein binding to the C1orf59 or PIWIL4 polypeptide can be obtained by preparing a cDNA library from cultured cells expected to express a protein binding to the C1orf59 or PIWIL4 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled C1orf59 or PIWIL4 polypeptide with the above filter, and detecting the plaques expressing proteins bound to the C1orf59 or PIWIL4 polypeptide according to the label. The polypeptide of the invention may be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to the C1orf59 or PIWIL4, or a peptide or polypeptide (for example, GST) that is fused to the C1orf59 or PIWIL4 polypeptide. Methods using radioisotope or fluorescence and such may be also used.

Alternatively, in another embodiment of the screening method of the present invention, a 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 and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").

In the two-hybrid system, a polypeptide of the invention 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 a protein binding to the polypeptide of the invention, 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 polypeptide of the invention is expressed in 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. Examples of suitable reporter genes include, but are not limited to, the Ade2 gene, lacZ gene, CAT gene, luciferase gene and such as can be used in addition to the HIS3 gene.

A substance binding to the polypeptide encoded by C1orf59 or PIWIL4 gene can also be screened using affinity chromatography. For example, the polypeptide of the invention may be immobilized on a carrier of an affinity column, and a test substance, containing a protein capable of binding to the polypeptide of the invention, is applied to the column. A test substance herein may be, for example, cell extracts, cell lysates, etc. After loading the test substance, the column is washed, and substances bound to the polypeptide of the invention can be prepared. When the test substance is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.

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 polypeptide of the invention and a test substance can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test substance using a biosensor such as BIAcore.

The methods of screening for molecules that bind when the immobilized C1orf59 or PIWIL4 polypeptide is exposed to synthetic chemical substances, or natural substance banks or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but chemical substances that bind to the C1orf59 or PIWIL4 protein (including agonist and antagonist) are well known to those skilled in the art.

Screening For A Substance That Suppresses The Biological Activity Of C1orf59 Or PIWIL4:
In the context of the present invention, the C1orf59 or PIWIL4 protein is characterized as having the activity of promoting proliferation in esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer cells. Using this biological activity as an index, the present invention provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method of screening for a substance for treating or preventing cancer, particularly wherein the cancer is esophageal cancer, cervical cancer, colon cancer, bile duct cancer and/or lung cancer. Thus, the present invention provides a method of screening for a substance for treating or preventing a C1orf59- and/or PIWIL4 -associated cancer, the method including the steps as follows:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of (corresponding to) the C1orf59 gene or the PIWIL4 gene;
(b) detecting the biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide encoded by the polynucleotide of C1orf59 or PIWIL4 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.

According to the present invention, the therapeutic effect of the test substance on suppressing the activity to promote cell proliferation, or a candidate substance for treating or preventing a C1orf59- and/or PIWIL4 -associated cancer (e.g., esophageal, cervical, colon, bile duct and/or lung cancers) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing a C1orf59- and/or PIWIL4 -associated cancer, using the C1orf59 or PIWIL4 polypeptide or fragments thereof including the steps as follows:
(a) contacting a test substance with the C1orf59 or PIWIL4 polypeptide or a functional fragment thereof; and
(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 C1orf59 and/or PIWIL4, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of (corresponding to) the C1orf59 gene or the PIWIL4 gene;
(b) detecting the biological activity of the polypeptide 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 C1orf59 or PIWIL4 gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.

In the context of present invention, the therapeutic effect may be correlated with the biological activity of a C1orf59 or PIWIL4 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of a C1orf59 or PIWIL4 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 an C1orf59 or PIWIL4 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.

The method of the present invention is described in more detail below.

Any polypeptides can be used for screening so long as they retain a biological activity of a C1orf59 or PIWIL4 protein. Examples of such biological activity includes cell-proliferating activity of the C1orf59 or PIWIL4 protein. For example, C1orf59 or PIWIL4 protein can be used and polypeptides functionally equivalent to these proteins can also be used. Such polypeptides may be expressed endogenously or exogenously by cells.

The substance isolated by this screening is a candidate for antagonists of the polypeptide encoded by C1orf59 or PIWIL4 gene. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. This term also refers to molecules that reduce or inhibit expression of the gene encoding C1orf59 or PIWIL4. Moreover, a substance isolated by this screening is a candidate for substances that inhibit the in vivo interaction of the C1orf59 or PIWIL4 polypeptide with molecules (including DNAs and proteins).

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 C1orf59 or PIWIL4 polypeptide, 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 survival cells or the colony forming activity. The substances that reduce the speed of proliferation of the cells expressed C1orf59 or PIWIL4 are selected as candidate substance for treating or preventing esophageal, cervical, colon, bile duct and/or lung cancer.

More specifically, the method includes the step of:
(a) contacting a test substance with cells expressing C1orf59 or PIWIL4;
(b) measuring cell-proliferating activity; and
(c) selecting the test substance that reduces the cell-proliferating activity in the comparison with the cell-proliferating activity in the absence of the test substance.
In preferable embodiments, the method of the present invention may further include the steps of:
(d) selecting the test substance that have no effect to the cells no or little expressing C1orf59 or PIWIL4.

Furthermore, C1orf59 protein has the methylation activity of piR1 (Fig. 4), and the protein has the activity of promoting the expression level of piR1 and piR2 (Fig. 4). Using these biological activities, the present invention provides a method for screening a substance that suppresses the proliferation of cancer cells associated with the overexpression of C1orf59 gene, and a method for screening a candidate substance for treating or preventing such cancer. Thus, the present invention provides a method including the steps as follows:
(a) contacting a test substance with a polypeptide derived from C1orf59 gene (i.e., C1orf59 polypeptide);
(b) detecting the biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity as compared to the biological activity in the absence of the test substance as 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 C1orf59 associated cancer 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 C1orf59 associated cancer, using the C1orf59 polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with the C1orf59 polypeptide or a functional fragment thereof; and
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.

In the present invention, the therapeutic effect may be correlated with the biological activity of C1orf59 polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of C1orf59 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 C1orf59 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test substance, the test substance may be identified as the substance having no significant therapeutic effect.

The screening methods of the present invention are described in more detail below.
Any polypeptides can be used for screening so long as they retain the biological activity of C1orf59 protein. Such biological activity includes piRNA (e.g., piR1) methylation activity and the activity of promoting the piRNAs (e.g., piR1 and piR2) expression. For example, C1orf59 protein can be used and polypeptides functionally equivalent to C1orf59 protein can also be used. Such polypeptides may be expressed endogenously or exogenously by cells.

In another aspect, the present invention also provides a screening method following the method described in "Screening For A C1orf59 OR PIWIL4 Binding Substance", including the steps of:
a) contacting a test substance with C1orf59 or PIWIL4 polypeptide ;
b) detecting the binding between the polypeptide and the test substance;
c) selecting the test substance that binds to the polypeptide;
d) contacting the test substance selected in step c) with C1orf59 or PIWLIL4 polypeptide;
e) comparing the biological activity of the polypeptide with the biological activity detected in the absence of the test substance; and
f) selecting the test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.

The substances isolated by this screening are candidates for antagonists of the polypeptide encoded by C1orf59 or PIWLIL4 gene. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. Said term also refers to molecules that reduce or inhibit the expression of C1orf59 or PIWLIL4 gene. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of C1orf59 or PIWLIL4 polypeptide with molecules (including DNAs and proteins).

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 C1orf59 and/or PIWLIL4 polypeptide, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring survival cells or the colony forming activity. The compounds that reduce the speed of proliferation of the cells expressed C1orf59 and/or PIWLIL4 are selected as candidate compound for treating or preventing cancer.

More specifically, the method includes the step of:
(a) contacting a test compound with cells overexpressing C1orf59 and/or PIWLIL4;
(b) measuring cell-proliferating activity; and
(c) selecting the test compound that reduces the cell-proliferating activity in the comparison with the cell-proliferating activity in the absence of the test compound.

In preferable embodiments, the method of the present invention may further include the steps of:
(d) selecting the test compound that have no effect to the cells no or little expressing C1orf59 and/or PIWLIL4.

When the biological activity to be detected in the present method is anti-apoptosis, it can be determined by usual methods performed by those skilled in the art such as measuring the number of sub-G1 cells, TUNEL method or LM-PCR method using various commercially available kits. For example, the number of sub-G1 cells can be determined by using FACS. Apoptosis can be also examined by TUNEL method using Apotag Direct (oncor) or LM-PCR using an ApoAlert LM-PCR ladder assay kit (Clontech) according to the attached manual.

When the polypeptide is C1orf59 polypeptide and the biological activity to be detected in the present method is the methylation activity, it can be determined by contacting a C1orf59 polypeptide with a substrate (e.g., piRNAs) under a suitable condition for methylation of the substrate and detecting the methylation level of the substrate.

More specifically, the method includes the steps of:
(a) contacting a C1orf59 polypeptide with a substrate to be methylated in the presence of the test substance under the condition capable of methylation of substrate.
(b) detecting the methylation level of the substrate; and
(c) selecting the test substance that decreases or reduces the methylation level of the substrate as compared to the methylation level detected in the absence of the test substance as the candidate substance.

Alternatively, in some embodiments, the present invention also 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 C1orf59, the method including steps of:
(a) contacting a C1orf59 polypeptide with a substrate to be methylated in the presence of the test substance under the condition capable of methylation of substrate
(b) detecting the methylation level of the substrate; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance decreases the methylation level of the substrate as compared to the methylation level detected in the absence of the test substance as the candidate substance.
Preferably, a substrate to be methylated by a C1orf59 polypeptide is a piRNA such as piR1 having the ribonucleotide sequence shown in SEQ ID NO: 9.

In the present invention, the methylation activity of a C1orf59 polypeptide can be determined by methods known in the art. For example, the C1orf59 polypeptide and a substrate can be incubated with a labeled methyl donor, under suitable assay conditions. A piRNA such as piR1, and S-adenosyl-[methyl-¹⁴C]-L-methionine or S-adenosyl-[methyl-³H]-L-methionine preferably can be used as a substrate and a methyl donor, respectively. Transfer of the radiolabel to the piRNA can be detected, for example, by polyacrylamide gel electrophoresis (PAGE), denaturing polyacrylamide gel electrophoresis (DPAGE) or fluorography following isolation of the piRNA form the reaction mixture. piRNA can be isolated, for example, by phenol extraction and ethanol precipitation. Alternatively, following the reaction, the substrate can be separated from the methyl donor by filtration, and the amount of radiolabel retained on the filter quantitated by scintillation counting. Other suitable labels that can be attached to methyl donors, such as chromogenic and fluorescent labels, and methods of detecting transfer of these labels to a substrate, are known in the art.

Alternatively, methylation activity of a C1orf59 polypeptide may be determined using a mass spectrometry or reagents that selectively recognize a methylated substrate.

Alternatively, when the biological activity to be detected is the activity of promoting piRNAs (e.g., piR1 or piR2 (a piRNA having the ribonucleotide sequence shown in SEQ ID NO: 10 )) expression level, it can be detected, for example, by shown in Fig. 4. For this method, a test compound is contacted with cells expressing C1orf59 gene, such as cancer cells.
More specifically, the method includes the steps of:
(a) contacting a test substance with cells expressing C1orf59 gene;
(b) measuring the expression level of piR1 or piR2 ; and
(c) selecting the test substance that reduces the expression level of piR1 or piR2 as compared to the expression level in the absence of the test substance as a candidate substance.

Alternatively, in some embodiments, the present invention also 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 C1orf59, the method including steps of:
(a) contacting a test substance with a cell expressing C1orf59; and
(b) detecting the expresstion level of piRNA (e.g. piR1 or piR2) 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 of piRNA (e.g. piR1 or piR2) in comparison with the expression level detected in the absence of the test substance.

Cells expressing C1orf59 gene include, for example, cell lines established from cancer (e.g., TE1, TE2, TE3, TE4, TE5, TE6, TE7, TE8, TE9, TE10 and TE11 for esophageal cancer), and purified cells from clinical cancer tissues, such cells can be used for the present screening method.

Measurement of he expression level of piR1 or piR2 can be carried out by methods described in "Method Of Detecting Or Diagnosing Cancer"
In the present invention, methods for preparing polypeptides functionally equivalent to a given protein are well-known by a person skilled in the art and include known methods of introducing mutations into the protein. Generally, it is known that modifications of one or more amino acid in a protein do not influence the function of the protein (Mark DF et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller MJ & Smith M, Nucleic Acids Res 1982, 10: 6487-500; Wang A et al., Science 1984, 224:1431-3; Dalbadie-McFarland G et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). In fact, mutated or modified proteins, proteins having amino acid sequences modified by substituting, deleting, inserting, and/or adding one or more amino acid residues of a certain amino acid sequence, 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 which alter a single amino acid or a small percentage of amino acids, or those considered to be "conservative modifications", wherein the alteration of a protein results in a protein with similar functions, are contemplated in the context of the instant invention.

In the present invention, it is revealed that suppressing the expression of C1orf59 or PIWIL4, reduces cell growth. Thus, by screening for candidate compounds that inhibits the biological activity of the C1orf59 or PIWIL4 polypeptide, candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic substance for cancers. For example, when a compound binding to C1orf59 or PIWIL4 protein inhibits described above activities of the cancer, it may be concluded that such compound has the C1orf59 or PIWIL4 specific therapeutic effect.

In the preferred embodiments, control cells which do not overexpress C1orf59 and/or PIWIL4 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 C1orf59 and/or PIWIL4 associating cancer, using the C1orf59 or PIWIL4 polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express a C1orf59 and/or PIWIL4 polypeptide or a functional fragment thereof, and control cells that do not express a C1orf59 and/or PIWIL4 polypeptide or a functional fragment thereof in the presence of the test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of said test substance.
In the context of the instant invention, the phrase "suppress the biological activity" encompasses at least 10% suppression of the biological activity of C1orf59 or PIWIL4 in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at least 90% suppression.

Screening For A Substance Altering The Expression Of C1orf59 Or PIWIL4:
In the present invention, a decrease in the expression of C1orf59 or PIWIL4 by siRNA results in the inhibition of cancer cell proliferation. Accordingly, the present invention provides a method of screening for a substance that inhibits the expression of C1orf59 or PIWIL4. A substance that inhibits the expression of C1orf59 or PIWIL4 is expected to suppress the proliferation of cancer cells, and thus is useful for treating or preventing cancer relating to C1orf59 and/or PIWIL4 overexpression. Examples of cancers relating to C1orf59 and/or PIWIL4 include esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer. Therefore, the present invention also provides a method for screening a substance that suppresses the proliferation of cancer cells, and a method for screening a substance for treating or preventing cancer relating to C1orf59 or PIWIL4 overexpression. 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 C1orf59 or PIWIL4; and
(b) selecting the test substance that reduces the expression level of C1orf59 or PIWIL4 as compared to a control.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cells growth in a cancer associated with over-expression of C1orf59 or PIWIL4, the method including steps of:
(a) contacting a candidate substance with a cell expressing C1orf59 or PIWIL4; and;
(b) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a substance reduces the expression level of C1orf59 or PIWIL4 as compared to a control.

The method of the present invention are described in more detail below.

Cells expressing the C1orf59 or PIWIL4 include, for example, cell lines established from esophageal, cervical, colon, bile duct and/or lung cancer cells or cell lines transfected with C1orf59 or PIWIL4 expression vectors; any of such cells can be used for the above screening of the present invention. The expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining and flow cytometry analysis. "Reduce the expression level" as defined herein are preferably at least 10% reduction of expression level of C1orf59 or PIWIL4 in comparison to the expression level in absence of the substance, more preferably at least 25%, 50% or 75% reduced level and most preferably at least 95% reduced level. The substance herein includes chemical substance, double-strand nucleotide, and so on. The preparation of the double-strand nucleotide is in aforementioned description. In the method of screening, a substance that reduces the expression level of C1orf59 or PIWIL4 can be selected as candidate substances to be used for the treatment or prevention of esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer.

Alternatively, the screening method of the present invention may include the following steps:
(a) contacting a candidate substance with a cell into which a vector, including the transcriptional regulatory region of C1orf59 or PIWIL4 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the candidate substance that reduces the expression or activity of said reporter gene.

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 a C1orf59- and/or PIWIL4- associated 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 a C1orf59 and/or PIWIL4 associated disease.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cells growth in a cancer associated with over-expression of C1orf59 and/or PIWIL4, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of C1orf59 or PIWIL4 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said 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 or activity of said reporter gene.

In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test substance with a cell into which a vector, composed of the transcriptional regulatory region of the C1orf59 or PIWIL4 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 said reporter gene; and
c) correlating the expression level 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 expression or activity of said reporter gene. For example, when the test substance reduces the expression or activity of said 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 said 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.

Suitable reporter genes and host cells are well known in the art. Illustrative reporter genes include, but are not limited to, luciferase, green fluorescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chlorolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of C1orf59 or PIWIL4. The transcriptional regulatory region of C1orf59 or PIWIL4 herein includes the region from transcriptional start site to at least 500bp upstream, preferably 1000bp, more preferably 5000 or 10000bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of any one of these genes. 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).

The vector containing the said reporter construct is 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, absorption spectrometer, flow cytometer and so on). "reduces the expression or activity" as defined herein are preferably at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% reduction and most preferably at least 95% reduction.

Screening Using The Binding Of C1orf59 And PIWIL4 Or SAM As An Index:
In the present invention, the direct interaction of C1orf59 with the PIWIL4 protein was shown by pull-down assay (Fig.6A). On the other hand, a mutant recombinant C1orf59 protein, which was mutated in SAM binging motif, lost the growth promotive effect (Fig. 5D). This result demonstrates that the binding of between C1orf59 and SAM is important for the growth promoting activity of C1orf59. Accordingly, the present invention provides a method of screening for a substance that inhibits the binding between C1orf59 and PIWIL4 or SAM.

Substances that inhibit the binding between a C1orf59 protein and PIWIL4 protein or SAM can be screened by detecting a binding level between a C1orf59 protein and PIWIL4 protein or SAM as an index. Therefore, the present invention provides a method for screening a substance for inhibiting the binding between a C1orf59 protein and PIWIL4 protein or SAM using a binding level between a C1orf59 protein and PIWIL4 protein or SAM as an index. Substances that inhibit binding between a C1orf59 protein and PIWIL4 protein or SAM are expected to be suppressing cancer cell proliferation. Accordingly, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a growth of cancer cells expressing the C1orf59 gene, e.g., esophageal cancer cell, cervical cancer cell, colon cancer cell, bile duct cancer cell or lung cancer cell, and therefore, a candidate substance for treating or preventing cancers, e.g. esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Further, substances obtained by the present screening method may be also useful for inhibiting cellular invasion.

Of particular interest to the present invention are the following methods of [1] to [9]:
[1] A method of screening for a substance that interrupts a binding between a C1orf59 polypeptide and a PIWIL4 polypeptide, said method including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with a PIWIL4 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[2] A method of screening for a substance useful in connection with the treatment or prevention of cancer or a post-operative recurrence thereof, or capable of inhibiting cancer cell growth, said method including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with a PIWIL4 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[3] The method of [1] or [2], wherein the functional equivalent of C1orf59 polypeptide includes the PIWIL4 binding domain of C1orf59 polypeptide;
[4] The method of [1] or [2], wherein the functional equivalent of PIWIL4 polypeptide includes the C1orf59-binding domain of PIWIL4 polypeptide;
[5] A method of screening for a substance that interrupts a binding between a C1orf59 polypeptide and SAM, said method including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with SAM in the presence of a test substance;
(b) detecting a binding level between the polypeptide and SAM;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[6] A method of screening for a substance useful in connection with the treatment or prevention of cancer or a post-operative recurrence thereof, or capable of inhibiting cancer cell growth, said method including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with SAM in the presence of a test substance;
(b) detecting a binding level between the polypeptide and SAM;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level;
[7] The method of [5] or [6], wherein the functional equivalent of C1orf59 polypeptide includes the SAM binding domain of C1orf59 polypeptide;
[8] The method of [7], wherein the SAM binding domain includes the amino acid sequence from the 53th amino acid residue to the 57th amino acid reside of SEQ ID NO: 2; and
[9] The method of any one of [1] to [8], wherein the cancer is selected from the group consisting of esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer, or inhibiting cancer cell growth , the method including steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with a PIWIL4 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduce the binding level.

Also, 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 cell growth , the method including steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with SAM in the presence of a test substance;
(b) detecting a binding level between the polypeptide and SAM;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance reduce the binding level.

Further, in another embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer, or f inhibiting cancer cell growth , the method including steps of:
(a) contacting a polypeptide having a PIWIL4-binding domain of a C1orf59 polypeptide with a polypeptide having a C1orf59-binding domain of a PIWIL4 polypeptide in the presence of a test substance;
(b) detecting binding between the polypeptides; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance inhibits binding between the polypeptides.

In another embodiments, the present invention further provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer, or capable of inhibiting cancer cell growth, the method including steps of:
(a) contacting a polypeptide having a SAM-binding domain of a C1orf59 polypeptide with SAM in the presence of a test substance;
(b) detecting binding between the polypeptide and SAM; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance inhibits binding between the polypeptides.

In the context of the present invention, functional equivalents of a C1orf59 and PIWIL4 polypeptide are polypeptides that have a biological activity equivalent to a C1orf59 polypeptide (SEQ ID NO: 2), PIWIL4 (SEQ ID NO: 4) polypeptide, respectively. Particularly, the functional equivalent of C1orf59 polypeptide is a polypeptide fragment of C1orf59 polypeptide containing the binding domain to PIWIL4 polypeptide or SAM. Similarly, the functional equivalent of PIWIL4 polypeptide is a polypeptide fragment of PIWIL4 polypeptide containing the C1orf59-binding domain.

Those of skill in the art will recognize that any of a number of standard methods may be used to screen for substances that inhibit the binding of C1orf59 polypeptide to PIWIL4 polypeptide or SAM.
Any polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof so long as they retain the aforementioned binding activity. Any test substance aforementioned can be used for screening.

Likewise, those of skill in the art will readily recognize that a number of conventional methods may be used to detect the binding between a C1orf59 protein and PIWIL4 protein or SAM. Examples of such methods include, for example, immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography and a biosensor using the surface plasmon resonance phenomenon.

In some embodiments, the present screening method may be carried out in a cell-based assay using cells expressing both of a C1orf59 protein and a PIWIL4 protein or SAM. Cells expressing C1orf59 protein and PIWIL4 protein or SAM include, for example, cell lines established from cancer cells, e.g. esophageal cancer cells, cervical cancer cells, colon cancer cells, bile duct cancer cells or lung cancer cells. Alternatively, the cells may be prepared by transforming cells with nucleotides encoding C1orf59 gene and PIWIL4 gene. Such transformation may be carried out using an expression vector encoding both C1orf59 gene and PIWIL4 gene, or expression vectors encoding either C1orf59 gene or PIWIL4 gene. The present screening can be conducted by incubating such cells in the presence of a test substance. The binding of C1orf59 protein to PIWIL4 protein can be detected by immunoprecipitation assay using an anti-C1orf59 antibody or anti- PIWIL4 antibody.

According to the present invention, the therapeutic effect of a candidate substance on inhibiting the cell growth or a candidate substance suitable for use in connection with the treatment or prevention of cancer associated with C1orf59 (e.g., esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance suitable for use in connection with the treatment or prevention of cancer (e.g., esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer), using a C1orf59 polypeptide or functional equivalent thereof including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with a PIWIL4 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the binding level of (c) with the therapeutic effect of the test substance.

Also, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance suitable for use in connection with the treatment or prevention of cancer (e.g., lung and esophageal cancers), using a C1orf59 polypeptide or functional equivalent thereof including the steps of:
(a) contacting a C1orf59 polypeptide or functional equivalent thereof with SAM in the presence of a test substance;
(b) detecting a binding level between the polypeptide and SAM;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the binding level of (c) with the therapeutic effect of the test substance.

In the present invention, the therapeutic effect may be correlated with the binding level between a C1orf59 polypeptide and a PIWIL4 polypeptide or SAM. For example, when the test substance suppresses the binding level between the polypeptides 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 binding level between the polypeptides 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.

Screening Using The Binding Of PIWIL4 And CBX5, SUV39H1 Or SUV39H2 As An Index:
In the context of the present invention, the direct interaction of PIWIL4 with CBX5, SUV39H1 or SUV39H2 protein was shown by pull-down assay (Fig.6B). Accordingly, the present invention provides a method of screening for a substance that inhibits the binding between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein.

Substances that inhibit the binding between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein can be screened by detecting a binding level between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein as an index. Therefore, the present invention provides a method for screening a substance for inhibiting the binding between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein using a binding level between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein as an index. Substances that inhibit binding between PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein are expected to be suppressing cancer cell proliferation. Accordingly, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a growth of cancer cells expressing PIWIL4 gene, e.g., esophageal cancer cell, cervical cancer cell, colon cancer cell, bile duct cancer cell or lung cancer cell, and therefore, a candidate substance for treating or preventing cancers, e.g. esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer. Further, substances obtained by the present screening method may be also useful for inhibiting cellular invasion.

Of particular interest to the present invention are the following methods of [1] to [5]:
[1] A method of screening for a substance that interrupts a binding between a PIWIL4 polypeptide and a CBX5, SUV39H1 or SUV39H2 polypeptide, said method including the steps of:
(a) contacting a PIWIL4 polypeptide or functional equivalent thereof with a CBX5, SUV39H1 or SUV39H2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduce the binding level.
[2] A method of screening for an substance useful in treating or preventing cancers, or inhibiting cancer cell growth, said method including the steps of:
(a) contacting a PIWIL4 polypeptide or functional equivalent thereof with a CBX5, SUV39H1 or SUV39H2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) selecting the test substance that reduces the binding level.
[3] The method of [1] or [2], wherein the functional equivalent of PIWIL4 polypeptide including the CBX5, SUV39H1 or SUV39H2 binding domain of the PIWIL4 polypeptide.
[4] The method of [1] or [2], wherein the functional equivalent of CBX5, SUV39H1 or SUV39H2 polypeptide including the PIWIL4-binding domain of the CBX5, SUV39H1 or SUV39H2 polypeptide.
[5] The method of [1], wherein the cancer is selected from the group consisting of esophageal cancer, cervical cancer, colon cancer, bile duct cancer and lung cancer.
In the context of the present invention, functional equivalents of a PIWIL4 and CBX5, SUV39H1 or SUV39H2 polypeptide are polypeptides that have a biological activity equivalent to a PIWIL4 polypeptide (SEQ ID NO: 4), CBX5 (SEQ ID NO: 25), SUV39H1 (SEQ ID NO: 27) or SUV39H2 (SEQ ID NO: 29) polypeptide, respectively. In preferred embodiments, functional equivalents of those polypeptide retain the aforementioned binding activity. Particularly, the functional equivalent of PIWIL4 is a polypeptide fragment of PIWIL4 polypeptide containing the binding domain to CBX5, SUV39H1 or SUV39H2 polypeptide. Similarly, the functional equivalent of CBX5, SUV39H1 or SUV39H2 polypeptide is a polypeptide fragment of CBX5, SUV39H1 or SUV39H2 polypeptide containing the PIWIL4-binding domain.

As a method of screening for substances that inhibits, the binding of PIWIL4 protein to CBX5, SUV39H1 or SUV39H2 protein, many methods well known by one skilled in the art can be used.
A polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. Any test substance aforementioned can be used for screening.

As a method of detecting the binding between a PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein, any methods well known by a person skilled in the art can be used. Such a detection can be conducted using, for example, an immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography and a biosensor using the surface plasmon resonance phenomenon.

In some embodiments, the present screening method may be carried out in a cell-based assay using cells expressing both of a PIWIL4 protein and a CBX5, SUV39H1 or SUV39H2 protein. Cells expressing PIWIL4 protein and CBX5, SUV39H1 or SUV39H2 protein include, for example, cell lines established from cancer cells, e.g. esophageal cancer cells, cervical cancer cells, colon cancer cells, bile duct cancer cells or lung cancer cells. Alternatively the cells may be prepared by transforming cells with nucleotides encoding PIWIL4 and CBX5, SUV39H1 or SUV39H2 gene. Such transformation may be carried out using an expression vector encoding both PIWIL4 gene and CBX5, SUV39H1 or SUV39H2 gene, or expression vectors encoding either PIWIL4 gene or CBX5, SUV39H1 or SUV39H2 gene. The present screening can be conducted by incubating such cells in the presence of a test substance. The binding of PIWIL4 protein to CBX5, SUV39H1 or SUV39H2 protein can be detected by immunoprecipitation assay using an anti-PIWIL4 antibody or anti- CBX5, SUV39H1 or SUV39H2 antibody.

According to the present invention, the therapeutic effect of a candidate substance on inhibiting the cell growth or a candidate substance for treating or preventing cancer relating to PIWIL4 (e.g., esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer) may be evaluated. Therefore, the present invention also provides a method of screening for a candidate substance for suppressing the cell proliferation, or a candidate substance for treating or preventing cancer (e.g., esophageal cancer, cervical cancer, colon cancer, bile duct cancer or lung cancer), using a PIWIL4 polypeptide or functional equivalent thereof including the steps of:
(a) contacting a PIWIL4 polypeptide or functional equivalent thereof with a CBX5, SUV39H1 or SUV39H2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test substance; and
(d) correlating the binding level of (c) with the therapeutic effect of the test substance;

Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting a cancer associated with over-expression of PIWIL4, the method including steps of:
(a) contacting a polypeptide having a CBX5, SUV39H1 or SUV39H2-binding domain of a PIWIL4 polypeptide with a polypeptide having a PIWIL4 -binding domain of a CBX5, SUV39H1 or SUV39H2 polypeptide in the presence of a test substance;
(b) detecting a binding between the polypeptides; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance inhibits the binding between the polypeptides.

In the present invention, the therapeutic effect may be correlated with the binding level between a PIWIL4 polypeptide and a CBX5, SUV39H1 or SUV39H2 polypeptide. For example, when the test substance suppresses the binding level between the polypeptides 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 binding level between the polypeptides 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.

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.

Materials and Methods
Cell lines and tissue samples.
The human esophageal carcinoma cell lines used in this study were as follows; 10 SCC cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, TE10, and TE11) and one ADC cell line (TE7) (Nishihira T et al. J Cancer Res Clin Oncol 1993;119:441-9.). All cells were grown in monolayer in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37 degrees Cin humidified air with 5% CO₂. Primary ESCC samples had been obtained earlier. This study and the use of all clinical materials mentioned were approved by individual institutional Ethical Committees. Clinical stage was judged according to the UICC TNM classification (Sobin L and Wittekind Ch. 6th ed. New York: Wiley-Liss; 2002.). Formalin-fixed primary 190 ESCCs (stage I-IVA; 19 female and 171 male patients; median age of 62 with a range of 42 - 81 years)and adjacent normal esophageal tissue samples had been obtained from patients undergoing curative surgery at Keiyukai Sapporo Hospital (Sapporo, Japan). And 61 ESCCs (stage I-IVB; 9 female and 52 male patients; median age of 63 with a range of 38 - 82 years)and adjacent normal esophageal tissue samples had also been obtained from patients undergoing curative surgery Hokkaido University and its affiliated hospitals (Sapporo, Japan). This study and the use of all clinical materials were approved by individual institutional ethical committees.

Semiquantitative RT-PCR.
A total of 3 micro g of mRNA aliquot from each sample were reverse transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen). Semiquantitative reverse transcription-PCR (RT-PCR) experiments were carried out with the following sets of synthesized primers specific for human C1orf59, p16 or p21, or with beta-actin (ACTB)-specific primers as an internal control: C1orf59, 5'- CTGAAACCTCGGGATCTGAA-3' (SEQ ID NO: 16) and 5'- TCCCCGACACCAGTAAACTC-3' (SEQ ID NO: 17); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO: 18) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 19); P16, 5'-GTGGACCTGGCTGAGGAG-3' (SEQ ID NO: 30) and 5'-CTTTCAATCGGGGATGTCTG-3' (SEQ ID NO: 31);
P21, 5'-CGAAGTCAGTTCCTTGTGGAG-3' (SEQ ID NO: 32) and
5'-CATGGGTTCTGACGGACAT-3' (SEQ ID NO: 33) . PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.

Northern-blot analysis.
Human multiple-tissue blots (BD Biosciences Clontech, Palo Alto, CA) were hybridized with a ³²P-labeled PCR product of C1orf59. The cDNA probes of C1orf59 were prepared by RT-PCR using the following primers: 5'- AAGCTTACAGCAGGAAAGGTTCT-3' (SEQ ID NO: 20), and 5'- ATATCTCAAGCAGGTCTGTCCAA-3' (SEQ ID NO: 21). Pre-hybridization, hybridization, and washing were performed according to the supplier's recommendations. The blots were autoradiographed at -80 degrees C for 1 week with intensifying screens.

Anti-C1ORF59 antibodies.
Two kinds of synthetic peptides (position: 292-308, 381-393 a.a. of SEQ ID NO:2) were inoculated into rabbits; the immune sera were purified on affinity columns according to standard methods with a synthetic peptide (381-393 a.a.). The affinity-purified anti-C1orf59 polyclonal antibodies were used for Western blotting and immunostaining. It was confirmed that the antibody was specific to C1orf59 on Western blots using lysates from cell lines that had been transfected with C1orf59 expression vector and those from esophageal cancer cell lines, either of which expressed C1orf59 endogenously or not.

Western-blotting.
Tumor cells were lysed in lysis buffer; 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and Protease Inhibitor Cocktail Set III (Calbiochem). The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as a standard. Ten micrograms of each lysate were resolved on 12% denaturing polyacrylamide gels (with 3% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane (GE Healthcare Biosciences). After blocking with 5% nonfat dry milk in TBST, the membrane was incubated with a rabbit polyclonal anti-human C1orf59 antibody (generated to synthetic peptides; please see above) for 1 hour at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences).

Immunocytochemistry.
Cultured cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 3 minutes at room temperature. Cells were covered by CASBLOCK (ZYMED) for 10 minutes at room temperature to block nonspecific binding. Cells were then incubated for 60 minutes at room temperature with primary antibodies diluted in PBS containing 1% BSA. After being washed with PBS, the immunocomplexes were stained with a goat anti-rabbit secondary antibody conjugated to Alexa 488 (Invitrogen). Each specimen was mounted with Vectashield (Vector Laboratories,Inc, Burlingame, CA) containing 4',6'-diamidine-2'-phenylindoldihydrochloride(DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS: Leica Microsystems, Wetzlar, Germany).

Immunohistochemistry and Tissue-microarray analysis.
Tumor tissue microarrays were constructed as published previously, using formalin-fixed ESCCs (Chin SF et al. Mol Pathol 2003;56:275-9., Callagy G et al. Diagn Mol Pathol 2003;12:27-34., Callagy G et al. J Pathol 2005;205:388-96.). Tissue areas for sampling were selected based on visual alignment with the corresponding H&E stained sections on slides. Three, four, or five tissue cores (diameter, 0.6 mm; height, 3-4 mm) taken from donor tumor blocks were placed into recipient paraffin blocks using a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). A core of normal tissue was punched from each case. Five-micrometer sections of the resulting microarray block were used for immunohistochemical analysis. Positivity for C1orf59 was assessed semiquantitatively by three independent investigators without prior knowledge of the clinicopathologic data, each of whom recorded staining positive or negative. Esophageal cancers were decided as positive only if all reviewers defined them as such. To investigate the significance of C1orf59 overexpression in clinical ESCCs, tissue sections were stained using ENVISION+ kit/horseradish peroxidase (HRP; DakoCytomation, Glostrup, Denmark). C1orf59 antibody (please see above) was added after blocking of endogenous peroxidase and proteins, and each section was incubated with HRP-labeled anti-rabbit IgG as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.

Statistical analysis.
It was used contingency tables to analyze the relationship between C1orf59 expression and clinicopathologic variables in ESCC patients. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to ESCC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for C1orf59 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, the preset inventors analyzed associations between death and possible prognostic factors including age, gender, pT-classification, and pN-classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced C1orf59 expression into the model, along with any and all variables that satisfied an entry level of a P value less than 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05. The expected sample numbers assigned in this analysis was calculated as follows; a difference in survival rate after following up for 2500 days was 22% (35-57%) with an 80% power for a two-sided significance level at 5%. 243 assessable patients were expected to be required.

RNA interference assay.
Small interfering RNA (siRNA) duplexes (Sigma Aldrich Japah) (100 nM) were transfected into an ESCC cell line TE1 and TE5, using 30 micro-L of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. The transfected cells were cultured for 7 days, the number of colonies was counted by Giemsa staining; and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8 solution; Dojindo Laboratories, Kumamoto, Japan). To confirm suppression of C1orf59 mRNA expression, semiquantitative RT-PCR experiments were carried out with synthesized primers specific to C1orf59 described above. The target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (Luciferase/LUC: Photinus pyralis luciferase gene), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 22); control 2 (Enhanced Green Fluorescence Protein/EGFP); 5'-GAAGCAGCACGACUUCUUC-3' (SEQ ID NO: 23); siRNA-C1ORF59-1, 5'-CAGUUUAAACCUCCACUAU-3' (RNA sequence of SEQ ID NO: 5); siRNA-C1ORF59-2, 5'-GUGGAAAGCUUAAGAGUGA-3' (RNA sequence of SEQ ID NO: 6); siRNA- PIWIL4-1, 5'- GUUACAAAGU CCUCCGGAA -3' (RNA sequence of SEQ ID NO: 7); siRNA- PIWIL4-2, 5'- GUCAGUAUGC UCACAAGCU-3' (RNA sequence of SEQ ID NO: 8).

Cell growth assay.
COS-7 and HEK293T cells were plated at densities of 5 x 10⁵ cells/100 mm dish, transfected with plasmids designed to express C1ORF59 (pcAGGSn3FC-C1ORF59-Flag) or mock plasmids. Cells were selected in medium containing 0.6 mg/mL of geneticin (Invitrogen) for 7 days, and cell numbers were assessed by MTT assay (cell counting kit-8 solution; Dojindo Laboratories).

Real time PCR for piRNA.
Small RNA fraction was extracted from cultured cells using QIAzol reagent (QIAGEN) according to the manufacturer's protocol. Extracted RNAs were reverse transcribed with miScript Reverse Transcription Kit (QIAGEN). Real time-PCR experiments were carried out with miScript SYBR Green PCR Kit (QIAGEN), and with primers for piR1, piR2 or U6 snRNA as an internal control which were originally designed by QIAGEN in Human miScript Assay kit.

Generating GST-fused recombinant protein.
The gene encoding full-length C1orf59 was cloned into the pEU-E01-GST-TEV-MCS-N1 vector with a N-terminal GST tag. And the protein was generated and purified by CellFree Sciences Co., Ltd.

Methyltransferase assay.
The in vitro methyltransferase assay was performed as described (Mui Chan C et al. Proc Natl Acad Sci U S A 2009; 106:17699-17704.) with slight modification. The methylation assays were carried out in a reaction mixture of 20 micro-L scale containing 25mM Tris-HCl (pH 8.0), 50mM KCl, 2.5mM MgCl₂, 0.05 mM EDTA, 2.5% glycerol, 5mM DTT, 2mM MnCl₂, 20 micro-M S-adenosyl-L-[methyl-¹⁴C] methionin (PerkinElmer Life & Analytical Sciences), and 10micro-M single-stranded RNA and 3 micro-M protein. The reaction mixtures were incubated at 37 degrees C for 40 min. Phenol extraction was carried out after the reaction. The aqueous layer was recovered, and RNA was purified by ethanol precipitation. The purified RNA was dissolved in 10 micro-L of TE buffer, and 10 micro-L of denaturing PAGE (DPAGE) loading buffer was added. The sample was heated at 95 degrees C for 2 min and then analyzed by a 15% DPAGE.

Northern blot for piRNA.
PIWIL4-3FH was overexpressed inTE1 cells, and PIWIL4 protein was immunoprecipitated with flag tag. Total RNA derived from IP product, and was loaded in urea gel. Membrane for NB was generated by transfer and EDC cross linking (Pall GS et al. Nucleic Acids Res 2007; 35:e60.). RNA probe was generated with probe construction kit (ambion).

Confirmation of interaction C1orf59 and PIWIL4 by immunoprecipitation.
Cell extracts from TE1-co-transfected with C1orf59 or mock, and with PIWIL4 or mock were precleared by incubation at 4 degrees C for 1 hour with 100 micro-L of protein G-agarose beads in a final volume of 1 mL of immunoprecipitation buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl) in the presence of proteinase inhibitor. After centrifugation at 1000 rpm for 1 min at 4 degrees C, the supernatant was incubated at 4 degrees C with anti-Flag M2 agarose beads for 2 hours. The beads were then collected by centrifugation at 5000 rpm for 1 min and washed six times with 1 mL of each immunoprecipitation buffer. The washed beads were resuspended in 20 micro-L of Laemmli sample buffer and boiled for 5 min, and the proteins were separated in 12% SDS polyacrylamide gel electrophoresis (PAGE) gels (BIO RAD). After electrophoresis, western blotting analysis system was done using a anti-C1orf59 antibody (see above) and anti-Flag M2 monoclonal antibody. (Catalog No. F3165, SIGMA-ALDRICH).

Results
C1ORF59 expression in several cancers and normal tissues.
To identify target molecules for the development of novel therapeutic substances and/or biomarkers for esophageal cancers, genome-wide expression profile analysis of ESCC was performed using a cDNA microarray (NPL 3-9). Among 27,648 genes screened, elevated expression (3-fold or higher) of C1orf59 transcript in the great majority of the esophageal cancer samplesexamined was identified. Its over-expression was confirmed by means of semi-quantitative RT-PCR experiments in 10 of 10 ESCC tissues, and in 7 of 11 ESCC cell lines (Fig. 1A). Furthermore, C1orf59 was highly expressed in cervical, colon, bile duct, and lung squamous cell cancers (Figs. 1B, C). Furthermore, a high level of C1orf59 in esophageal cancer cell lines was confirmed by Western blot analyses using anti-C1orf59 antibody (Fig. 1D). Immunofluorescence analysis was conducted to examine the subcellular localization of endogenous C1orf59 in esophageal cancer cell line TE1 and found that C1orf59 was located in the nucleus and cytoplasm (Fig. 1E). Northern-blot analysis using C1orf59 cDNA as a probe identified a strong signal corresponding to a 2-kb transcript only in the testis among 23 tissues examined (Fig. 2A). Furthermore, C1orf59 protein expressions in six normal tissues (liver heart, kidney, lung, esophagus and testis) was compared with those in esophageal cancers using anti-C1orf59 polyclonal antibodies by immunohistochemistry. C1orf59 expressed abundantly in testis (mainly in nucleus and cytoplasm of primary spermatocytes) and esophageal cancer; however, its expression was hardly detectable in the remaining five normal tissues (Fig. 2B). C1orf59 was strongly expressed in esophageal tumors in spite of no expression in normal esophagus, detected by semi-quantitative RT-PCR (Fig. 7A), by Immunohistochemical staining (Fig. 7B), C1orf59 was strongly expressed in tumor in spite of no expression in normal esophagus, detected. C1orf59 was strongly expressed in several kind of tumors in spite of no expression in normal tisseus, detected by semi-quantitative RT-PCR (Fig. 7C). Expression of PIWIL1, PIWIL2, PIWIL3, PIWIL4 was investigated and only PIWIL4 was expressed in esophageal cancers (Figs. 8A, B).

Association of C1ORF59 expression with poor prognosis for ESCC patients.
Then, correlations of the C1orf59 expression in surgically resected ESCCs with various clinicopathologic variables was examined. To verify the clinicopathological significance of C1orf59, the expression of C1orf59 protein by means of tissue microarrays containing 297 ESCC patients who underwent surgical resection was examined additionally. Positive staining was observed in 172 of 251 (68.5%) esophagealcancers, whereas no staining was observed in any of the normalportions of the same tissues. Then, a correlation of C1orf59 expression (positive versus negative) with various clinicopathological parameters was examined and found its significant correlation with tumor size (higher in pT2-T3; P =0.0005 by Fisher's exact test; Table 1A). The Kaplan-Meier method indicated significant association between C1ORF59 status (positive versus negative) in ESCCs and tumor-specific survival rate (shorter survival periods in C1orf59-positive cases; P = 0.0029 by the log-rank test; Fig. 2D). By univariate analysis, tumor size (pT1 versus pT2-3), lymph node metastasis (pN0 versus pN1-2), gender (female versus male), and C1orf59 positivity (negative versus positive) were significantly related to poor tumor-specific survival of ESCC patients (Table 1B). Furthermore, multivariate analysis using the Cox proportional hazard model indicated that pT stage, pN stage, gender, and positive C1orf59 staining were independent prognostic factors for ESCCs patients (Table 1B).

Effect of C1ORF59 or PIWIL4 on cell growth.
To assess whether upregulation of C1orf59 plays a role in growth or survival of lung-cancer cells, the present inventors transfected siRNA against C1orf59 (si-1 and -2), along with two different control (siRNAs for LUC and, EGFP) into TE1 and TE5 cells to suppress expression of endogenous C1orf59 (Fig. 3A). The level of C1orf59 expression in the cells transfected with si-1, si-2 was significantly reduced, in comparison with two control siRNAs (Fig. 3A). Cell viability and colony numbers measured by MTT and colony-formation assays were reduced significantly in the cells transfected with si-1 or si-2 in comparison with those transfected with control siRNA (Figs. 3B, C).

To further examine a potential role of C1orf59 in tumorigenesis, plasmids designed to express C1orf59 (pcAGGSn3FC-C1orf59) were prepared and were transfected them into COS-7 and HEK293 cells. After confirmation of C1orf59 expression by western-blot analysis (Fig.3D), MTT and colony-formation assays were carried out, and found that growth of the C1orf59-COS-7 and C1orf59-HEK293 cells were promoted at a significant degree in comparison to the COS-7 and HEK293 cells transfected with the mock vector (Fig. 3E). Expression of PIWIL4 in response to si-PIWIL4s (si-1 and -2) or control siRNAs (LUC and EGFP) in TE1 cells was analyzed. The level of PIWIL4 expression in the cells transfected with si-1, si-2 was significantly reduced, in comparison with two control siRNAs (Fig. 8C). Cell viability and colony numbers measured by MTT and colony-formation assays were reduced significantly in the cells transfected with si-1 or si-2 in comparison with those transfected with control siRNA (Figs. 8D, E).

Identification of piRNA having same sequence as partial mRNA sequence of cancer-testis antigen.
Many piRNAs have same sequence on a mRNA. In mouse, mRNA-like piRNA gene which was expressed only in testis was reported (Kim M et al. RNA 2008; 14:1005-1011.). It was found that some piRNAs have sequences as partial mRNA of cancer-testis antigen. The present inventors reported many cancer-testis antigen which highly expressed in esophageal cancers, so those mRNAs were screened, and found 7 piRNAs (shown in Table 2).

Detection of piRNA in cancer cells.
It was thought that enzymatic activity of C1orf59 may contribute to carcinogenesis and cell growth. At first, the existence of piRNAs in esophageal cancer cells as well as testis tissue was confirmed. Using primers for piR1, the piRNAs in esophageal clinical cancers were detected by real time PCR (Fig. 4B), and in cell lines by northern blot. (Fig. 4C).

C1orf59 Methylates 2'-OH of 3' terminal nucleotide of piRNA.
To elucidate the biological mechanism of C1orf59 in carcinogenesis, it was attempted to confirm the methyltransferase activity by in vitro methyltransferase assay. GST-fused C1orf59 protein could methylate the synthesized piR1, nevertheless, pre-methylated piRNA on 2'-OH of 3' terminal nucleotide was not methylated (Fig. 4A).

C1orf59 Methylates and may stabilize piRNA.
To investigate the role of methylation for piRNA, the expression of piR1,piR2 by knock down of C1orf59 was confirmed. When C1orf59 was knocked down, expression of piRNA was decreased in TE1, TE5 (Fig. 4D). Furthermore, expression of piRNA in HEK293T was increased by C1orf59 overexpression (Fig. 4E).

Mutation analysis.
The mutant recombinant protein, and expression vector were generated (Fig. 5A). Mutant recombinant C1orf59 was deactivated in piRNA methylation activity (Fig. 5B). After confirmation of C1orf59 expression by western-blot analysis(Fig. 5C), the present inventors carried out MTT assays, and found that growth of the C1orf59-HEK293T cells were promoted at a significant degree in comparison to HEK293T cells transfected with the mock vector. On the other hands, mutant recombinant C1orf59 lost the growth promotive effect (Fig. 5D).

Identification of downstream of C1orf59 in cancer cells.
HEN1 homologs and PIWI protein were thought to be interacted (Horwich MD et al. Curr Biol 2007; 17:1265-1272). It was confirmed the expression of human PIWI proteins, and only PIWIL4 was highly expressed in esophageal cancers (Fig. 8B). The results of immunoprecipitation analysis indicated that C1orf59 was interacted with PIWIL4 (Fig. 6A). PIWIL4 was thought to be associated with Histone methylation, so the present inventors confirmed the interaction of PIWIL4 and H3K9 methylate associating proteins, CBX5 (HP1 subfamiy), SUV39H1, and SUV39H2 (Fig. 6B, C, D). By knock down of C1orf59, trymethylation status of H3K9 was decreased and by overexpression of C1orf59, trymethylation status of H3K9 was increased (Fig. 6E). Furthermore, suppresser genes, p16, p21 expression were let gone by knock down of C1orf59 (Fig. 6G).

Discussion
It has been shown here that C1orf59 is frequently overexpressed in clinical esophageal cancers samples, and cell lines, and that the gene product is indispensable for survival/growth of cancer cells.
C1orf59 protein encodes a 393-amino-acid protein with SAM dependent methyltransferase dmain. C1orf59 was thought to be homolog of HEN1, which is the methyltransferase for microRNA in plants (Park W et al. Curr Biol 2002; 12:1484-1495.). Methylation for miRNA may contribute the stability of miRNA (Ramachandran V and Chen X. Science 2008; 321:1490-1492.). Some homologs of HEN1 were reported as methyltransferase for Piwi interacting RNA (piRNA) in mouse, Drosophila and so on (Horwich MD et al. Curr Biol 2007; 17:1265-1272., Kirino Y and Mourelatos Z. Nucleic Acids Symp Ser (Oxf) 2007; 51:417-418., Kirino Y and Mourelatos Z. RNA 2007; 13:1397-1401., Saito K et al. Genes Dev 2007; 21:1603-1608). Because the methyltransferase activity of C1orf59 for piRNA, and the existence of piRNA in cancer cells were confirmed, so it was thought that piRNAs played important role in cencers cells, and that piRNAs need the methyl-modification by C1orf59 to exist stably in cells. By further analysis, the present inventors confirmed that only PIWIL4 was highly expressed in esophageal cancer cells among PIWIL family genes, and C1oed59 protein and PIWIL4 protein were interacted. PIWI in Drosophila melanogaster and PIWIL4 in human were reported to be associated with methylation of H3K9 (Yin H and Lin H. Nature 2007; 450:304-308., Sugimoto K et al. Biochem Biophys Res Commun 2007; 359:497-502). PIWIL4 was interacted with CBX5, SUV39H1, and SUV39H2, and the present inventors confirmed that knock down of C1orf59 resulted in the reduction of trimethylation of H3K9. In cancer cells, some suppresser genes are known to be suppressed, the present inventors searched the suppresser genes which deactivated by effect of C1orf59, and identified p16 and p21. p16, p21 expression were let gone by knock down of C1orf59. p16 gene was reported to be hypermethylated in esophageal cancer (Taghavi N et al, BMC Cancer 2010;10:138).

It was speculated that C1orf59 contributed to deactivation of suppresser genes such as p16, p21 through trimethylation of H3K9 because RISC with PIWI protein couldn't work as a methylating factor without methylated piRNA.
In the present study, the present inventors demonstrated that C1orf59 gene was frequently overexpressed in cervical, colon, bile duct, lung squamous cell, and esophageal cancers, and might play an important role in the development of those cancers. Knockdown of C1orf59 expression by siRNA in esophageal cancer cells resulted in suppression of cell growth. Moreover, clinicopathological evidence obtained through our tissue-microarray experiments indicated that ESCC patients with C1orf59-positive tumors had shorter cancer-specific survival periods than those with C1orf59-negative tumors. The results obtained by in vitro and in vivo assays strongly suggested that C1orf59 is likely to be an important growth factor and be associated with a more malignant phenotype of esophageal cancer cells. By further investigations of C1orf59 function, C1orf59 contributed to oncogenes activation in carcinogenesis through the methylation of piRNA which is testis and cancer specific small RNA. Because C1orf59 should be classified as one of the typical cancer testis antigens, selective inhibition of C1orf59 activity by molecular targeted substances could be a promising therapeutic strategy that is expected to have a powerful biological activity against cancer with a minimal risk of adverse events.

In summary, C1orf59 might play an important role in the growth of esophageal cancer. C1orf59 overexpression in resected specimens may be a useful index for application of adjuvant therapy to the patients who are likely to have poor prognosis. In addition, the data strongly imply the possibility of designing new anti-cancer drugs and cancer vaccines to specifically target the C1orf59 for human cancer treatment.

The gene-expression analysis of cancers described herein has identified specific genes as a target for cancer prevention and therapy. Based on the expression of this differentially expressed gene, i.e., C1orf59 and PIWIL4, the present invention provides novel molecular diagnostic markers for identifying and detecting cancers as well as assessing the prognosis. Further, piRNA, identified as substances methylated by C1orf59, was confirmed overexpression in cancers. Therefore, the present invention also provides a novel diagnostic strategy using piRNA.

Furthermore, as described herein, C1orf59 and PIWIL4 are involved in cancer cell survival. Therefore, the present invention also provides novel molecular targets for treating and preventing cancer. They may be useful for developing novel therapeutic drugs and preventative agents without adverse effects. Further, CBX5, SUV39H1, and SUV39H2 was identified as the genes that is interacted with PIWIL4. Therefore, the present invention also provides a novel screening strategy using PIWIL4, CBX5, SUV39H1, and SUV39H2.

The methods described herein are also useful for the identification of additional molecular targets for prevention, diagnosis, prognosis, and treatment of cancers. The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic or prognostic 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 provides indicators for developing novel strategies for diagnosis, prognosis, treatment, and ultimately prevention of cancers.

All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims

A method for diagnosing cancer, said method comprising the steps of:
(a) determining the expression level of the C1orf59 gene, the PIWIL4 gene or piRNA in a subject-derived biological sample by a method selected from the group consisting of:
(i) detecting an mRNA of the C1orf59 gene and/or the PIWIL4 gene,
(ii) detecting a protein encoded by the C1orf59 gene and/or the PIWIL4 gene,
(iii) detecting a biological activity of the protein encoded by the C1orf59 gene and/or the PIWIL4 gene, and
(iv) detecting a piR1 and/or piR2;
(b) correlating an increase in the expression level determined in step (a) as compared to a normal control level of the C1orf59 gene, the PIWIL4 gene or piRNA to the presence of cancer.
The method of claim 1, wherein the expression level determined in step (a) is determined by detecting the binding of an antibody against the protein encoded by the C1orf59 gene or PIWIL4 gene.
The method of claim 1, wherein the subject-derived biological sample comprises a biopsy specimen.
A method for assessing or determining the prognosis of a patient with cancer, which method comprises the steps of:
(a) detecting an expression level of the C1orf59 gene in a patient-derived biological sample;
(b) comparing the expression level detected in step (a) to a control level; and
(c) assessing or determining the prognosis of the patient based on the comparison of step (b).
The method of claim 4, wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level is determined as poor prognosis.
The method of claim 4, wherein the expression level is determined by a method selected from the group consisting of:
(a) detecting an mRNA of the C1orf59 gene;
(b) detecting a protein encoded by the C1orf59 gene; and
(c) detecting a biological activity of a protein encoded by the C1orf59 gene.
The method of claim 4, wherein the patient derived biological sample comprises a biopsy specimen.
A kit for diagnosing cancer or assessing or determining the prognosis of a patient with cancer, which comprises a reagent selected from the group consisting of:
(a) a reagent for detecting an mRNA of the C1orf59 gene and/or the PIWIL4 gene;
(b) a reagent for detecting a protein encoded by the C1orf59 gene and/or the PIWIL4 gene;
(c) a reagent for detecting a biological activity of a protein encoded by the C1orf59 gene and/or the PIWIL4 gene; and
(d) a reagent for detecting a piR1 and/or piR2;
The kit of claim 8, wherein the reagent is a probe that binds to the mRNA of the C1orf59 gene or gene PIWIL4 gene, piR1 or piR2.
The kit of claim 8, wherein the reagent is an antibody against and binding to a protein encoded by the C1orf59 gene or the PIWIL4 gene.
An isolated double-stranded molecule that, when introduced into a cell, inhibits expression of the C1orf59 gene or the PIWIL4 gene as well as cell proliferation, said molecule comprising a sense strand and an antisense strand complementary thereto, said strands hybridized to each other to form the double-stranded molecule.
The double-stranded molecule of claim 11, wherein the sense strand comprises a sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8.
The double-stranded molecule of claim 11 or 12, wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length.
The double-stranded molecule of any one of claims 11 to 13, which consists of a single polynucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
The double-stranded molecule of claim 14, which has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A], wherein [A] is the sense strand comprising a sequence corresponding to a target sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7 and 8, [B] is the intervening single-strand consisting of 3 to 23 nucleotides, and [A'] is the antisense strand comprising a complementary sequence to [A].
A vector encoding the double-stranded molecule of any one of claims 11 to 15.
A method for treating or preventing a cancer expressing at least one gene selected from the group consisting of the C1orf59 gene and the PIWIL4 gene, wherein the method comprises the step of administering at least one isolated double-stranded molecule of any one of claims 11 to 15 or a vector of claim 16.
A composition for treating or preventing a cancer expressing at least one gene selected from the group consisting of the C1orf59 gene and the PIWIL4 gene, wherein composition comprised at least one isolated double-stranded molecule of any one of claims 11 to 15 or a vector of claim 16.
A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene , or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene and/or the PIWIL4 gene;
(b) detecting the binding activity between the polypeptide and the test substance; and
(c) selecting a substance that binds to the polypeptide.
A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene or the PIWIL4 gene , or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene or the PIWIL4 gene;
(b) detecting a biological activity of the polypeptide of step (a); and
(c) selecting the test substance that suppresses the biological activity of the polypeptide as compared to the biological activity of said polypeptide detected in the absence of the test substance.
The method of claim 20, wherein the biological activity is the facilitation of the cell proliferation.
A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing the C1orf59 gene and/or the PIWIL4 gene and
(b) selecting the test substance that reduces the expression level of the C1orf59 gene and/or the PIWIL4 gene in comparison with the expression level detected in the absence of the test substance.
A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene and/or the PIWIL4 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell into which a vector, comprising the transcriptional regulatory region of the C1orf59 gene or the PIWIL4 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression or activity of said reporter gene; and
(c) selecting the test substance that reduces the expression or activity level of said reporter gene as compared to a control.
A method of screening for a candidate substance for treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide encoded by a polynucleotide corresponding to the C1orf59 gene with a substrate to be methylated in the presence of a test substance under a condition capable of methylation of the substrate;
(b) detecting the methylation level of the substrate; and
(c) selecting the test substance that decreases the methylation level of the substrate compared to a control level.
The method of claim 24, wherein the substrate is piRNA.
The method of claim 25, wherein the piRNA is piR1 or piR2.
A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising a PIWIL4-binding domain of a C1orf59 polypeptide with a polypeptide comprising a C1orf59-binding domain of a PIWIL4 polypeptide in the presence of a test substance;
(b) detecting binding between the polypeptides; and
(c) selecting the test substance that inhibits binding between the polypeptides.
The method of claim 27, wherein the polypeptide comprising the PIWIL4-binding domain comprises a C1orf59 polypeptide.
The method of claim 27, wherein the polypeptide comprising the C1orf59-binding domain comprises a PIWIL4 polypeptide.
A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising an S-adenosylmethionine (SAM)-binding domain of a C1orf59 polypeptide with SAM in the presence of a test substance;
(b) detecting binding between the polypeptide and SAM; and
(c) selecting the test substance that inhibits the binding.
The method of claim 30, wherein the polypeptide comprising the SAM-binding domain comprises a C1orf59 polypeptide.
A method of screening for a candidate substance for treating or preventing a cancer associated with over-expression of the C1orf59 gene, or inhibiting said cancer cells growth, said method comprising the steps of:
(a) contacting a test substance with a cell expressing the C1orf59 gene and
(b) selecting the test substance that reduces the expression level of piRNA in comparison with the expression level detected in the absence of the test substance.
The method of claim 32, wherein the piRNA is piR1 or piR2.
A method of screening for a candidate substance useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a polypeptide comprising a CBX5, SUV39H1 or SUV39H2-binding domain of a PIWIL4 polypeptide with a polypeptide comprising a PIWIL4 -binding domain of a CBX5, SUV39H1 or SUV39H2 polypeptide in the presence of a test substance;
(b) detecting a binding between the polypeptides; and
(c) selecting the test substance that inhibits the binding between the polypeptides.
The method of claim 34, wherein the polypeptide comprising the CBX5, SUV39H1 or SUV39H2 -binding domain comprises a PIWIL4 polypeptide.
The method of claim 34, wherein the polypeptide comprising the PIWIL4-binding domain comprises a CBX5, SUV39H1 or SUV39H2 polypeptide.