WO2012023290A1

WO2012023290A1 - Rasef as tumor marker and therapeutic target for cancer

Info

Publication number: WO2012023290A1
Application number: PCT/JP2011/004628
Authority: WO
Inventors: Yataro Daigo; Yusuke Nakamura; Takuya Tsunoda
Original assignee: Oncotherapy Science, Inc.
Priority date: 2010-08-20
Filing date: 2011-08-19
Publication date: 2012-02-23

Abstract

The present invention is based on the finding that RASEF genes are overexpressed in cancer, and involved in cancer cell survival. The present invention is also based on the finding that RASEF interacts with ERK1/2. The present invention features methods for diagnosing cancer or assessing/determining the prognosis of a subject with cancer, using RASEF genes as diagnostic markers. The present invention also features a double-stranded molecule against a RASEF gene and inhibitory polypeptide for ERK1/2 as well as vector encoding them, a method or composition for treating and/or preventing cancer using such double-stranded molecule and inhibitory polypeptide. Also, disclosed are methods of identifying a candidate substance for treating and preventing lung cancer, using RASEF or the interaction between RASEF and ERK1/2 as molecular targets.

Description

RASEF AS TUMOR MARKER AND THERAPEUTIC TARGET FOR CANCER

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 lung cancer as well as methods for treating and preventing lung cancer. Moreover, the present invention relates to methods for screening a substance for treating and/or preventing lung cancer.

Priority
The present application claims the benefit of U.S. Provisional Application No. 61/375,459, filed on August 20, 2010, and Japanese Patent Application No. 2010-201362, filed on August 22, 2010, the contents of which are hereby incorporated herein by reference in their entirety for all purposes.

Lung cancer is leading cause of death in the world (NPL1) and the world's most common cause of cancer-related death (NPL2). In spite of the use of modern surgical techniques combined with various adjuvant treatment modalities, such as radiotherapy and chemotherapy, the overall 5-year survival rate of lung cancer patients still remains at 20% (NPL3). In the last few decades cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC, however, those regimens provide a limited survival benefit compared with cisplatin-based therapies (NPL 4). In addition to these cytotoxic drugs, several molecular-targeted agents such as monoclonal antibodies against VEGF (i.e., bevacizumab/anti-VEGF) or EGFR (i.e., cetuximab/anti-EGFR) as well as inhibitors for EGFR tyrosine kinase (i.e., gefitinib and erlotinib) were developed and are now widely used in clinical practice (NPL 5-7), but fatal adverse events such as interstitial pneumonia by gefitinib or severe hemorrhage by bevacizumab were reported (NPL 8, 9). Therefore, further development of new agents targeting cancer specific molecules without adverse effect is urgently needed. Each of the new regimens can provide survival benefits to a limited proportion of the patients. Well-organized classification of clinical and pathological stage has been most reliable source of clinical decision for selecting treatment modalities, such as adjuvant chemotherapy, but considering the fact that about 30% of stage I non-small-cell lung cancer patients, who were not candidates of adjuvant chemotherapy, will have recurrent disease.
Hence, new therapeutic strategies, such as the development of more effective molecular-targeted agents applicable to the great majority of patients with less toxicity, are eagerly awaited. It is important to develop more predictive biomarkers for selecting patients who should be treated with additional therapies.

Systematic analysis of expression levels of thousands of genes using a cDNA microarray technology is an effective approach for identifying molecules involved in pathways of carcinogenesis or those associated with efficacy to anticancer therapy; some of such genes or their gene products may be good target molecules for the development of novel therapies and/or cancer biomarkers. To identify such molecules, particularly oncoantigens, a genome-wide expression profile analysis of 120 clinical lung cancer samples, coupled with enrichment of tumor cells by laser microdissection was performed and then compared the expression profile data with those in 31 normal human tissues (27 adult and 4 fetal organs) (NPL10-15). To verify the clinicopathologic significance of the respective gene products, a screening system by a combination of the tumor-tissue microarray analysis of clinical lung cancer materials and RNA interference technique was established (NPL16- 43). This systematic approach revealed that a RAS and EF-hand domain containing protein (RASEF) is a novel molecule that is overexpressed commonly in primary lung cancers and is essential for cell growth/survival of cancer cells.

RAS and EF-hand domain containing protein (RASEF) was first described as a gene in genomic locus, 9q, which was commonly deleted lesion in acute myeloid leukemia patients (NPL44). RASEF was also reported to be down-regulated in malignant melanoma and primary uveal melanoma, but not suppressed in breast cancers (NPL45, 46). RASEF contains a Rab GTPase domain so that it is considered as a protein in Rab GTPase protein family, but unlike other Rab containing protein, RASEF contains two EF-hand domains which bind calucium ions in the N-terminal side and coiled-coil motif in internal lesion, as well as Rab GTPase motif in the C-terminal side of RASEF (NPL47).
The present inventors here report the first evidence that RASEF plays a significant role in pulmonary carcinogenesis and tumor progression through the interaction with extracellular signal-regulated kinase (ERK) 1/2, and show that RASEF is a useful prognostic biomarker and therapeutic target for lung cancer.

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In the course of screening for novel molecular targets for diagnosis, treatment and prevention of human cancers, genome-wide expression profile analyses of 120 clinical lung cancers was performed on cDNA microarray, coupled with enrichment of tumor cells by laser microdissection (Kikuchi T, et al. Oncogene. 2003 Apr 10;22(14):2192-205.; Kikuchi T, et al. Int J Oncol. 2006 Apr;28(4):799-805; Kakiuchi S, et al., Mol Cancer Res. 2003 May;1(7):485-99; Kakiuchi S, et al., Hum Mol Genet. 2004 Dec 15;13(24):3029-43. Epub 2004 Oct 20; Taniwaki M, et al., Int J Oncol. 2006 Sep; 29(3):567-75.). The results demonstrate that the RASEF gene is frequently over-expressed in the great majority of primary lung cancers. Also, the interaction of RASEF protein with ERK1 protein or ERK2 protein and phosphorylation by them were demonstrated.
Furthermore, siRNAs against the gene suppressed cancer cell proliferation effectively. These results demonstrate that RASEF gene is a good molecular target for diagnosis or treatment of cancer.
Thus, the present invention relates to cancer-related gene RASEF, which is commonly up-regulated in tumors, and strategies for the development of molecular targeted drugs for cancer treatment using RASEF.

In one aspect, the present invention provides a method for diagnosing cancer, e.g., a cancer over-expressing a RASEF gene, e.g., lung cancer, using the expression level of the RASEF gene as an index. In the methods of the present invention, the mRNA of RASEF gene can be detected by appropriate primers or probes or, alternatively, the RASEF protein can be detected by anti- RASEF antibody in order to determine the expression level of the gene. In some embodiments, the cancer is mediated or promoted by a RASEF gene. In some embodiments, the cancer is lung cancer. In one embodiment, the cancer is a non small cell lung cancers (NSCLC) including lung squamous cell carcinoma (SCC), adenocarcinoma (ADC) and large cell carcinoma (LCC), or a small-cell lung cancers (SCLC).
The present invention also provides a method for predicting the progress of a subject with cancer, e.g., lung cancer, using the expression level of the RASEF as an index.
Furthermore, the present invention provides a method for predicting the prognosis of the cancer, e.g., lung cancer, patient using the expression level of the RASEF gene or biological activity of the RASEF protein as an index.
In another embodiment, the present invention provides a method for screening a candidate substance for treating or preventing cancer, e.g., lung cancer, using the binding to the RASEF polypeptide, the expression level of the RASEF gene or reporter gene surrogating the RASEF gene, or biological activity of the RASEF polypeptide as an index.

In another embodiment, the present invention provides a method for screening a candidate substance for treating or preventing cancer, e.g., lung cancer, using the interaction between RASEF polypeptide and ERK1 and/or ERK2 polypeptide as an index.
In another embodiment, the present invention provides a method for screening a candidate substance for treating or preventing cancer, e.g., lung cancer, using the phosphorylation of RASEF polypeptide by ERK1 and/or ERK2 polypeptide as an index.
In a further embodiment, the present invention provides double-stranded molecules, e.g., siRNA, against the RASEF gene, that inhibits the expression of the gene, and vectors encoding the double stranded molecule. The double-stranded molecules of the present invention are useful for treating or preventing cancers, e.g., a cancer mediated by a RASEF or resulting from overexpression of a RASEF, e.g., lung cancer.

In another embodiment, the present invention provides methods of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against RASEF gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the gene.
In another embodiment, the present invention provides compositions for treating or preventing cancer, comprising a double-stranded molecule against a RASEF, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the gene, and a pharmaceutically acceptable carrier.
In addition, the present invention arises, in part, from the discovery that inhibition of the binding between a RASEF polypeptide and an ERK1/2 protein by a RASEF-derived peptide, which corresponded to the binding domain to the ERK1/2 protein, effectively suppressed growth of lung cancer cells. Accordingly, the present invention also provides a polypeptide including the amino acid sequence of SEQ ID NO: 16 or variant polypeptide thereof, wherein the polypeptide inhibits a biological activity of the RASEF polypeptide. In preferred embodiments, the polypeptide is modified with a cell-membrane permeable substance.

In another aspect, the present invention provides methods for treating and/or preventing cancer, wherein the method including the step of administering the aforementioned polypeptide to a subject.
In anther aspect, the present invention provides compositions for treating and/or preventing cancer, wherein the composition including the aforementioned polypeptide and a pharmaceutically acceptable carrier.

More specifically, the present invention provides the following [1] to [49]:
[1] A method of detecting or diagnosing lung cancer in a subject, comprising determining a expression level of RASEF in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing lung cancer, or the presence of lung cancer in said subject, wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of a RASEF gene,
(b) detecting a protein encoded by the RASEF gene, and
(c) detecting biological activity of a protein encoded by the RASEF gene.
[2] The method of [1], wherein said increase is at least 10% greater than said normal control level.
[3] The method of [1], wherein the subject-derived biological sample is a biopsy sample.
[4] A kit for use in diagnosis or detection of lung cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the RASEF gene.
[5] A method for assessing prognosis of a subject with lung cancer, wherein the method comprises steps of:
(a) detecting an expression level of RASEF in a subject-derived biological sample;
(b) comparing the detected expression level to a control level; and
(c) determining prognosis of the patient based on the comparison of (b).
[6] The method of [5], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis.
[7] The method of [6], wherein the increase is at least 10% greater than said control level.
[8] The method of [5], wherein said expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of a RASEF gene;
(b) detecting a protein encoded by the RASEF gene; and
(c) detecting a biological activity of the protein encoded by the RASEF gene.
[9] A kit for assessing a lung cancer prognosis, wherein the kit comprises any one component selected from the group consisting of:
(a) a reagent for detecting the presence of an mRNA encoding the amino acid sequence of SEQ ID NO: 16 .
(b) a reagent for detecting the presence of a protein comprising the amino acid sequence of SEQ ID NO: 16 , and
(c) a reagent for detecting the biological activity of a protein comprising the amino acid sequence of SEQ ID NO: 16 .
[10] The method of [5 ]to [8], and the kit of [9], wherein the lung cancer is NSCLC.
[11] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a RASEF polypeptide, or fragment thereof;
(b) detecting binding activity between the polypeptide or fragment thereof, and the test substance; and
(c) selecting the test substance that binds to the polypeptide or fragment thereof.
[12] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a cell expressing a RASEF gene;
(b) detecting an expression level of the RASEF gene; and
(c) selecting the test substance that reduces the expression level of the RASEF gene in comparison with the expression level detected in absence of the test substance.
[13] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a RASEF polypeptide or fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment thereof of step (a); and
(c) selecting the test substance that suppresses a biological activity of the polypeptide or fragment thereof in comparison with a biological activity detected in the absence of the test substance.
[14] The method of [13], wherein the biological activity is cell proliferation enhancing activity.
[15] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a cell into which a vector comprising a transcriptional regulatory region of a RASEF gene and a reporter gene that is expressed under control of transcriptional regulatory region has been introduced,
(b) measuring expression or activity of said reporter gene; and
(c) selecting the test substance that reduces an expression or activity level of said reporter gene, in comparison with the level detected in the absence of the test substance.
[16] A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 polypeptide or functional equivalent thereof and/or an ERK2 polypeptide or functional equivalent thereof in the presence of a test substance ;
(b) detecting a binding 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 or inhibits the binding level in comparison with the level detected in the absence of the test substance .
[17] The method of [16], wherein the functional equivalent of the RASEF polypeptide comprises a ERK1 and/or ERK2-binding domain.
[18] The method of [17] wherein the ERK1 and/or ERK2-binding domain is comprised in residues 553-575 of SEQ ID NO: 16.
[19] The method of [17] wherein the ERK1 and/or ERK2-binding domain is comprised in residues 520-575 of SEQ ID NO: 16.
[20] The method of [17] wherein the ERK1 and/or ERK2-binding domain is comprised in residues 455-740 of SEQ ID NO: 16.
[21] The method of [18], [19] or [20], wherein the functional equivalent of the ERK1 polyeptide and/or the ERK2 polypeptide comprises a RASEF -binding domain.
[22] A method of screening for a candidate substance for treating or preventing a disease associated with overexpression of a RASEF gene, or inhibiting proliferation of a cell expressing the RASEF gene, said method comprising the steps of:
(a) contacting a RASEF polypeptide or a fragment thereof with an ERK1 polypeptide or functional equivalent thereof and/or an ERK2 polypeptide or a fragment thereof in the presence of a test substance under a suitable condition for phosphorylation;
(b) detecting the phosphorylation level of the RASEF polypeptide or fragment thereof;
(c) comparing the phosphorylation level with that detected in the absence of the test substance; and
(d) selecting the test substance that reduces the phosphorylation level of the RASEF polypeptide or fragment thereof as compared to the phosphorylation level detected in the absence of the test substance.
[23] The method of [22], the fragment of the RASEF polypeptide comprises the Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16.
[24] The merhod of [22], the fragment of the ERK1 polypeptide and/or the ERK2 polypeptide is a fragment retaining kinase activity.
[25] The method of [22], wherein the step (a) comprises incubating a RASEF polypeptide or a fragment thereof and an ERK1 polypeptide or a fragment thereof and/or an ERK2 polypeptide or a fragment thereof in the presence of a phosphate donor in the incubation mixture.
[26] The method of [25], wherein the phosphate donor is ATP.
[27] The method of [22], the disease associated with overexpression of RASEF is cancer.
[28] A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10, and the antisense strand comprises a nucleotide sequence complementary to said target sequence, wherein said sense strand and said antisense strand hybridize to each other to form the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits the expression of said gene.
[29] The double-stranded molecule of [28] wherein the sense strand hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
[30] The double-stranded molecule of [ 28] or[ 29] wherein said double-stranded molecule is a single polynucleotide comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
[31] The double-stranded molecule of [ 30] wherein said polynucleotide has a general formula
5'-[A]-[B]-[A']-3'
wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
[32] A vector comprising each or both of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 and 10, and wherein the antisense strand comprises a nucleotide sequence which is complementary to said sense strand, wherein 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 a RASEF gene, inhibits expression of said RASEF gene.
[33] A vector comprising each or both of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 and 10 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing a RASEF gene, inhibits the cell proliferation.
[34] The vector of [32] or[ 33], wherein the polynucleotide is a polynucleotide of between about 19 and about 25 nucleotides in length.
[35] The vector of [ 32] or [33], wherein said double-stranded molecule is a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
[36] The vector of [34], wherein said polynucleotide has a general formula
5'-[A]-[B]-[A']-3'
wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
[37] A method of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against a RASEF gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the RASEF gene.
[38] A method of [37], wherein the double stranded molecule is that of any one of [ 28] to [31].
[39] A method of [38], wherein the vector is that of any one of [32] to [36].
[40] A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against a RASEF gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the RASEF gene, and a pharmaceutically acceptable carrier.
[41] The composition of [40], wherein the double stranded molecule is that of any one of [28] to [31].
[42] The composition of [41], wherein the vector is that of any one of [31] to [35].
[43] A polypeptide comprising the amino acid sequence of (a) or (b) below:
(a) the amino acid sequence of SEQ ID NO: 43;
(b) the amino acid sequence in which one, two or several amino acid is substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43;
wherein the polypeptide inhibits a biological activity of the RASEF polypeptide.
[44] The polypeptide of [43], wherein the biological activity of the RASEF polypeptide is a binding activity to the ERK1/2 protein.
[45] The polypeptide of [43] or [44], which is modified with a cell-membrane permeable substance.
[46] The polypeptide of [45], which has the following general formula:
[R]-[D];
wherein [R] represents the cell-membrane permeable substance; and [D] represents a polypeptide comprising the amino acid sequence of (a) or (b) below:
(a) the amino acid sequence of SEQ ID NO: 43;
(b) the amino acid sequence in which one, two or several amino acid is substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43,
wherein [R] and [D] are linked directly or indirectly through a linker.
[47] A composition for treating and/or preventing cancer expressing RASEF gene, wherein the composition comprises the polypeptide of any one of [43] to [46] and a pharmaceutically acceptable carrier.
[48] The composition of [47], wherein the cancer to be treated is lung cancer.
[49] A method for treating and/or preventing cancer expressing RASEF gene, wherein the method comprises the step of administering the polypeptide of any one of [43]to [46] to a subject.
[50] The method of [49], wherein the cancer to be treated is lung cancer.

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 which follows:
Figure 1 depicts the analysis of RASEF expression in tumor tissues, cell lines and normal tissue. A, Expression of RASEF in 15 clinical lung cancer and normal lung tissue samples (top panels) [lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) and small cell lung carcinoma (SCLC); top] and 22 lung cancer cell lines (bottom panels) detected by semiquantitative RT-PCR analysis. B, Expression of RASEF in clinical lung cancers and adjacent normal lung, and confirmed its overexpression. C, Western blot analysis of RASEF protein by specific anti-RASEF antibody. D, Immunoblotting after phosphatase treatment of RASEF protein in NCI0H2170 cells. Shifted band in phosphatase-treated samples indicates that RASEF is phosphorylated. E, Expression and subcellular localization of endogenous RASEF protein in RASEF-positive and -negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in NCI-H2170 and A549 cells, whereas no staining was observed in SBC3 and bronchial epithelia derived BEAS-2B cell lines. F, Expression and subcellular localization of endogenous RASEF protein in RASEF-positive and -negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in NCI-H1373 and NCI-H2170 cells, whereas no staining was observed in NCI-H226 and and bronchial epithelia derived BEAS-2B cell lines.

Figure 2 depicts the expression of the RASEF protein in normal and lung cancer tissues. A, Northern blot analysis of the RASEF transcript in 16 normal adult human tissues. A weak signal was observed in only prostate and testis. B, Comparison of RASEF protein expression between normal tissues and lung cancers by immunohistochemistry. Almost no staining in normal organs except prostate but strong RASEF staining in cytoplasm of cancer tissues. Original magnification, X200. C, Immunohistochemistry of lung tumor tissues and adjacent normal tissues. Original magnification, X100.

Figure 3 depicts the prognostic significance of high RASEF expression in surgically treated NSCLC patients by tissue microarray. A, Examples for strong, weak, and absent RASEF expression in lung cancer tissues and a normal tissue. Original magnification, x100. B, Kaplan-Meier analysis of tumor-specific survival of NSCLC patients (n=341) who underwent curative resection according to expression levels of RASEF (P < 0.0001 by log-rank test). Vertical lines on survival curves mean censored cases.

Figure 4 depicts the inhibition of growth of lung cancer cells by siRNAs against RASEF. A, Gene knockdown effect on RASEF expression in A549 cells (left) and LC319 cells (right) by si-RASEFs (#1 and #2) and control siRNAs (si-LUC/Luciferase, si-SCR/Scramble chloroplast Euglena gracilis gene coding for the 5S and 16S rRNA), analyzed by semiquantitative RT-PCR. B, C, Effect of knocking down of RASEF on cell growth by MTT assay (B) and Colony formation assay (C). D, Statistical analysis of colony formation assay in C. Columns, relative absorbance of triplicate assays; bars, SD. E, F, G, Inhibition of growth of lung cancer cells by siRNAs against RASEF. Knockdown effect on RASEF protein expression in NCI-H2170 and NCI-H1373 cells by si-RASEFs (#1 and #2) and control siRNAs (si-EGFP and si-LUC), analyzed by Western blotting (E). Effect of knocking down of RASEF on cell growth by MTT assay (F) and Colony formation assay (G). Columns, relative absorbance of triplicate assays; bars, SD.

Figure 5 depicts the promotion of cell proliferation in COS-7 and DMS114 cells exogenously overexpressing RASEF. A, Detection of transient RASEF expression by western blotting. B, MTT assays of COS-7 and DMS114 cells 120 hours after transfection of RASEF-expressing vector. Columns, relative absorbance of triplicate assays; bars, SD.

Figure 6 depicts RASEF is a substrate for ERK. A, in vitro kinase assay of RASEF and ERK. 4 or 8 ul of RASEF recombinant proteins were reacted with recombinant active ERK2 in the presence of [gamma-³²P] ATP. The products were analyzed by autoradiography. The amount of RASEF that was subjected to SDS-PAGE was assessed by Coomassie brilliant blue staining (bottom panel). B, In vitro kinase assay of RASEF and ERK. Immunoprecipitated RASEF (RASEF-IP) was reacted with recombinant active ERK2 in the presence of [gamma-³²P] ATP. The products were analyzed by autoradiography. The amount of RASEF that was subjected to SDS-PAGE was assessed by Coomassie brilliant blue (CBB) staining (bottom panel). C, Identification of ERK-dependent phosphorylation sites of RASEF. COS-7 cells that were transfected with Flag-taged RASEF or co-transfected with Flag-taged RASEF and myc-taged ERK2 were lysed 8 minutes after 100 ng/ml EGF stimulation. The cell lysates were immunoprecipitated with Flag, and the proteins were electrophoresed on SDS-PAGE gel. The gels were stained with CBB. The RASEF bands were excised selectively to serve for analysis by mass spectrometry.

Figure 7. A, Identification of ERK1/2-dependent phosphorylation site on RASEF. COS-7 cells transfected with Flag-taged RASEF expression vector and myc-taged ERK2 expression vector were lysed 10 minutes after 50 ng/ml EGF stimulation with or without MEK inhibitor U0126. Immunoprecipitation assay with anti-Flag M2 agarose antibody and electrophoressis on SDS-PAGE gel were performed. The gels were stained with colloidal Coomassie brilliant blue (CBB). The RASEF bands were excised selectively to serve for analysis by mass spectrometry. Activity of ERK1/2 was evaluated by Western blotting with anti-phospho-ERK antibody. B, Confirmation of ERK1/2-dependent phosphorylation site of RASEF, S520, by in vitro kinase assay. Wild type RASEF (RASEF-WT) or RASEF-S520A immunoprecipitant was reacted with recombinant active ERK2 in the presence of [gamma-³²P] ATP. The amount of immunoprecipitates that were subjected to SDS-PAGE was assessed by Ponceau staining (bottom panel). C, The effect of phospho-mimicking or phospho-defective RASEF constructs on lung cancer cell growth. Expression of each constructs was detected by Western blot analysis (upper panel). Cell viability of DMS114 cells transfected with mock vector or wild type RASEFor RASEF-S520E or RASEF-S520A expression vector was quantified by the MTT assay at 6 days after transfection (bottom panel). Columns, relative absorbance of triplicate assays; bars, SD.

Figure 8 depicts the direct interaction of the RASEF and ERK1/2 proteins. A, Interaction of exogenous RASEF with endogenous ERK1/2. Extracts from COS-7 transfected with Flag-tagged RASEF or mock were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody or anti-ERK1/2 antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with anti-ERK1/2 antibody or anti-Flag antibody. B, Interaction of exogenous RASEF and exogenouse ERK2. Extracts from COS-7 transfected with Flag-tagged RASEF and/or ERK2 were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody or anti-myc antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with anti-myc antibody or anti-Flag antibody. C, Direct interaction of the RASEF and ERK1/2. The immunoprecipitates using anti-RASEF rabbit polyclonal antibody (Proteintech Group, Inc.) were subjected to Western blotting with anti-ERK1/2 mouse monoclonal antibody (Cell Signaling Technology). IP, immunoprecipitation. D, ERK1/2-dependent phosphorylation site Ser-520 of RASEF could be important for RASEF-ERK1/2 binding. Cell extracts from wild type RASEF or RASEF-S520A induced DMS114 cells were immunoprecipitated with anti-ERK1/2 rabbit polyclonal antibody (Santa Cruz Biotechnology) or anti-Flag M2 mouse monoclonal antibody (Sigma-Aldrich). Western blotting with indicated antibodies was performed.

Figure 9. RASEF-ERK1/2 interaction promotes ERK1/2 activity. A, B, RASEF could positively mediate ERK1/2 activity in lung cancer cells. DMS114 cell transfected with RASEF or Mock vector (A) and NCI-H2170 treated with siRNA for RASEF (#2) or LUC (B) were lysed, and Western blotting with indicated antibodies was performed. C, ERK1/2 activity promoting effect of RASEF is decreased by phospho-defective mutation at Ser-520. DMS114 cells transfected either with wild type RASEF or with RASEF-S520A or with mock vector were lysed, and Western blotting with indicated antibodies was performed. The signal intensity corresponding phospho-ERK1/2 protein was quantified by image J (bottom panel).

Figure 10 A, Schematic representation of the constructs of RASEF. B, Determination of the ERK1/2 binding regions of RASEF by immunoprecipitation. The Flag-tagged RASEF and various constructs of RASEF were pulled down by immunoprecipitation with anti-ERK1/2 antibody and then immunoblotted with anti-Flag antibody. Identification of the ERK1/2-interacting regions of RASEF. C, Schematic representation of various constructs of RASEF. D, E, Determination of the ERK1/2-binding regions of RASEF by immunoprecipitation experiments using DMS114 cells. The RASEF 520-575 construct was indicated to be ERK1/2-binding region.

Figure 11. A, Schematic drawing of three cell-permeable peptides of RASEF covering RASEF520-575 that corresponds to the ERK-interacting region in RASEF. B, Inhibition of interaction between exogenous RASEF and endogenous ERK using cell-permeable peptide, detected by immunoprecipitation assay. COS-7 cells transfected RASEF expression vector were lysed after treatment with 20 micro-M cell-permeable peptides for 5 hours. C, Inhibition of binding between endogenous RASEF and ERK1/2 using cell-permeable peptide, detected by immunoprecipitation assay. NCI-H2170 cells were lysed after treatment either with 11R-RASEF 553-575 or with scramble peptide for 4 hours (upper panel). The immunoprecipitates with anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. The signal intensity corresponding ERK1/2 protein was quantified by image J (lowert panel). D, Inhibition of ERK1/2-dependent phosphorylation of RASEF using cell-permeable peptide, detected by In vitro kinase assay. RASEF and cell-permeable peptide were reacted with recombinant active ERK2 in the presence of [gamma-³²P] ATP. The amount of immunoprecipitates and peptides that were subjected to SDS-PAGE was assessed by Ponceau staining. The signal intensity corresponding phosphorylated RASEF was quantified by image J (bottom panel). E, F, Growth suppressive effect of dominant-negative peptides in lung cancer cells. 11R-RASEF 553-575 showed dose-dependent growth suppressive effect in RASEF-positive cells. Columns, relative absorbance of triplicate assays; bars, SD; *, P < 0.05; **, P < 0.0001; N.S., Not significant.

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, GenBank Accession, 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.
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.

Definitions
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free from at least one substance that can be included in the natural source. Thus, an isolated or purified antibody refers to antibodies that are substantially free of cellular material for example, carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.

Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, in some embodiments it is also substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In one embodiment, proteins including antibodies of the present invention are isolated or purified.

In the context of the present invention, the phrase "RASEF gene" encompasses polynucleotides that encode the human RASEF or any of the functional equivalents of the human RASEF gene. Also, the phrase "ERK1 gene" encompasses polynucleotides that encode the human ERK1 or any of the functional equivalents of the human ERK1 gene. Additionally, the phrase "ERK2 gene" encompasses polynucleotides that encode the human ERK2 or any of the functional equivalents of the human ERK2 gene.
The RASEF gene, the ERK1 gene and the ERK2 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that functions similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to 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 can be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The gene, polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA or a combination thereof.
As used herein, the term "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). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, for example, a nutrient broth or gel in which an organism has been propagated, which contains cellular components, for example, proteins or polynucleotides.

The term "ERK1/2" refers to the protein consisting of ERK1 and ERK2. The polynucleotide and polypeptide sequences of the genes are shown in, but not limited to, SEQ ID NO: 18, 20 or 22 for ERK1 and: SEQ ID NO: 25 for ERK2. Furthermore, the sequence data are also available via Genbank accession numbers NM_002746, NM_001040056 or NM_001109891 for ERK1, and NM_002745 or NM_138957 for ERK2.
Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the RASEF gene, such as lung cancer, including non small-cell lung cancer (NSCLC) small-cell lung cancer (SCLC). NSCLC includes lung squamous cell carcinoma (SCC), adenocarcinoma (ADC) and large cell carcinoma (LCC).

(1) Genes and Polypeptides
Ras and EF-hand containing (RASEF) was first described as a gene in genomic locus, 9q, which was commonly deleted lesion in acute myeloid leukemia patients. RASEF was also reported to be down-regulated in malignant melanoma and primary uveal melanoma, but not suppressed in breast cancers. RASEF contains Rab GTPase domain so that it is considered as a protein in Rab GTPase protein family, but unlike other Rab containing protein, RASEF contains two EF-hands domain which binds calucium ions in the N-terminal side and coiled-coil motif in internal lesion, as well as Rab GTPase motif in the C-terminal side of RASEF.
The nucleotide sequence of human RASEF gene is shown in SEQ ID NO: 15 and is also available as GenBank Accession No. NM_152573.2. Herein, the phrase "RASEF gene" encompasses the human RASEF gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the RASEF gene.
The amino acid sequence encoded by the human RASEF gene is shown as SEQ ID NO: 16. In the present invention, the polypeptide encoded by the RASEF gene is referred to as "RASEF", and sometimes as "RASEF polypeptide" or "RASEF protein".

ERK1 is a member of the MAP kinase family. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act in a signaling cascade that regulates various cellular processes such as proliferation, differentiation, and cell cycle progression in response to a variety of extracellular signals. This kinase is activated by upstream kinases, resulting in its translocation to the nucleus where it phosphorylates nuclear targets. Alternatively spliced transcript variants encoding different protein isoforms have been described (GenBank Accession No.: NM_002746, NM_001040056, NM_001109891). The variant (1) (NM_002746) represents the most common transcript and encodes isoform 1. The nucleotide sequence of human ERK1 gene is shown in SEQ ID NO: 17, 19 or 21. Herein, the phrase "ERK1 gene" encompasses the human ERK1 gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the ERK1 gene. The amino acid sequence encoded by the human ERK1 gene is shown as SEQ ID NO: 18, 20 or 22. In the present invention, the polypeptide encoded by the ERK1 gene is referred to as "ERK1", and sometimes as "ERK1 polypeptide" or "ERK1 protein".

ERK2 is a member of the MAP kinase family. MAP kinases, also known as extracellular signal-regulated kinases (ERKs), act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. The activation of this kinase requires its phosphorylation by upstream kinases. Upon activation, this kinase translocates to the nucleus of the stimulated cells, where it phosphorylates nuclear targets. Two alternatively spliced transcript variants encoding the same protein, but differing in the UTRs, have been reported for this gene. This variant 1 (NM_002745) represents the longer transcript. Both variants 1 (NM_002745) and 2 (NM_138957) encode the same protein. The nucleotide sequence of human ERK2 gene is shown in SEQ ID NO: 23 or 24. Herein, the phrase "ERK2 gene" encompasses the human ERK2 gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the ERK2 gene. The amino acid sequence encoded by the human ERK2 gene is shown as SEQ ID NO: 25. In the present invention, the polypeptide encoded by the ERK2 gene is referred to as "ERK2", and sometimes as "ERK2 polypeptide" or "ERK2 protein".
The molecular weights of ERK1 and ERK2 protein are 44kDa and 42kDa, respectively.

According to an aspect of the present invention, functional equivalents are also included in the RASEF, ERK1 and ERK2. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptide that retains at least one biological activity of RASEF, ERK1 or ERK2 can be used as such a functional equivalent in the present invention. For example, functional equivalents of RASEF retain enhancing activity of cell proliferation and the functional equivalents of ERK1 and ERK2 retain kinase activity. In addition, the biological activity of RASEF contains binding activity to ERK1 and/or ERK2. Therefore, in a typical embodiment, a functional equivalent of RASEF can contain an ERK1 and/or ERK2 binding region. Also, the biological activity of ERK1 and/or ERK2 contains binding activity to RASEF. Therefore, in some embodiments, a functional equivalent of ERK1 and/or ERK2 can contain a RASEF binding region. In addition, functional equivalents of RASEF retain the property that is phosphorylated by the kinases such as ERK1 and ERK2.

Functional equivalents of RASEF include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the RASEF protein. Also, functional equivalents of ERK1 and ERK2 include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the ERK1 and ERK2 protein, respectively.
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 U S A. 1984 Sep;81(18):5662-6; Zoller MJ & Smith M. Nucleic Acids Res. 1982 Oct 25;10(20):6487-500; Wang A, et al., Science. 1984 Jun 29;224(4656):1431-3; Dalbadie-McFarland G, et al., Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409-13). One of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence which alters a single amino acid or a small percentage of amino acids is a "conservative modification" wherein the alteration of a protein results in a protein with similar functions.

Examples of properties of amino acid side chains are hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine), hydrophilic amino acids (arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (glycine, alanine, valine, leucine, isoleucine, praline); a hydroxyl group containing side-chain (serine, threonine, tyrosine); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (aspartic acid, asparagine, glutamic acid, glutamine); a base containing side-chain (arginine, lysine, histidine); and an aromatic containing side-chain (histidine, phenylalanine, tyrosine, tryptophan). Furthermore, conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
(1) Alanine (A), Glycine (G);
(2) Aspartic acid (D), Glutamic acid (E);
(3) Aspargine (N), Glutamine (Q);
(4) Arginine (R), Lysine (K);
(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
(7) Serine (S), Threonine (T); and
(8) Cysteine (C), Methionine (M)
(see, e.g., Thomas E. Creighton, Proteins Publisher: New York: W.H. Freeman, c1984).

Such conservatively modified polypeptides are included in the RASEF, ERK1 or ERK2 protein. However, the present invention is not restricted thereto and the RASEF, ERK1 or ERK2 protein includes non-conservative modifications so long as they retain any one of the biological activity of the RASEF, ERK1 or ERK2 protein. The number of amino acids to be mutated in such a modified protein is generally 10 amino acids of less, for example, 6 amino acids of less, for example, 3 amino acids or less.

An example of a protein modified by addition of one or more amino acids residues is a fusion protein of the RASEF, ERK1 or ERK2 protein. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the RASEF, ERK1 or ERK2 gene with a DNA encoding another peptide or protein, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. The "other" component of the fusion protein is typically a small epitope composed of several to a dozen amino acids. There is no restriction as to the peptides or proteins fused to the RASEF, ERK1 or ERK2 protein so long as the resulting fusion protein retains any one of the objective biological activities of the RASEF, ERK1 or ERK2 proteins. Exemplary fusion proteins contemplated by the instant invention include fusions of the RASEF, ERK1 or ERK2 protein and other small peptides or proteins such as FLAG (Hopp TP, et al., Biotechnology 6: 1204-10 (1988)), a polyhistidine (His-tag) such as 6xHis containing six His (histidine) residues or 10xHis containing 10 His residues, Influenza aggregate or agglutinin (HA), human c-myc fragment, Vesicular stomatitis virus glycoprotein (VSV-GP), p18HIV fragment, T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage), SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, and the like. Other examples of proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such.
Furthermore, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.

Methods known in the art to isolate functional equivalent proteins include, for example, hybridization techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Lab. Press, 2001). One skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human RASEF DNA sequences (e.g., SEQ ID NO: 15) encoding the human RASEF protein, and isolate functional equivalent proteins to the human RASEF protein from the isolated DNA. Also, one skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human ERK1 DNA sequences (e.g., SEQ ID NO: 17, 19 or 21) encoding the human ERK1 protein, and isolate functional equivalent proteins to the human ERK1 protein from the isolated DNA. Additionally, one skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human ERK2 DNA sequences (e.g., SEQ ID NO: 23 or 24) encoding the human ERK2 protein, and isolate functional equivalent proteins to the human ERK2 protein from the isolated DNA.

Thus, the proteins used for the present invention include those that are encoded by DNA that hybridize under stringent conditions with a whole or part of the DNA sequence encoding the human RASEF protein, human ERK1 protein or the human ERK2 protein and are functional equivalent to the human RASEF protein, human ERK1 protein or the human ERK2 protein. These proteins include mammal homologues corresponding to the proteins derived from human or mouse (for example, proteins encoded by monkey, rat, rabbit or bovine genes).

The conditions of hybridization for isolating a DNA encoding a protein functional equivalent to the human RASEF gene, human ERK1 gene or human ERK2 gene can be routinely selected by a person skilled in the art. 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 differ under 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 Centigrade lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength 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 can also be achieved with the addition of destabilizing agents for example, formamide. For selective or specific hybridization, a positive signal is at least two times of background, for example, 10 times of background hybridization.

For example, hybridization can be performed by conducting prehybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degreesC for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. A low stringent condition is, for example, 42 degrees C, 2x SSC, 0.1% SDS, for example, 50 degreesC, 2x SSC, 0.1% SDS. In some embodiments, high stringent condition is used. A high stringent condition is, for example, 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 degreesC for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors for example, temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein functional equivalent to the human RASEF, ERK1 or ERK2 gene, using a primer synthesized based on the sequence information of the DNA (SEQ ID NO: 15 for RASEF, 17, 19 and 21 for ERK1, 23 and 24 for ERK2) encoding the human RASEF, ERK1 or ERK2 protein (SEQ ID NO: 16 for RASEF, 18, 20 and 22 for ERK1 or 25 for ERK2), examples of primer sequences are pointed out in Semi-quantitative RT-PCR in [EXAMPLE].

Proteins that are functionally equivalent to the human RASEF, ERK1 or ERK2 protein encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology (also referred to as sequence identity) to the amino acid sequence of the human RASEF, ERK1 or ERK2 protein. "High homology" (also referred to as "high sequence identity") typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences). Typically, high homology or sequence identity refers to homology of 40% or higher, for example, 60% or higher, for example, 80% or higher, for example, 85%, 90%, 95%, 98%, 99%, or higher. The degree of homology or identity between two polypeptide or polynucleotide sequences can be determined by following the algorithm (Wilbur WJ & Lipman DJ. Proc Natl Acad Sci U S A. 1983 Feb; 80 (3):726-30).

Additional examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.

The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
A protein useful in the context of the present invention can 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 any one of the biological activity of the RASEF protein (SEQ ID NO: 16), ERK1 protein (SEQ ID NO: 18, 20 or 22) or ERK2 protein (SEQ ID NO: 25), it is useful in the present invention.

The present invention also encompasses the use of partial peptides of the RASEF protein, the ERK1 protein and the ERK2 protein. A partial peptide has an amino acid sequence specific to the RASEF protein, the ERK1 protein or the ERK2 protein and consists of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.

A partial RASEF peptide used for the screenings of the present invention suitably contains at least a binding domain of RASEF. Furthermore, a partial RASEF peptide used for the screenings of the present invention suitably contains ERK1 and/or ERK2 binding region or phosphorylation site. Such partial peptides are also encompassed by the phrase "functional equivalent" of the RASEF protein. Also, a partial peptide of ERK1 or ERK2 used for the screenings of the present invention suitably contains at least a binding domain of ERK1 and/or ERK2. Furthermore, a partial ERK1 or ERK2 peptide used for the screenings of the present invention suitably contains RASEF binding region. Such partial peptides are also encompassed by the phrase "functional equivalent" of the ERK1 or ERK2 protein.
The polypeptide or fragments used for the present method can be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
(1) Peptide Synthesis, Interscience, New York, 1966;
(2) The Proteins, Vol. 2, Academic Press, New York, 1976;
(3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
(4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
(5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
(6) WO99/67288; and
(7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.

Alternatively, the protein can be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison DA., et al., J Bacteriol. 1977 Oct;132(1):349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983;101:347-62). For example, first, a suitable vector comprising a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence comprising a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the RASEF, ERK1 or ERK2 is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.

A promoter can be used for the expression. Any commonly used promoters can be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF- alpha promoter (Kim DW, et al. Gene. 1990 Jul 16;91(2):217-23), the CAG promoter (Niwa H, et al., Gene. 1991 Dec 15;108(2):193-9), the RSV LTR promoter (Cullen BR. Methods Enzymol. 1987;152:684-704), the SR alpha promoter (Takebe Y, et al., Mol Cell Biol. 1988 Jan;8(1):466-72), the CMV immediate early promoter (Seed B & Aruffo A. Proc Natl Acad Sci U S A. 1987 May;84(10):3365-9), the SV40 late promoter (Gheysen D & Fiers W. J Mol Appl Genet. 1982;1(5):385-94), the Adenovirus late promoter (Kaufman RJ, et al., Mol Cell Biol. 1989 Mar;9(3):946-58), the HSV TK promoter, and such.

The introduction of the vector into host cells to express the RASEF, ERK1 or ERK2 gene can be performed according to any methods, for example, the electroporation method (Chu G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26), the calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52), the DEAE dextran method (Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25;12(14):5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug;4(8):1641-3), the Lipofectin method (Derijard B, et al., Cell. 1994 Mar 25;76(6):1025-37; Lamb BT, et al., Nat Genet. 1993 Sep;5(1):22-30; Rabindran SK, et al., Science. 1993 Jan 8;259(5092):230-4), and such.
The proteins can also be produced in vitro by using an in vitro translation system.

(2) Antibodies
The terms "antibody" as used herein is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide thereof. An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Furthermore, an antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An "antibody" indicates all classes (e.g. IgA, IgD, IgE, IgG and IgM).
The present invention may use antibodies against RASEF protein or ERK1 and/or ERK2 protein. These antibodies can be useful for diagnosing lung cancer. Furthermore, the present invention may use antibodies against partial peptides of RASEF polypeptides or ERK1 and/or ERK2 polypeptides.

In the screening methods described bellow, antibodies against the ERK1 and/or ERK2 binding region of RASEF polypeptides or the RASEF binding region of the ERK1 and/or ERK2 polypeptides may be used. These antibodies can be useful for inhibiting and/or blocking an interaction, e.g. binding, between RASEF polypeptides and ERK1 and/or ERK2 polypeptides and can be useful for treating and/or preventing cancer (over)expressing RASEF, ERK1 and/or ERK2, e.g. lung cancer. These antibodies will be provided by known methods. For techniques for the production of the antibodies used in the present invention, conventional methods can be used.

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

As used herein, the term "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 ribonucleotide corresponding to a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), a ribonucleotide corresponding to an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand") or both. The siRNA can 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 can either be a dsRNA or shRNA.

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

As used herein the term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the region is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting 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 can also be referred to as an "intervening single-strand".

As used herein, the term "siD/R-NA" refers to a double-stranded 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 an oligonucleotide composed of DNA and an oligonucleotide 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 can contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand") or both. The siD/R-NA can 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 can 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 comprising 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 can comprise 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, comprising a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting 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 can also be referred to as "intervening single-strand".

(i) Target sequences
As used herein, the term "target sequence" is a nucleotide sequence within an mRNA or cDNA sequence of a target gene, which will result in suppress of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene. A nucleotide sequence within an mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the target gene in a cell expressing the gene. The double stranded polynucleotide by which suppresses the gene expression may consist of the target sequence and 3' overhang (e.g., uu).
When a target sequence is shown by cDNA sequence, the sense strand of the double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.

Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s. For example, when a target sequence is shown in the DNA sequence of SEQ ID NO: 9 and the sense strand of the double-stranded molecule has the 3' side half region composed of DNA, "a sequence corresponding to a target sequence" is "5'- GUUAGUACCTTGTACCAAA-3'"
Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand. For example, when a target sequence is shown in the DNA sequence of SEQ ID NO: 9 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA, "a complementary sequence to a target sequence" is "3'- CAAUCAUGGAACATGGTTT -5'".

On the other hand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence of SEQ ID NO: 9 is the DNA sequence of SEQ ID NO: 9, and the complementary sequence corresponding to a target sequence of SEQ ID NO: 9 is the RNA sequence of "3'- CAAUCAUGGAACAUGGUUU -5'" .
A double-stranded molecule may has one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
A double-stranded molecule against RASEF gene, which molecule hybridizes to a RASEF mRNA, inhibits or reduces production of RASEF protein encoded by the gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the RASEF protein.
The expression of RASEF gene in cancer cell lines, was inhibited by each two double-stranded molecules (Fig. 4).
Therefore, the present invention provides isolated double-stranded molecules having the property to inhibit or reduce the expression of RASEF gene in cancer cells when introduced into a cell. The target sequences of double-stranded molecules may be designed by siRNA design algorithm mentioned below.

In some embodiments, target sequences for RASEF include, for example,
5'- GTTAGTACCTTGTACCAAA -3' (SEQ ID NO: 9) or
5'- CTTCATCCGTGAGATCAGA -3' (SEQ ID NO: 10).
In other words, the present invention also provides a double-stranded molecule whose target sequence comprises or consisting of SEQ ID NO: 9 or 10.

Specifically, the present invention provides the following double-stranded molecules [1] to [18]:
[1] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a RASEF gene and cell proliferation, wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10;
[2] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a RASEF gene and cell proliferation, wherein the double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 ;.
[3] The double-stranded molecule of [1] or [2], wherein the target sequence comprises from about 19 to about 25 contiguous nucleotide from the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO:10.
[4] The double-stranded molecule of any one of [1] to [2], which has a length of less than about 100 nucleotides.
[5] The double-stranded molecule of [4], which has a length of less than about 75 nucleotides.
[6] The double-stranded molecule of [5], which has a length of less than about 50 nucleotides.
[7] The double-stranded molecule of [6] which has a length of less than about 25 nucleotides.
[8] The double-stranded molecule of [7], which has a length of between about 19 and about 25 nucleotides.
[9] The double-stranded molecule of any one of [1] to [8], which consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[10] The double-stranded molecule of [9], which has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein
[A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10
[B] is an intervening single-strand; and
[A'] is an antisense strand comprising a nucleotide sequence corresponding to a sequence complementary to the target sequence selected in [A].
[11] The double-stranded molecule of any one of [1] to [10], which comprises RNA.
[12] The double-stranded molecule of any one of [1] to [11], which comprises both DNA and RNA.
[13] The double-stranded molecule of [12], which 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 made of DNA and RNA, respectively.
[15] The double-stranded molecule of [12], which is a chimera of DNA and RNA.
[16] The double-stranded molecule of [15], wherein a 5'-end region of the target sequence in the sense strand, and/or a 3'-end region of the complementary sequence of the target sequence in the antisense strand consists of RNA.
[17] The double-stranded molecule of [16], wherein the RNA region consists of 9 to 13 nucleotides; and
[18] The double-stranded molecule of any one of [1] to [2], which contains one or two 3' overhang(s).

The double-stranded molecule of the present invention will be 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 Pat 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 (on the worldwide web at ambion.com/techlib/misc/siRNA_finder.html).
The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.

Design 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. recommend to avoid designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these can be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes can 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: on the worldwide web at ncbi.nlm.nih.gov/BLAST/, is used (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.

Preferred target sequences for RASEF gene designed in "Examples" include
5'- GTTAGTACCTTGTACCAAA -3' (SEQ ID NO: 9) or
5'- CTTCATCCGTGAGATCAGA -3' (SEQ ID NO: 10).
Specifically, the present invention provides the double-stranded molecules targeting any one of above-mentioned target sequences that were respectively examined for their ability to inhibit and reduce the growth of cancer cells expressing the target genes. The growth of cancer cells expressing RASEF gene, were inhibited and reduced by double-stranded molecules of the present invention (Fig. 4).
Therefore, the present invention provides double-stranded molecules targeting a target sequence for RASEF gene selected from the group consisting of
5'- GTTAGTACCTTGTACCAAA -3' (SEQ ID NO: 9) and
5'- CTTCATCCGTGAGATCAGA -3' (SEQ ID NO: 10).

The double-stranded molecules of the present invention targeting the above-mentioned target sequence of RASEF gene include isolated polynucleotide(s) that comprises any one of the nucleic acid sequences of target sequences and/or complementary sequences to the target sequences. Examples of a double-stranded molecule targeting RASEF gene include an oligonucleotide comprising the sequence corresponding to SEQ ID NO: 9 or 10, and complementary sequences thereto. 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 RASEF gene. Herein, "minor modification" in a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.

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

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

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

In the present invention, double-stranded molecules targeting the RASEF gene may have a sequence selected from among SEQ ID NOs: 9 and 10 as a target sequence. Accordingly, examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 9 or 10 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 9 or 10 and a complementary sequence thereto.

According to the present invention, a double-stranded molecule of the present invention can be tested for its ability to inhibit gene expression using the methods utilized in the Examples (see, RNA interference assay in "EXAMPLES"). In the Examples, the double-stranded molecules comprising sense strands and antisense strands complementary thereto of various portions of mRNA of RASEF genes were tested in vitro for their ability to decrease production of RASEF gene product in cancers cell lines (e.g., using A549 and LC319) according to standard methods. Furthermore, for example, reduction in RASEF 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 RASEF gene mRNA mentioned (see,(b)) Semi-quantitative RT-PCR in "EXAMPLES"). Sequences which decrease the production of RASEF gene product in vitro cell-based assays can then be tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assays can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of RASEF 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 comprises modified nucleotides and/or non-phosphodiester linkages, these polynucleotides can also bind each other in the same manner. Generally, complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. Furthermore, the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization. In one embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In some embodiments, where the strands of the duplex are fully complementary, such duplexes contain no mismatches.

The polynucleotide of the invention are typically less than 500, 200, 100, 75, 50, or 25 nucleotides in length. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against RASEF gene or preparing template DNAs encoding the double-stranded molecules. When the polynucleotides are used for forming double-stranded molecules, the sense strand of the polynucleotide can be longer than 19 nucleotides, for example, longer than 21 nucleotides, for example, between about 19 and 25 nucleotides.
Accordingly, the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence. In some embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.

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

The double-stranded molecules of the invention can contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which can be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxyabasic residue incorporation (US Pat Appl. No. 20060122137).

In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).

In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides can be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents for example, US Pat Appl. No.20060234970 are available. The present invention is not limited to these examples and any known chemical modifications can be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.

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

In some embodiments, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may have the structure either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. In order to enhance stability of the double-stranded molecule, in some embodiments, the molecule 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. In one example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA.

The upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in some embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention comprise 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 can be a domain of about 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, 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 (US Pat Appl. No. 20050004064).

In the present invention, the double-stranded molecule can form a hairpin, for example, a short hairpin RNA (shRNA) and short hairpin made 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 comprises the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.

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. 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 can be selected from the group consisting of, for example, SEQ ID NO: 9 and SEQ ID NO: 10..

The present invention is not limited to these examples, and the target sequence in [A] can be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted RASEF gene and result in inhibits or reduces the cell expressing these genes. The region [A] hybridizes to [A'] to form a loop comprising the region [B]. The intervening single-stranded portion [B], i.e., the loop sequence can be 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from group consisting of following sequences (on the worldwide web at 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.

Exemplary double-stranded molecules having hairpin loop structure of the present invention are shown below. In the following structure, the loop sequence can be selected from group consisting of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:
GUUAGUACCUUGUACCAAA-[B]- UUUGGUACAAGGUACUAAC (for target sequence of SEQ ID NO: 9);
CUUCAUCCGUGAGAUCAGA-[B]-UCUGAUCUCACGGAUCAAG (for target sequence of SEQ ID NO: 10);
Furthermore, in order to enhance the inhibition activity of the double-stranded molecules, several nucleotides can be added to 3'end of the sense strand and/or the antisense strand, as 3' overhangs. The number of nycleotides to be added is at least 2, generally 2 to 10, for example, 2 to 5. The added nucleotides form single strand at the 3'end of sense strand and/or the antisense strand of the double-stranded molecule. The nucleotides for 3' overhang are preferably "u" or "t", but are not limited to. When the double-stranded molecule has a harpin loop structure, a 3' overhang is added to the 3' end of the antisense strand.

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

Alternatively, the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence. The regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules will be described below.

(ii) Vector
Also included in the invention is a vector containing one or more of the double-stranded molecules described herein, and a cell containing the vector. A vector of the present invention 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. In one embodiment, the vector includes regulatory elements necessary for expression of the double-stranded molecule. Accordingly, in one embodiment, the expression vector encodes the double-stranded molecule of the present invention and is adapted for expression of the double-stranded molecule. Such vectors of the present invention can be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.

Alternatively, the present invention provides vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 or 10, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the RASEF gene, inhibits expression of said gene. In some embodiments, the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 15). More usually, the combination of polynucleotide comprises a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence. More preferably, the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein [A] is a nucleotide sequence comprising SEQ ID NO: 9 or 10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].

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

The vectors of the present invention can also be equipped so as to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Pat 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 (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Pat No. 5,922,687).

The vectors of the present invention can be, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, for example, vaccinia or fowlpox (see, e.g., US Pat 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) vectors. 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.

(iii) Methods of Inhibiting or Reducing a Growth of Cancer Cells and Treating or Preventing Cancer Using Double-Stranded Molecules
In the present invention, double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to inhibit or reduce the growth of cells (over)expressing the target genes. The growth of cancer cells (over)expressing RASEF gene, was inhibited or reduced by double-stranded molecules of the present invention (Fig. 4).
Therefore, the present invention provides methods for inhibiting cell growth, i.e., cancerous cell growth of a cell from a cancer resulting from overexpression of a RASEF gene, or that is mediated by a RASEF gene, by inhibiting the expression of the RASEF gene. RASEF gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention which specifically target the expression of RASEF gene or the vectors of the present invention that can express any of the double-stranded molecules of the present invention.

The ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cells indicates that they can be used for methods for treating cancer, a cancer resulting from overexpression of a RASEF gene, or that is mediated by a RASEF gene. Thus, the present invention provides methods to treat patients with a cancer resulting from overexpression of RASEF gene, or that is mediated by a RASEF gene by administering a double-stranded molecule, i.e., an inhibitory nucleic acid, against a RASEF gene or a vector expressing the molecule without adverse effect because those genes were hardly detected in normal organs.

Specifically, the present invention provides the following methods [1] to [23]:
[1] A method for inhibiting or reducing a growth of a cell (over)expressing a RASEF gene or a method for treating or preventing cancer (over)expressing RASEF gene, wherein said method comprising the step of administering to a subject at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said RASEF gene.
[2] The method of [1], wherein said double-stranded molecule acts at mRNA which shares sequence identity with or is complementary to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10.
[3] The method of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein said sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10.
[4] The method of any one of [1] to [3], wherein more than one of the double-stranded molecules are administered;
[5] The method of [4], wherein the double-stranded molecules target the same gene;
[6] The method of any one of [1] to [5], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[7] The method of [6], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[8] The method of [7], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[9] The method of [8], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[10] The method of any one of [1] to [9], wherein the sense strand hybridize with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length;
[11] The method of any one of [1] to [10], wherein said double-stranded molecule consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[12] The method of [11], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10.;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[13] The method of any one of [1] to [12], wherein the double-stranded molecule comprises RNA.
[14] The method of any one of [1] to [12], wherein the double-stranded molecule comprises both DNA and RNA.
[15] The method of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
[16] The method of [15] wherein the sense and antisense strand polynucleotides a made of DNA and RNA, respectively.
[17] The method of [14], wherein the double-stranded molecule is a chimera of DNA and RNA.
[18] The method of [17], wherein a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides a made of RNA.
[19] The method of [18], wherein the flanking region consists of 9 to 13 nucleotides.
[20] The method of any one of [1] to [19], wherein the double-stranded molecule contains one or two 3' overhang(s).
[21] The method of any one of [1] to [20], wherein the double-stranded molecule is encoded by a vector.
[22] The method of [21], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[23] The method of any one of [1] to [22], wherein the double-stranded molecule is contained in a composition which comprises in addition to the molecule a transfection-enhancing agent and/or cell permeable agent.

The method of the present invention will be described in more detail below.
The growth of cells (over)expressing a RASEF gene is inhibited by contacting the cells with a double-stranded molecule against RASEF gene, a vector expressing the molecule or a composition comprising 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 compared to a cell not exposed to the molecule. Cell growth can be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
The growth of any kind of cell can 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 cancers cells.

Thus, patients suffering from or at risk of developing disease related to RASEF gene can be treated by administering 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 comprising at least one of the molecules. For example, patients of cancers can be treated according to the present methods. The type of cancer can be identified by standard methods according to the particular type of tumor to be diagnosed. In some embodiments, patients treated by the methods of the present invention are selected by detecting the (over)expression of a RASEF gene in a biopsy from the patient by RT-PCR, hybridization or immunoassay. In some embodiments, before the treatment of the present invention, the biopsy specimen from the subject is confirmed for RASEF gene over-expression by methods known in the art, for example, immunohistochemical analysis, hybridization or RT-PCR (see, Semi-quantitative RT-PCR, Western-blotting or Immunohistochemistry in "EXAMPLES").

According to the present method to inhibit or reduce cell growth and thereby treating cancer, when administering more than one of the double-stranded molecules (or vectors expressing or compositions containing the same), each of the molecules can direct to the different target sequence of same gene, or different target sequences of different gene. For example, the method can utilize different double-stranded molecules directing to RASEF gene transcript. Alternatively, for example, the method can utilize double-stranded molecules directed to one, two or more target sequences selected from same gene.

For inhibiting cell growth, a double-stranded molecule of present invention can 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 can be introduced into cells as a vector. For introducing the double-stranded molecules and vectors into the cells, transfection-enhancing agent, for example, FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), can be employed.

A treatment is determined efficacious if it leads to clinical benefit for example, reduction in expression of a RASEF 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.
It is understood that the double-stranded molecule of the invention degrades the target mRNA (gene transcripts) 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, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.

One skilled in the art can readily determine an effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors for example, 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 comprises an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, for example, from about 2 nM to about 50 nM, for example, 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 present methods can be used to inhibit the growth or metastasis of cancer; for example, a cancer resulting from overexpression of a RASEF gene or that is mediated by a RASEF gene, e.g., lung cancer. In particular, a double-stranded molecule directed to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 for RASEF find use for the treatment of cancers.

For treating cancer, e.g., a cancer promoted by a RASEF gene, 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, for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
In the 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 which 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. In one embodiment, the delivery reagent is a liposome.

Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, for example, retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, for example, cholesterol. The selection of lipids is generally guided by consideration of factors for example, 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.

In some embodiments, the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site. Ligands which bind to receptors prevalent in tumor or vascular endothelial cells, for example, monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, find use.
In some embodiments, 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 comprise 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 can be water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, for example, 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 for example, 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, for example, ganglioside GM₁. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.

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

Vectors expressing a double-stranded molecule of the 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 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 (for example, 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 comprising a porous, non-porous, or gelatinous material); and inhalation. In some embodiments, injections or infusions of the double-stranded molecule or vector can be given at or near the site of 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 agent can be directly into the tissue or near the site of cancer. Multiple injections of the agent into the tissue at or near the site of cancer can be administered.

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, for example, from about seven to about ten days. In one exemplary dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. When a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.

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

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

The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
According to the present invention, the expression level of RASEF in cancer cell in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of RASEF 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 RASEF. Those skilled in the art can prepare such probes utilizing the sequence information of RASEF. For example, the cDNA of RASEF may be used as the probes. If necessary, the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.

Furthermore, the transcription product of RASEF may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers may be prepared based on the available sequence information of the gene.
Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of RASEF. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to 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 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their 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 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the quantity of RASEF protein (SEQ ID NO: 16) may be determined. Methods for determining the quantity of the protein as the translation product include 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 or modified antibody retains the binding ability to the RASEF 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 RASEF gene based on its translation product, the intensity of staining may be measured via immunohistochemical analysis using an antibody against the RASEF protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of RASEF gene.
The expression level of a target gene, i.e., the RASEF gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target 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 cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known. In addition, normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of RASEF gene in samples from subjects whose disease states are known. Furthermore, the control level can be derived from a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of RASEF gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. It is typical to use a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, it is preferred to use the standard value of the expression levels of RASEF gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.

In the context of the present invention, a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level".
When the expression level of RASEF gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.

(iv) Compositions
Furthermore, the present invention provides pharmaceutical compositions comprising at least one of the present double-stranded molecules or the vectors coding for the molecules.
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.
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. Before being formulated, the "active ingredient" may also be referred to as "bulk", "drug substance" or "technical product".

Specifically, the present invention provides the following compositions [1] to [24]:
[1] A composition for inhibiting or reducing a growth of cell expressing RASEF gene, or for treating or preventing a cancer expressing a RASEF gene, which comprises at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said gene.
[2] The composition of [1], wherein said double-stranded molecule acts at mRNA which matched a target sequence selected from the group SEQ ID NO: 9 and SEQ ID NO: 10 for RASEF.
[3] The composition of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein said sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10.
[4] The composition of any one of [1] to [3], wherein the cancer to be treated is lung cancer;
[5] The composition of [4], wherein the lung cancer is small cell lung cancer or non-small cell lung cancer;
[6] The composition of any one of [1] to [5], wherein the composition contains more than one of the double-stranded molecules;
[7] The composition of [6], wherein the the double-stranded molecules target the same gene;
[8] The composition of any one of [1] to [7], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[9] The composition of [8], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[10] The composition of [9], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[11] The composition of [10], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[12] The composition of any one of [1] to [11], wherein the sense strand hybridize with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length;
[13] The composition of any one of [1] to [12], wherein said double-stranded molecule consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[14] The composition of [13], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3' or 5'-[A']-[B]-[A]-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[15] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises RNA;
[16] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises DNA and RNA;
[17] The composition of [16], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[18] The composition of [17], wherein the sense and antisense strand polynucleotides are made of DNA and RNA, respectively;
[19] The composition of [18], wherein the double-stranded molecule is a chimera of DNA and RNA;
[20] The composition of [19], wherein at least a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides consists of RNA.
[21] The composition of [20], wherein the flanking region consists of 9 to 13 nucleotides;
[22] The composition of any one of [1] to [21], wherein the double-stranded molecule contains one or two 3' overhang(s);
[23] The composition of any one of [1] to [22], wherein the double-stranded molecule is encoded by a vector;
[24] The composition of any one of [1] to [23], which further comprising a transfection-enhancing agent, cell permeable agent or pharmaceutically acceptable carrier.

The composition of the present invention will be described in more detail below.
The double-stranded molecules of the invention can be 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. 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 comprise 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. Exemplary physiologically acceptable carrier media include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
According to the present invention, the composition can contain more than one of the double-stranded molecules, each of the molecules can be directed to the same target sequence, or different target sequences of RASEF gene. For example, the composition can contain double-stranded molecules directed to RASEF gene. Alternatively, for example, the composition can contain double-stranded molecules directed to target sequences selected from RASEF gene.

Furthermore, the present composition can contain a vector coding for one or plural double-stranded molecules. For example, the vector can encode one, two or several kinds of the present double-stranded molecules. Alternatively, the present composition can contain more than one of the vectors, each of the vectors coding for a different double-stranded molecule.
Moreover, the present double-stranded molecules can be contained as liposomes in the present composition. See under the item of "(iii) Methods Of Inhibiting Or Reducing A Growth Of Cancer Cells And Treating Or Preventing Cancer Using Double-Stranded Molecules r" for details of liposomes.

Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. 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 (for example, 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 grade 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 comprise any of the carriers and excipients listed above and 10-95%, for example, 25-75%, of one or more double-stranded molecule of the invention. A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, for example, 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 can contain other pharmaceutical active ingredients so long as they do not inhibit the in vivo function of the present double-stranded molecules. For example, the composition can contain chemotherapeutic agents conventionally used for treating cancers.

The present invention also provides the use of the double-stranded nucleic acid molecules of the present invention in manufacturing a pharmaceutical composition for treating a cancer (over)expressing the RASEF gene. For example, the present invention relates to the use of double-stranded nucleic acid molecule inhibiting the (over)expression of a RASEF gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10, for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the RASEF gene.
Alternatively, the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the RASEF gene.

The present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the RASEF gene, wherein the method or process comprises step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the (over)expression of a RASEF gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10 as active ingredients.
The present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the RASEF gene, wherein the method or process comprises 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 RASEF gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9 or 10.

(4) Method for Diagnosing RASEF -Mediated Cancers
The expression of RASEF gene were found to be specifically elevated in lung cancer tissues compared with corresponding normal tissues (Fig. 2A, B). Therefore, the genes identified herein as well as its transcription and translation products have diagnostic utility as markers for cancers mediated by RASEF gene and by measuring the expression of the RASEF gene in a sample derived from a patient suspected to be suffering from cancers, these cancers can be diagnosed. Specifically, the present invention provides a method for diagnosing cancers mediated by RASEF by determining the expression level of RASEF in a subject. The RASEF -promoted cancers that can be diagnosed by the present method include lung cancers. Lung cancers include non-small lung cancer (NSCLC), small-cell lung cancer (SCLC). NSCLC includes a lung adenocarcinoma (ADC), a lung squamous-cell carcinoma (SCC), and a lung large-cell carcinoma (LCC). According to the present invention, an intermediate result for examining the condition of a subject can be provided. Such intermediate result can be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention can 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.

Specifically, the present invention provides the following methods [1] to [15]:
[1] A method for diagnosing cancer mediated or promoted by a RASEF, wherein said method comprises the steps of:
(a) detecting the expression level of RASEF gene in a subject-derived biological sample; and
(b) relating an increase of the expression level compared to a normal control level of the gene to the disease.
[2] A method of detecting or diagnosing cancer in a subject, comprising determining a expression level of RASEF in a subject-derived biological sample, wherein an increase of the level compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing cancer, or the presence of cancer in the subject,
[3] The method of [1] or [2], wherein the expression level is at least 10 % greater than normal control level.
[4] The method of any one of [1] to [3], wherein the expression level is detected by any one of the method select from the group consisting of:
(a) detecting the mRNA encoding the RASEF polypeptide;
(b) detecting the RASEF polypeptide; and
(c) detecting the biological activity of the RASEF polypeptide.
[5] The method of any one of [1] to [3], wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of RASEF gene,
(b) detecting a protein encoded by RASEF gene, and
(c) detecting (a) biological activity(activities) of a protein encoded by RASEF gene.
[6] The method of any one of [1] to [5], wherein the cancer results from overexpression of a RASEF, or is mediated or promoted by a RASEF.
[7] The method of any one of [1] to [6], wherein the cancers is lung cancer.
[8] The method of [7], wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.
[9] The method of [4] or [5], wherein the expression level is determined by detecting a hybridization of probe to the gene transcript encoding the RASEF polypeptide.
[10] The method of [4] or [5], wherein the expression level is determined by detecting a binding of an antibody against the RASEF polypeptide.
[11] The method of any one of [1] to [10], wherein the biological sample comprises biopsy sample, sputum or blood.
[12] The method of any one of [1] to [10], wherein the subject-derived biological sample comprises an epithelial cell, serum or pleural effusion.
[13] The method of [1] to [12], wherein the subject-derived biological sample comprises a cancer cell.
[14] The method of [1] to [13], wherein the subject-derived biological sample comprises a cancerous epithelial cell.
[15] The method of [1] to [10], wherein the subject-derived biological sample comprises a lung tissue.

The method of diagnosing cancers will be described in more detail below.
A subject to be diagnosed by the present method is can be a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
In performing the present methods, a biological sample is collected from a subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it comprises the objective transcription or translation product of RASEF gene. The biological samples include, but are not limited to, bodily tissues and fluids, for example, blood, e.g. serum, sputum, urine and pleural effusion. In some embodiments, the biological sample contains a cell population comprising an epithelial cell, for example, a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell can 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 RASEFgene in the 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, the mRNA of RASEF gene can be quantified using probes by hybridization methods (e.g. Northern blot analysis). The detection can be carried out on a chip or an array. The use of an array can be for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including RASEF gene. Those skilled in the art can prepare such probes utilizing the sequence information of the RASEF (SEQ ID NO: 15; GenBank Accession No. NM_152573.2). For example, the cDNA of RASEF gene can be used as a probe. If necessary, the probe can be labeled with a suitable label, for example, dyes, fluorescent and isotopes, and the expression level of the gene can be detected as the intensity of the hybridized labels.

Furthermore, the transcription product of RASEF gene can be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers (SEQ ID NO: 1 and 2, 5 and 6 or 11 and 12 for RASEF) used in the Example can be employed for the detection by RT-PCR or Northern blot, 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 RASEF gene.

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

As another method to detect the expression level of RASEF based on its translation product, the intensity of staining can be observed via immunohistochemical analysis using an antibody against RASEF protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of RASEF (see, Immunohistochemistry and Tissue-microarray analysis in "EXAMPLES").
In the context of the present invention, methods for detecting or identifying cancer in a subject or cancer cells in a subject-derived sample begin with a determination of RASEF gene expression level. Once determined, using any of the aforementioned techniques, this value is as compared to a control level.

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 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 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 lung. An increase in the expression level of RASEF 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 lung cancer. In the context of the present invention, the subject-derived sample may be any tissues obtained from test subjects, e.g., patients suspected of having cancer. For example, tissues may include epithelial cells. More particularly, tissues may be epithelial cells collected from a suspected cancerous area. Alternatively, the expression level of RASEF in a sample can be compared to a cancer control level of RASEF 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.

The control level can 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 can be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of RASEF 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 a RASEF in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used. In some embodiments, the standard value of the expression levels of RASEF in a population with a known disease state is used. The standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
To improve the accuracy of the diagnosis, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in lung cancer may also be determined, in addition to the expression level of the RASEF gene. Furthermore, in the case where the expression levels of multiple cancer-related genes are compared, a similarity in the gene expression pattern between the sample and the reference that is cancerous indicates that the subject is suffering from or at a risk of developing lung cancer.

In the context of the present invention, 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 lung cancer marker genes including RASEF gene in a biological sample can be considered to be increased if it increases from a control level of the corresponding lung 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.
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.

Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung tissue sample, said method comprising the step of determining the expression level of the RASEF gene in a subject-derived biological 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 tissue.

Such result may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the RASEF gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic 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. 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.

Alternatively, the present invention provides use of a reagent for preapring a diagnostic reagent for diagnosing cancer. In some embodiments, the reagent can be selected from the group consisting of:
(a) a reagent for detecting mRNA of the RASEF gene;
(b) a reagent for detecting the RASEF protein; and
(c) a reagent for detecting the biological activity of the RASEF protein.
Specifically, such reagent is an oligonucleotide that hybridizes to the RASEF polynucleotide, or an antibody that binds to the RASEF polypeptide.
In other words, the present invention also provides a kit for use in diagnosis or detection of cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the RASEF gene.

In the present invention, it is revealed that RASEF is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting RASEF can be achieved by the present invention. In the present invention, the cancer treatment targeting RASEF refers to suppression or inhibition of RASEF activity and/or expression in the cancer cells. Any anti-RASEF agents may be used for the cancer treatment targeting RASEF. In the present agents may be used for the cancer treatment targeting RASEF. In the present invention, the anti-RASEF agents include following substance as active ingredient:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
Accordingly, in a typical embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of:
a) determining the expression level of RASEF in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of RASEF with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of RASEF is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).

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

(5) Method for Assessing the Prognosis of RASEF Mediated Cancer
The present invention is based, in part, on the discovery that RASEF (over)expression is significantly associated with poorer prognosis of patients with RASEF-mediated cancers, e.g., lung cancers, more typically NSCLC. Thus, the present invention provides a method for determining or assessing the prognosis of a patient with cancer, e.g., a cancer mediated by or resulting from overexpression of a RASEF, e.g, lung cancer, by detecting the expression level of the RASEF 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).

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 or 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, estimated time of survival, and the like). For example, a determination of the expression level of RASEF 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 for example, disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.

Specifically, the present invention provides the following methods [1] to [7]:
[1] A method for assessing prognosis of a subject with lung cancer, wherein the method comprises steps of:
(a) detecting an expression level of RASEF in a subject-derived biological sample;
(b) comparing the detected expression level to a control level; and
(c) determining prognosis of the patient based on the comparison of (b);
[2] The method of [1], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis;
[3] The method of [1], wherein the control level is a poor prognosis control level and a similar expression level to the control level indicates poor prognosis;
[4] The method of [2], wherein the increase is at least 10% greater than said control level; and
[5] The method of any one of [1] to [4], wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of RASEF gene;
(b) detecting a protein encoded by RASEF gene; and
(c) detecting a biological activity of the protein encoded by RASEF gene;
[6] The method of any one of [1] to [5], wherein the subject-derived biological sample comprises a lung cancer tissue or lung cancer cells.
[7] The method of [6], wherein the lung cancer is NSCLC or SCLC.

The patient-derived biological sample used for the method can be any sample derived from the subject to be assessed so long as the RASEF gene can be detected in the sample. In some embodiments, the biological sample comprises a lung cell (a cell obtained from lung ). Furthermore, the biological sample includes bodily fluids for example, sputum, blood, serum, plasma, pleural effusion, and so on. Moreover, the sample can be cells purified or obtained from a tissue. The biological samples can be obtained from a patient at various time points, including before, during, and/or after a treatment. For example, a lung cancer cell(s) obtained from a subject to be assessed is a preferable biological sample.

According to the present invention, it was shown that the higher the expression level of the RASEF gene measured in the patient-derived biological sample, the poorer the prognosis for post-treatment remission, recovery, and/or survival and the higher the likelihood of poor clinical outcome. Thus, according to the present method, the "control level" used for comparison can be, for example, the expression level of the RASEF 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 will be referred to as "good prognosis control level". Alternatively, the "control level" can be the expression level of the RASEF 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" is a single expression pattern derived from a single reference population or from a plurality of expression patterns. Thus, the control level can be determined based on the expression level of the RASEF 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. In some embodiments, the cancer is lung cancer. In some embodiments, the standard value of the expression levels of the RASEF gene in a patient group with a known disease state is used. The standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.

The control level can 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 can be determined by a statistical method based on the results obtained by analyzing the expression level of the RASEF 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 or patients. Moreover, according to an aspect of the present invention, the expression level of the RASEF gene in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used.

According to the present invention, a similarity in the expression level of the RASEF gene to the good prognosis control level indicates a more favorable prognosis of the patient and an increase in the expression level in comparison to the good prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. On the other hand, a decrease in the expression level of the RASEF gene in comparison to the poor prognosis control level indicates a more favorable prognosis of the patient and a similarity in the expression level to the poor prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. For example, a 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.
An expression level of the RASEF gene in a biological sample can be considered altered (i.e., increased or decreased) 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, can be used to normalize the expression levels of the RASEF gene.
The expression level can 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, for example, mRNA and protein.

For instance, the transcription product of the RASEF gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a RASEF gene probe to the gene transcript. The detection can be carried out on a chip or an array. An array can be used for detecting the expression level of a plurality of genes including the RASEF gene. As another example, amplification-based detection methods, for example, reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the RASEF gene can be employed for the detection (see (b) Semi-quantitative RT-PCR in [EXAMPLE]). The RASEF gene-specific probe or primers can be designed and prepared using conventional techniques by referring to the whole sequence of the RASEF (SEQ ID NO: 15). For example, the primers (SEQ ID NOs: 1 and 2, 5 and 6, 11 and 12) used in the Example can 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 RASEF 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 Centigrade 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 30degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60degrees Centigrade for longer probes or primers. Stringent conditions can also be achieved with the addition of destabilizing agents, for example, formamide.

Alternatively, the translation product can be detected for the assessment of the present invention. For example, the quantity of the RASEF protein can 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 RASEF protein. The antibody can be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to the RASEF protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.

As another method to detect the expression level of the RASEF gene based on its translation product, the intensity of staining can be observed via immunohistochemical analysis using an antibody against RASEF protein. Namely, the observation of strong staining indicates increased presence of the RASEF protein and at the same time high expression level of the RASEF gene.
Furthermore, the RASEF protein is known to have a cell proliferating activity. Therefore, the expression level of the RASEF gene can be determined using such cell proliferating activity as an index. For example, cells which express RASEF 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 RASEF gene, the expression level of other lung cell-associated genes, for example, genes known to be differentially expressed in lung cancer, can also be determined to improve the accuracy of the assessment. Such other lung cancer-associated genes include those described in WO 2004/031413 and WO 2005/090603.
The patient to be assessed for the prognosis of cancer according to the method can be a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.

Alternatively, according to the present invention, an intermediate result can also be provided in addition to other test results for assessing the prognosis of a subject. Such intermediate result can assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject. Additional information that can 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 RASEF gene is useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g. NSCLC). Therefore, the present invention also provides a method for detecting prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer including NSCLC, which comprises steps of:
a) detecting or determining an expression level of a RASEF 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 to the control level is indicative of potential or suspicion of poor prognosis (poor survival).

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

(6) Kits for Diagnosing Cancer or Assessing the Prognosis of Cancer
The present invention provides a kit for diagnosing cancer or assessing the prognosis of cancer. The present invention also provides a kit for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of a cancer treatment. In some embodiments, the cancer is mediated by RASEF, or resulting from overexpression of RASEF, e.g., lung cancer, more typically NSCLC. Specifically, the kit comprises at least one reagent for detecting the expression of the RASEF gene in a patient-derived biological sample, which reagent can be selected from the group of:
(a) a reagent for detecting mRNA of the RASEF gene;
(b) a reagent for detecting the RASEF protein; and
(c) a reagent for detecting the biological activity of the RASEF protein.

Suitable reagents for detecting mRNA of the RASEF gene include nucleic acids that specifically bind to or identify the RASEF mRNA, for example, oligonucleotides which have a complementary sequence to a part of the RASEF mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the RASEF mRNA. These kinds of oligonucleotides can be prepared based on methods well known in the art. If needed, the reagent for detecting the RASEF mRNA can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the RASEF mRNA can 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 including a RASEF sequence, or an anti sense strand nucleotide sequence of a nucleic acid including a RASEF sequence, or of a naturally occurring mutant of these sequences. In particular, for example, in a typical embodiment, an oligonucleotide having 5-50 in length can be used as a primer for amplifying the genes, to be detected. More usually, mRNA or cDNA of a RASEF gene can be detected with oligonucleotide probe or primer of a specific size, generally 15- 30b in length. In preferred embodiments, length of the oligonucleotide probe or primer can be selected from 15-25. 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 include 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 RASEF protein include antibodies to the RASEF protein. The antibody can be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used as the reagent, so long as the fragment retains the binding ability to the RASEF protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof. Furthermore, the antibody can 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 can be employed for the present invention. Moreover, more than one reagent for detecting the RASEF protein can be included in the kit.

Furthermore, the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed RASEF protein in the biological sample. For example, the cell is 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 RASEF mRNA can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the biological activity of the RASEF protein can be included in the kit.

The kit can comprise more than one of the aforementioned reagents. Furthermore, the kit can comprise a solid matrix and reagent for binding a probe against the RASEF gene or antibody against the RASEF protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the RASEF protein. For example, tissue samples obtained from patient with good prognosis or poor prognosis can serve as useful control reagents. A kit of the present invention can 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 can be comprised in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers can be formed from a variety of materials, for example, glass or plastic.

According to an aspect of the present invention, the kit of the present invention for diagnosing cancer may further include either of positive or negative controls sample, or both. The positive control sample of the present invention may be established lung cancer cell lines. In a preferred embodiment, such clell lines are selected from the group consisting of:
lung adenocarcinoma (ADC) cell lines such as A427, A549, LC319, PC-14, PC-3, PC-9, NCI-H1373, NCI-H1781, NCI-H358, and the like;
lung squamous cell carcinoma (SCC) cell lines such as NCI-H226, NCI-H520, NCI-H2170, NCI-H1703, EBC-1, RERF-LC-AI and the like;
small cell lung cancer (SCLC) cell lines such as DMS114, DMS273, SBC-3, SBC-5, NCI-H196, NCI-H446 and the like; and
large cell carcinoma (LCC) cell lines such as LX1 and the like.

Alternatively, the RASEF positive samples may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s), including lung adenocarcinoma, lung squamous cell carcinoma, SCLC, and/or large cell carcinoma. Alternatively, positive control samples may be prepared by determined a cut-off value and preparing a sample containing an amount of an RASEF mRNA or protein more than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a normal range and a cancerous range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an RASEF standard sample providing a cut-off value amount of an RASEF mRNA or polypeptide. On the contrary, negative control samples may be prepared from non-cancerous cell lines or non-cancerous tissues such as normal lung tissues, or may be prepared by preparing a sample containing an RASEF mRNA or protein less than cut-off value.

Likewise, the kit of the present invention for assessing the prognosis of cancer may further include either of a good prognosis control sample or a poor prognosis control sample, or both. As described in (5) Method for Assessing the Prognosis of RASEF Mediated Cancer, a good control may be an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment. Meanwhile, a poor control may be an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment.

In a preferred embodiment, a good prognosis control sample may also be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed good or positive prognosis of lung cancer, after the treatment. In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from a lung cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
Alternatively, a good prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an RASEF mRNA or protein less than the cut-off value. Herein, the phrase "cut-off value" refers to the value dividing between a good prognosis range and a poor prognosis range. For example, one skilled in the art may be determine a cut-off value using a receiver operating characteristic (ROC) curve. The present kit may include an RASEF standard sample providing a cut-off value amount of an RASEF mRNA or polypeptide.

On the contrary, a poor prognosis control sample may be a clinical lung cancer tissue(s) obtained from a lung cancer patient(s) who showed poor or negative prognosis of lung cancer, after the treatment. In a preferred embodiment, such lung cancer tissue may be an NSCLC tissue(s) obtained from a lung cancer patient(s). In a more preferred embodiment, such NSCLC tissue may be a lung adenocarcinoma (ADC) tissue(s), a lung squamous cell carcinoma (SCC) tissue(s), and/or a large cell carcinoma tissue(s).
Alternatively, a poor prognosis control sample may be prepared by determined a cut-off value and preparing a sample containing an amount of an RASEF mRNA or protein more than the cut-off value.

As an embodiment of the present invention, when the reagent is a probe against the RASEF mRNA, the reagent can be immobilized on a solid matrix, for example, a porous strip, to form at least one detection site. The measurement or detection region of the porous strip can include a plurality of sites, each containing a nucleic acid (probe). A test strip can also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a strip separated from the test strip. Optionally, the different detection sites can 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 RASEF mRNA present in the sample. The detection sites can 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 can further comprise a positive control sample or RASEF standard sample. The positive control sample of the present invention can be prepared by collecting RASEF positive blood samples and then those RASEF level are assayed. Alternatively, purified RASEF protein or polynucleotide can be added to RASEF free serum to form the positive sample or the RASEF standard. In the present invention, purified RASEF can be recombinant protein. The RASEF level of the positive control sample is, for example more than cut off value.

(7) Screening Methods
Using the RASEF gene, polypeptide encoded by the gene or fragment thereof, or transcriptional regulatory region of the gene, it is possible to screen substances that alter the expressions of the genes or the biological activities of polypeptides encoded by the genes. Such substances may be used as pharmaceuticals for treating or preventing cancer, in particular, lung cancer. Thus, the present invention provides methods of screening for candidate substances for treating or preventing cancer using the RASEF gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene.
Substances isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the RASEF gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, lung cancer). Namely, the substances screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.

(I) Test substances for screening
In the context of the present invention, substances to be identified through the present screening methods can be 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 can be a single substance or a combination of substances. When a combination of substances is used in the methods, the substances can 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 compounds, synthetic micro-molecular compounds (including nucleic acid constructs, for example, antisense DNA, siRNA, ribozymes, etc.) and natural compounds 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-compound" library method and
(5) synthetic library methods using affinity chromatography selection.

The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds can 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 2002-103360).

A substance in which a part of the structure of the substance screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the substances obtained by the screening methods of the present invention.
Furthermore, when the screened test substance is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein can be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein can be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test substance which is a candidate for treating or preventing cancer.
Test substances useful in the screening described herein can also be antibodies or non-antibody binding proteins that specifically bind to the RASEF protein or partial RASEF peptides that lack the activity to binding for partner. Such partial protein or antibody can be prepared by the methods described herein (see (1) Cancer-related genes and cancer-related protein, and functional equivalent thereof in Definition or Antibodies) and can be tested for their ability to block binding of the protein with its binding partners.

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

An example of the molecular modeling system described generally above 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 review computer modeling of drugs interactive with specific proteins, for example, 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 for example, 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 an inhibitor of the RASEF activity has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified inhibitor, as detailed below. The resulting library of candidate inhibitors, or "test substances" can be screened using the methods of the present invention to identify test substances of the library that disrupt the RASEF activity for the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer.

(ii) Combinatorial chemical synthesis
Combinatorial libraries of test substances can be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors of the RASEF activity. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries can 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 can 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 for example, hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology, 1990-2008, John Wiley Interscience; Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3^rd Ed., 2001, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

(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.

(II) Screening methods
(i) General screening Method
Using the RASEF gene, proteins encoded by the gene or transcriptional regulatory region of the gene, substances that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene can be identified. In the present invention, RASEF gene were found to be over-expressed in cancer, and demonstrated to be involved in cancer cell growth and/or survival. Therefore, substances that alter the expressions of these genes or the biological activities of polypeptides by those genes can be candidate therapeutics for cancer.

Antagonists that binds to the RASEF polypeptide may inhibit the biological activity to mediate cell proliferation of cancer, and thus, they are candidates for treating the cancer. Therefore, the present invention provides a method for identifying potential candidates for treating or preventing cancer expressing RASEF, by identifying substances that bind to RASEF polypeptide.
As a method of screening for substances that inhibit the binding between a RASEF protein and a binding partner thereof (e.g., RASEF and ERK1 and/or ERK2 each other), many methods well known by one skilled in the art can be used. For example, screening can be carried out as an in vitro assay system, for example, a cellular system. More specifically, first, either the RASEF protein or the binding partner thereof is bound to a support, and the other protein is added together with a test substance thereto. For instance, the RASEF is bound to a support, and the binding partner polypeptide is added together with a test substance thereto. Next, the mixture is incubated, washed and the other protein bound to the support is detected and/or measured.

In the context of the present invention, "inhibition of binding" between two proteins refers to at least reducing binding between the proteins. Thus, in some cases, the percentage of binding pairs in a sample in the presence of a test substance will be decreased compared to an appropriate (e.g., not treated with test substance or from a non-cancer sample, or from a cancer sample) control. The reduction in the amount of proteins bound can be, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 25%, 10%, 5%, 1% or less (e.g., 0%), than the pairs bound in a control sample.
Examples of supports that can be used for binding proteins include, for example, insoluble polysaccharides, for example, agarose, cellulose and dextran; and synthetic resins, for example, polyacrylamide, polystyrene and silicon; for example, commercial available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials can be used. When using beads, they can be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables one to readily isolate proteins bound on the beads via magnetism.

The binding of a protein to a support can be conducted according to routine methods, for example, chemical bonding and physical adsorption, for example. Alternatively, a protein can be bound to a support via antibodies that specifically recognize the protein. Moreover, binding of a protein to a support can be also conducted by means of avidin and biotin.
The methods of screening for molecules that bind when the immobilized polypeptide is exposed to synthetic chemical compounds, 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: 458-63 (1996); Verdine, Nature 384: 11-3 (1996)) to isolate not only proteins but chemical compounds that bind to the protein (including agonist and antagonist) are well known to one skilled in the art.

Furthermore, in the screening method of the present invention, substances that suppress the expression level of RASEF can be also identified as candidate therapeutics for cancer. The expression level of a polypeptide or functional equivalent thereof can be detected according to any method known in the art. For example, a reporter assay can be used. Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared by using the transcriptional regulatory region of RASEF gene. When the transcriptional regulatory region of the gene has been known to those skilled in the art, a reporter construct can be prepared by using the previous sequence information. When the transcriptional regulatory region remains unidentified, a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the gene. Specifically, the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of a RASEF gene of interest. The transcriptional regulatory region of a RASEF gene is the region from a start codon to at least 500bp upstream, for example, 1000bp, for example, 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. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., Chapter 17, 2001, Cold Springs Harbor Laboratory Press).
Furthermore, in the screening method of the present invention, substances that inhibit a biological activity of RASEF protein can be also identified as candidate therapeutics for cancer.

Various low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted for detection or measuring of a biological activity of the RASEF polypeptide. For high-throughput assays, a substrate can conveniently be immobilized on a solid support. Following the reaction, the substrate converted by the polypeptide can be detected on the solid support by the methods described above. Alternatively, the contact step can be performed in solution, after which a substrate can be immobilized on a solid support, and the substrate converted by the polypeptide can be detected. To facilitate such assays, the solid support can be coated with streptavidin and the substrate labeled with biotin, or the solid support can be coated with antibodies against the substrate. The skilled person can determine suitable assay formats depending on the desired throughput capacity of the screen.

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

In the present invention, it is revealed that suppression of the expression level and/or biological activity of RASEF lead to suppression of the growth of cancer cells. Therefore, when a substance suppresses the expression and/or activity of RASEF, the suppression is indicative of a potential therapeutic effect in a subject. In the present invention, a potential therapeutic effect refers to a clinical benefit with a reasonable expectation. In the present invention, such clinical benefit includes;
(a) reduction in expression of the RASEF gene,
(b) decrease in size, prevalence, or metastatic potential of the cancer in the subject,
(c) preventing cancers from forming, or
(d) preventing or alleviating a clinical symptom of cancer.

(ii) Screening for Substances that Bind to RASEF protein(s)
In present invention, over-expression of RASEF was detected in cancer, but not in normal tissues. Further, polypeptides encoded by these genes were demonstrated to be involved in cancer cell growth. Therefore, those genes can be good molecular targets for cancer therapy and diagnosis. Substances that bind to RASEF polypeptide may inhibit biological activities of these polypeptides. Such substances are used as pharmaceuticals for treating or preventing lung cancer or detecting agents for diagnosing lung cancer and assessing a prognosis of lung cancer patient.

Specifically, the present invention provides the method of screening for a substance useful in diagnosing, treating or preventing cancers using the RASEF polypeptide. An embodiment of this screening method comprises the steps of:
(a) contacting a test substance with a RASEF polypeptide or fragment thereof;
(b) detecting the binding level between the polypeptide or the fragment and the test substance;
(c) selecting the test substance that binds to the polypeptide or the fragment of step (a).

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 RASEF associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing cancer.
More specifically, the method includes the steps of:
(a) contacting a test substance with a RASEF polypeptide or fragment thereof;
(b) detecting the binding level between the polypeptide and said test substance;
(c) correlating the binding level of b) with the therapeutic effect of the test substance.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of RASEF, the method including steps of:
(a) contacting a substance with a polypeptide encoded by a polynucleotide of RASEF;
(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 the substance binds to the polypeptide.

In the present invention, the therapeutic effect may be correlated with the binding level of the RASEF protein. For example, when the test substance binds to RASEF protein, the test substance may be identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not bind to RASEF protein, the test substance may be identified as the substance having no significant therapeutic effect.
The method of the present invention will be described in more detail below.

The RASEF polypeptide to be used for screening can 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. In preferred embodiments, the polypeptide is isolated from cells expressing RASEF, or chemically synthesized to be contacted with a test substance in vitro.

As a method of screening for proteins, for example, that bind to RASEF polypeptide using RASEF polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, immunoprecipitation method. The gene encoding RASEF polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.

The promoter to be used for the expression can 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 RASEF gene can be expressed as a fusion protein comprising 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 which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available. Also, a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the RASEF polypeptide by the fusion is also reported. Epitopes, for example, 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 RASEF polypeptide (Experimental Medicine 13: 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex consists of the RASEF polypeptide, a polypeptide comprising the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the RASEF 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 RASEF gene is prepared as a fusion protein with an epitope, for example, GST, an immune complex can be formed in the same manner as in the use of the antibody against the RASEF polypeptide, using a substance specifically binding to these epitopes, for example, 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 RASEF polypeptide is difficult to detect by a common staining method, for example, 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-cysteine, 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.

As a method of screening for proteins binding to RASEF polypeptide using the polypeptide, for example, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, a protein binding to the RASEF polypeptide can be obtained by preparing a cDNA library from cultured cells (e.g., lung cancer cell line ) expected to express a protein binding to the RASEF 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 RASEF polypeptide with the above filter, and detecting the plaques expressing proteins bound to RASEF polypeptide according to the label. The polypeptide of the invention can be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to RASEF polypeptide, or a peptide or polypeptide (for example, GST) that is fused to RASEF polypeptide. Methods using radioisotope or fluorescence and such can be also used.

The terms "label" and "detectable label" are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS(trademark)), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, .35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels for example colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system utilizing cells can 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, the 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. As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.

A substance binding to the polypeptide encoded by RASEF gene can also be screened using affinity chromatography. For example, the polypeptide of the invention can 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 can 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 can 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 for example, BIAcore.

The methods of screening for molecules that bind when the immobilized RASEF 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 RASEF protein (including agonist and antagonist) are well known to one skilled in the art.

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

(iii) Screening for Substance that Suppress the Biological Activity of RASEF Protein
The present invention also provides a method for screening a candidate substance for treating or preventing cancer using a biological activity of the RASEF polypeptide, or fragment thereof as an index.

Specifically, the present invention provides the following methods of [1] to [3]:
[1] A method of screening for a substance useful in treating or preventing cancers expressing RASEF, said method comprising the steps of:
(a) contacting a test substance with a RASEF polynucleotide, or functional equivalent or fragment thereof;
(b) detecting a biological activity of the polypeptide of step (a);
(c) comparing the level detected in the step (b) with those detected in the absence of the test substance;
(d) selecting the test substance that reduces or inhibits the biological activity of the polypeptide.
[2] The method of [1], wherein the biological activity is a cell proliferation promoting activity;
[3] The method of [1], wherein the biological activity of RASEF polypeptide is binding activity to ERK1 and/or ERK2 polypeptide.

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 RASEF associating disease e.g., lung 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 RASEF associating disease, e.g., lung cancer, using the RASEF polypeptide or fragments thereof including the steps as follows:
a) contacting a test substance with the RASEF 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 RASEF, the method including steps of:
(a) contacting a test substance with a polypeptide encoded by a polynucleotide of RASEF 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 the test substance suppresses the biological activity of the polypeptide encoded by the polynucleotide of RASEF gene as compared to the biological activity of said polypeptide detected in the absence of the test substance.

In the present invention, the therapeutic effect may be correlated with the biological activity of RASEF polypeptide or a functional fragment thereof. For example, when the test substance suppresses or inhibits the biological activity of RASEF 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 RASEF 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 will be described in more detail below.
Any polypeptides can be used for screening so long as they comprise the biological activity of the RASEF protein. Such biological activity includes the cell proliferation enhancing activity or the binding activity to ERK1 and/or ERK2 polypeptide. For example, RASEF protein can be used and polypeptides functionally equivalent to these proteins can also be used. Such polypeptides can be expressed endogenously or exogenously by cells.

The substance isolated by this screening is a candidate for antagonists of the polypeptide encoded by RASEF 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 expression of the gene encoding RASEF. Moreover, a substance isolated by this screening is a candidate for substances which inhibit the in vivo interaction of the RASEF polypeptide with molecules (including DNAs and proteins).
In the present invention, the RASEF protein has the activity of promoting cell proliferation of cancer cells (Fig. 5). Therefore, in the screening method of the present invention, using this biological activity, a substance which inhibits a biological activity of these proteins can be screened. Such substances would be potential candidates for treating cancer, e.g., lung cancer.

When the biological activity to be detected in the present method is cell proliferation promoting activity, it can be detected, for example, by preparing cells which express the RASEF 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 the colony formation activity, e.g. MTT assay, colony formation assay or FACS shown in "EXAMPLES". In some embodiments, cells expressing RASEF gene is isolated and cultured cells exogenously or endogenously expresseing RASEF gene in vitro.
The term of "suppress the biological activity" as defined herein refers to at least 10% suppression of the biological activity of RASEF in comparison with in absence of the substance, for example, at least 25%, 50% or 75% suppression, for example, at least 90% suppression.

In the some embodiments, control cells which do not express RASEF 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 RASEF associating disease, e.g., lung cancer, using the RASEF polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express a RASEF polypeptide or a functional fragment thereof, and control cells that do not express a RASEF polypeptide or a functional fragment thereof in the presence of a test substance;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test substance 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 the test substance.

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

(iv) Screening for Substances that Alter the Expression of RASEF
In the present invention, the decrease of the expression of RASEF by a double-stranded molecule specific for RASEF caused inhibition of cancer cell proliferation (Fig. 4). Therefore, substances that can be used in the treatment or prevention of cancer can be identified through screenings that use the expression levels of RASEF as indices. In the context of the present invention, such screening can comprise, for example, the following steps:
(a) contacting a test substance with a cell expressing RASEF gene ;
(b) detecting the expression level of the RASEF gene; and
(c) selecting the test substance that reduces the expression level of RASEF gene as compared to that detected in the absence of the test substance.

According to the present invention, the therapeutic effect of the test substance on inhibiting the cell growth or a candidate substance for treating or preventing RASEF associating disease e.g., lung cancer, 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 RASEF associating disease e.g., lung cancer,.

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 the RASEF gene;
b) detecting the expression level of the RASEF gene; and
c) correlating the expression level of b) with the therapeutic effect of the test substance.

Alternatively, according to the present invention, the potential therapeutic effect of a test substance on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of RASEF, the method including steps of:
(a) contacting a test substance with a cell expressing the RASEF gene;
(b) detecting the expression level of the RASEF gene of step (a); and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when the test substance reduces the expression level of the RASEF gene.

In the present invention, the therapeutic effect may be correlated with the expression level of the RASEF gene. For example, when the test substance reduces the expression level of the RASEF gene as compared to a level detected in the absence of the test substance, the test substance may identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level of the RASEF 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.

The method of the present invention will be described in more detail below.
Cells expressing the RASEF include, for example, cell lines established from lung cancer; such cells can be used for the above screening of the present invention (e.g., A549 and LC319). 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, ELISA or flow cytometry analysis. The term of "reduce the expression level" as defined herein refers to at least 10% reduction of expression level of RASEF in comparison to the expression level in absence of the substance, for example, at least 25%, 50% or 75% reduced level, for example, at least 95% reduced level. The substance herein includes chemical compound, 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 RASEF can be selected as candidate substances to be used for the treatment or prevention of cancers, e.g. lung cancer. In some embodiments, cells expressing RASEF gene is isolated and cultured cells exogenously or endogenously expresseing RASEF gene in vitro.

Alternatively, the screening method of the present invention can comprise the following steps:
(a) contacting a candidate substance with a cell into which a vector, comprising the transcriptional regulatory region of RASEF and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression level or activity of said reporter gene; and
(c) selecting the candidate substance that reduces the expression level 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 RASEF associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate substance that suppresses the proliferation of cancer cells, and a method for screening a candidate substance for treating or preventing RASEF associating disease.

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

Alternatively, in some embodiments, the present invention 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 RASEF, the method including steps of:
(a) contacting a test substance with a cell into which a vector, including the transcriptional regulatory region of RASEF and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression level 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 level or activity of said reporter gene.

In the present invention, the therapeutic effect may be correlated with the expression level or activity of said reporter gene. For example, when the test substance reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test substance, the test substance may be identified or selected as the candidate substance having the therapeutic effect. Alternatively, when the test substance does not reduce the expression level or activity of said reporter gene 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.

Suitable reporter genes and host cells are well known in the art. For example, reporter genes are luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of CX. The transcriptional regulatory region of CX herein is the region from start codon to at least 500bp upstream, for example, 1000bp, for example, 5000 or 10000bp upstream, but not restricted. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).

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 refers to at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the substance, for example, at least 25%, 50% or 75% reduction, for example, at least 95% reduction.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

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

(v) Screening using the binding of RASEF and ERK1/2 as an index
In the present invention, it was confirmed that the RASEF protein interacts with ERK1/2 that consisting of ERK1 protein and ERK2 protein (Fig. 8). The direct interaction between exogenous RASEF protein and endogenous ERK1/2 was demonstrated as shown in Fig. 8A. Additionally, the interaction between exogenous RASEF and exogenous ERK2 using COS-7 co-transfected with RASEF and ERK2 was confirmed (Fig. 8B). Furthermore, the direct interaction between endogenous RASEF and ERK1/2 was confirmed by immunoprecipitation assay using extracts from lung cancer cell line NCI-H2170. The direct binding between endogenous RASEF and ERK1/2 was demonstrated as shown in Fig. 8C.

To further investigate the biological importance of the interaction of these two proteins, either of 3 partial constructs of RASEF with Flag sequence at its NH2-terminal (RASEF1-240, RASEF170-520, RASEF 455-750) was transfected into COS-7 cells, subsequently immunoprecipitation with polyclonal anti-ERK1/2 antibody indicated that the COOH-term side construct (RASEF 455-750) including RAB domain was able to interact with endogenous ERK1/2 (Fig. 10B). To further define the minimal and high-affinity ERK1/2-binding site in RASEF520-740, either of 4 additional constructs of RASEF was transfected into DMS114 cells (RASEF520-575, RASEF575-630, RASEF630-685 and RASEF685-740; Fig. 10C) and the immunoprecipitation assay was perfprmed with anti-Flag M2 agarose antibody. Western blotting with anti-ERK1/2 antibody revealed that RASEF520-575 was able to bind with ERK1/2, but other constructs were not (Fig. 10E). These experiments determined that the 55-amino acid polypeptide in RASEF should be important for the binding with ERK1/2.

Thus, a substance that inhibits the binding between RASEF protein and ERK1 and/or ERK2 protein can be screened using such a binding of RASEF protein and ERK1 and/or ERK2 protein as an index. Such substances may have potential therapeutic effect on cancer treatment as those proteins are involved in cancer cell growth. Therefore, the present invention provides a method for screening a substance for inhibiting the binding between RASEF protein and ERK1 and/or ERK2 protein using such a binding of RASEF protein and ERK1 and/or ERK2 protein as an index. Furthermore, the present invention also provides a method for screening a candidate substance for inhibiting or reducing a growth of cancer cells expressing RASEF and ERK1 and/or ERK2 gene, e.g. lung cancer cell, and a candidate substance for treating or preventing cancers, e.g. lung cancer.

Specifically, the present invention provides the following methods of [1] to [7]:
[1] A method of screening for a substance that interrupts a binding between a RASEF polypeptide and an ERK1 and/or ERK2 polypeptide, said method comprising the steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding 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 or inhibits the binding level.
[2] A method of screening for a candidate substance for useful in treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting a binding 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 or inhibits the binding level.
[3] The method of [1] or [2], wherein the functional equivalent of RASEF polypeptide comprising the ERK1 and/or ERK2-binding domain.
[4] The method of [3], wherein the ERK1 and/or ERK2-binding domain of RASEF is comprised in the region of 520-575 of SEQ ID NO: 16.
[5] The method of [3], wherein the ERK1 and/or ERK2-binding domain of RASEF is comprised in the region of 455-740 of SEQ ID NO: 16.
[6] The method of [1] or [2], wherein the functional equivalent of ERK1 and/or ERK2 polypeptide comprising the RASEF-binding domain
[7] The method of [1], wherein the cancer is lung cancer.

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

More specifically, the method includes the steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 and/or ERK2 polypeptide or functional equivalent thereof in the presence of a test substance;
(b) detecting the level of binding 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 cancer, the method including steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 and/or ERK2 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.

In the present invention, the therapeutic effect may be correlated with the binding level of the RASEF and ERK1 and/or ERK2 proteins. For example, when the test substance reduces the binding level of RASEF and ERK1 and/or ERK2 proteins 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 binding level of RASEF and ERK1 and/or ERK2 proteins as compared to a level detected in the absence of the test substance, the test substance may identified as the substance having no significant therapeutic effect.

In the context of the present invention, a functional equivalent of a RASEF polypeptide is a polypeptide that has a biological activity equivalent to a RASEF polypeptide (SEQ ID NO: 16), respectively (see, (1) Genes and Polypeptides). More specifically, a functional equivalent of a RASEF polypeptide is a polypeptide comprises the ERK1 and/or ERK2-binding domain that is within the residues 455-740 of SEQ ID NO: 16. Alternatively, the functional equivalent of ERK1 polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 18, 20 or 22 comprising the RASEF-binding domain. Also, the functional equivalent of ERK2 polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 25 comprising the PIF1-binding domain.

As a method of screening for substances that inhibits the binding of RASEF to ERK1 and/or ERK2, many methods well known by one skilled in the art can be used. Such a screening 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 (see (i) General screening Method and (ii) Screening for Substances that Bind to RASEF protein(s)).

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. In preferred embodiments, the polypeptide is isolated from cells expressing RASEF, or chemically synthesized to be contacted with an ERK1 and/or ERK2 polypeptide in vitro. Also, in preferred embodiments, the polypeptide is isolated from cells expressing ERK1 and/or ERK2, or chemically synthesized to be contacted with a RASEF polypeptide in vitro.
Any aforementioned test substance can be used (see (1) Test substances for screening).

In some embodiments, this method further comprises the step of detecting the binding of the candidate substance to RASEF protein or ERK1 and/or ERK2 protein, or detecting the level of binding RASEF proteinto ERK1 and/or ERK2 protein. Cells expressing RASEF protein and ERK1 and/or ERK2 protein include, for example, cell lines established from cancer, e.g. lung cancer, such cells can be used for the above screening of the present invention so long as the cells express these genes. Alternatively cells can be transfected both or either of expression vectors of RASEF and ERK1 and/or ERK2 protein, so as to express these two genes. The binding of RASEF protein to ERK1 and/or ERK2 protein can be detected by immunoprecipitation assay using an anti-Flag antibody and ERK1 and/or ERK2 antibody (Fig. 8A).

In the present invention, it is revealed that suppressing the binding between RASEF protein and ERK1 and/or ERK2 protein, reduces cell growth. Thus, by screening for candidate substances that inhibits the binding between RASEF protein and ERK1 and/or ERK2 protein, candidate substances that have the potential to treat or prevent cancers can be identified. Potential of these candidate substances to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a substance inhibits the binding between RASEF protein and ERK1 and/or ERK2 protein inhibits activities of cancer, it may be concluded that such substance has RASEF specific therapeutic effect.

(vi) Screening using the phosphorylation level of RASEF by ERK1/2 as an index
According to the present invention, the sequence of RASEF contains several consensus sites for ERK phosphoryloation. The phosphorylation of RASEF was increased in the presence of ERK2 protein (Fig.6A, 6B), indicating that RASEF could be phosphorylated by ERK2. Furthermore, phosphoproteome analysis using Mass spectrometry and mammalian cells that were transfected with both RASEF and ERK2 expression vectors identified several ERK-dependent serine phosphorylation sites that appeared to be important for cell proliferation (Fig.7C, 10A). The RASEF protein treated with ERK2 transfection showed additional phosphorylated serine sites (Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523 and Serine-719), compared with untreated RASEF protein. Thus, the present invention provides a method for screening a substance that reduces phosphorylation level of a RASEF polypeptide by an ERK1 and/or ERK2 polypeptide. Among substances identified in the screening, substances that reduces phosphorylation level of RASEF polypeptide may become candidate substances for treating or preventing cancer, or inhibiting cancer cell growth.

Specifically, the present invention provides the following methods of [1] to [8]:
[1] A method of screening for a candidate substance for treating or preventing a disease associated with overexpression of RASEF gene, or inhibiting proliferation of a cell expressing RASEF gene, said method comprising the steps of:
(a) contacting a RASEF polypeptide or a fragment thereof with an ERK1 and/or ERK2 polypeptide or a fragment thereof in the presence of a test substance under a suitable condition for phosphorylation;
(b) detecting the phosphorylation level of the RASEF polypeptide or fragment thereof;
(c) comparing the phosphorylation level with that detected in the absence of the test substance; and
(d) selecting the test substance that reduces the phosphorylation level as compared to that detected in the absence of the test substance as a candidate substance for treating or preventing the disease.
[2] The method of [1], the fragment of a RASEF polypeptide is a fragment comprising serine residue corresponding to Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16.
[3] The merhod of [1], the fragment of an ERK1 and/or ERK2 polypeptide is a fragment retaining kinase activity.
[4] The method of [1], the serine residue of which the phosphorylation level is detected, is a serine residue corresponding to Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16.
[5] The method of [1] , wherein the step (a) comprises incubating a RASEF polypeptide or a fragment thereof and an ERK1 and/or ERK2 polypeptide in the presence of a phosphate donor in the incubation mixture.
[6] The method of [5], wherein the phosphate donor is ATP.
[7] The method of [1], the disease associated with overexpression of RASEF is cancer.
[8] The method of [7], the cancer is lung cancer.

Alternatively, in some embodiments, the present invention may provide a method for evaluating or estimating a therapeutic effect of a test substance in connection with the treatment and/or prevention of cancer or the inhibition of a cancer associated with over-expression of RASEF, the method including steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 and/or ERK2 polypeptide in the presence of the test substance under the condition capable of phosphorylation of RASEF polypeptide by ERK1 and/or ERK2 polypeptide
(b) detecting the phosphorylation level of the RASEF polypeptide; and
(c) correlating the potential therapeutic effect and the test substance, wherein the potential therapeutic effect is shown, when a test substance decreases the phosphorylation level of the RASEF polypeptide as compared to the phosphorylation level detected in the absence of the test substance as the candidate substance.

In the context of the present invention, the therapeutic effect may be correlated with a phosphorylation level of a RASEF polypeptide by ERK1 and/or ERK2 polypeptide. For example, when a test substance reduces the phosphorylation level of a RASEF polypeptide 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 phosphorylation level of the substrate 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.

RASEF polypeptide and ERK1 and ERK2 polypeptide to be used for the screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. Such polypeptides can be prepared by methods well known in the art (see "Genes and Polypeptides"). Preferably, the polypeptides are purified or isolated.

In some embodiment, the polypeptides may be added commercially available epitopes to the N- and/or C- terminus. Examples of such epitopes include, but are not limited to, 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. In preferred embodiments, the polypeptide is isolated from cells expressing RASEF, or chemically synthesized to be contacted with an ERK1 and/or ERK2 polypeptide in vitro. Also, in preferred embodiments, the polypeptide is isolated from cells expressing ERK1 and/or ERK2, or chemically synthesized to be contacted with a RASEF polypeptide in vitro.

In addition to purified or isolated polypeptides, cells that express RASEF polypeptide and an ERK1 and/or ERK2 polypeptide may be also used for the screening method of the present invention. Herein, any cell can be used, so long as it expresses the RASEF polypeptide or a functional equivalent thereof (see, the "Genes and Polypeptides" section and definitions above). The cell used in the present screening can be a cell naturally expressing the RASEF polypeptide including, for example, cells derived from and cell-lines established from lung cancer. Cell-lines of lung cancer cell, A549, NCI-H2170, LC319 and so on, can be employed.
Alternatively, the cell used in the screening can be a cell that naturally does not express the RASEF polypeptide and which is transfected with a RASEF polypeptide or a RASEF functional equivalent-expressing vector. Such recombinant cells can be obtained through known genetic engineering methods (e.g., Morrison DA., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymologist (eds. Wu et al.) 1983, 101: 347-62) as mentioned above.

In the present invention, the phosphorylation level of RASEF polypeptide can be determined by methods known in the art. For example, RASEF polypeptide may be incubated with an ERK1 and/or ERK2 polypeptide and a phosphate donor, under a suitable condition for phosphorylation. For example, a suitable phosphate donor is ATP. The ATP may contain [gamma-32P]ATP.

The phosphorylation level of RASEF polypeptide can be determined based on the radioactivity after incubation. The radioactivity in the RASEF polypeptide may be detected, for example, by SDS-polyacrylamide gel electrophoresis and autoradiography. Alternatively, following the incubation the RASEF polypeptide may be separated from phosphate donor by conventional methods such as gel filtration and immunoprecipitation, and the radioactivity in the substrate may be measured by methods well-known in the art. Other suitable labels that can be attached to phosphate group in a RASEF polypeptide, such as chromogenic and fluorescent labels, and methods of detecting these labels, are known in the art.

Alternatively, phosphorylation level of RASEF polypeptide may be determined using a mass spectrometry or reagents that selectively recognize a phosphorylated site. For example, antibodies against the phosphoserine may be used as such reagents. Any immunological techniques using such antibodies can be used for the detection of phosphorylation level of the RASEF polypeptide. In the present invention, the RASEF polypeptide is phosphorylated at Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16. Antibodies against a phosphoserine may be used. For example, ELISA or Immunoblotting with antibodies recognizing a phosphoserine may be used for the present invention.

"Suppress the phosphorylation level" as defined herein are typically at least 10% suppression in comparison with in absence of the substance, more preferably at least 25%, 50% or 75% suppression and most preferably at 90% suppression.
In the some embodiments, control cells which do not express RASEF
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 RASEF associating disease, using the RASEF polypeptide or
fragments thereof including the steps as follows:
a) culturing cells which express a RASEF polypeptide or a functional
fragment thereof, and control cells that do not express a RASEF polypeptide or a functional fragment thereof in the presence of a test substance;
b) detecting the phosphorylation level of the RASEF polypeptide of the cells which express the protein and control cells; and
c) selecting the test substance that reduces the phosphorylation level in the cells
which express the protein as compared to the phosphorylation level detected in the control cells and in the absence of said test substance.

(8) A kit for detecting the ability of a test substance to reduce the phosphorylation level of RASEF polypeptid by ERK1/2.
The present invention further provides a kit for detecting the ability of a test substance to reduce a phosphorylation level of a RASEF polypeptide.
The above kits of the present invention find a use for identifying a substance that reduces a phosphorylation level of a RASEF polypeptide by an ERK1 and/or ERK2 polypeptide. Furthermore, the kits of the present invention are useful for screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth.

Specifically, the present invention provides the following kits of [1] to [5]:
[1] A kit for detecting for the ability of a test substance to reduce phosphorylation level of RASEF polypeptide by an ERK1 and/or ERK2 polypeptide, wherein the kit comprises the following components:
(a) a RASEF polypeptide or a fragment thereof;
(b) an ERK1 and/or ERK2 polypeptide;
(c) a phosphate doner, and
(d) a reagent for detecting phosphorylation level of at least one serine residue in the RASEF polypeptide or fragment thereof.
[2] The kit of [1], wherein the fragment of a RASEF polypeptide is a fragment comprising a serine residue corresponding to Serine-377, Serine-386, Serine-402, Serine406, Serine-520, Serine-523 or Serine-719 in the amino acid sequence of SEQ ID NO: 16.
[3] The kit of [1], wherein the fragment thereof of the ERK1 and/or ERK2 polypeptide comprises a fragment of the ERK1 and/or ERK2 polypeptide having a kinase activity.
[4] The kit of [1], wherein the phosphate donor is an ATP.
[5] The kit of [1], the reagent is an antibody against a phosphoserine.
Details of the kits of the present invention will be described bellow.

RASEF polypeptide contained in the kits of the present invention may either the full length of RASEF polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 16, or a functionally equivalent thereof such as a fragment of the full length of RASEF polypeptide. Herein, the functionally equivalent of RASEF polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of RASEF polypeptide, capable of being phosphorylated by an ERK1 and/or ERK2 polypeptide. Preferably, the functionally equivalents of RASEF polypeptide retains at least one phosphorylation site capable to be phosphorylated by ERK1 and/or ERK2 polypeptide. Such phosphorylation site includes Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16. Thus, typical examples of the functionally equivalent of RASEF polypeptide include a fragment of a RASEF polypeptide retaining a phosphorylated serine residue corresponding to the Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16. Such fragments may contain a contiguous sequence of the amino acid sequence of SEQ ID NO: 16 including the phosphorylated Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16., having more than 10 amino acid residues. More preferably, the fragments may have more than 15, 20, 25, 30, 50, 75, 100, 150, 200, 250, 300, 350 or 400 amino acid residues.

ERK1 and/or ERK2 polypeptide contained in the kits of the present invention may either the full length of ERK1 and/or ERK2 polypeptide such as a polypeptide containing an amino acid sequence of SEQ ID NOs: 18, 20, 22 or 25, or a functionally equivalent thereof such as a fragment of the full length of ERK1 and/or ERK2 polypeptide. Herein, the functionally equivalent of ERK1 and/or ERK2 polypeptide refers to a modified polypeptide, a fragment or a modified fragment of the full length of ERK1 and/or ERK2 polypeptide, having kinase activity for RASEF polypeptide.

Reagents for detecting the phosphorylation level of the RASEF polypeptide may be any reagents that are able to be used for detection of phosphorylation level of the RASEF polypeptide. For example, antibodies against a phosphorylated RASEF polypeptide, in particular antibodies against a phosphorylated Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16 may be used as such reagent. The anti-phosphorylated RASEF 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 phosphorylated RASEF polypeptide. Methods to prepare these kinds of antibodies 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. For example, radiolabels, chromogenic labels, fluorescent labels and such may be used for labeling the antibody.

When the kit contains a labeled phosphate donor, the reagents for detecting the phosphorylation level of the RASEF polypeptide may be reagents for detecting signal generated by the label. For example, when the RASEF polypeptide is labeled with a radiolabel, the reagents for the detection of phosphorylation level may be liquid scintillators, reagents for autoradiography and the like.

The kit may contain more than one of the aforementioned reagents. Furthermore, the kit may include a solid matrix for binding an anti-phosphoserine antibody, a medium or buffer and container for incubating the polypeptides under suitable condition for phosphorylation, positive and negative control samples. The 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 substances and such may be included 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.

Dominant negative polypeptides
As demonstrated herein, a fragment of RASEF polypeptide that has the amino acid sequence of SEQ ID NO: 43 effectively suppresses growth of lung cancer cells (Figs. 11E and 11F). Accordingly, the present invention also provides polypeptides that have the amino acid sequence of SEQ ID NO: 43, which has the function to inhibit an activity of the RASEF polypeptide.
The polypeptides of the present invention can be of any length, so long as the polypeptides retain the function to inhibit an activity of the RASEF polypeptide. Specifically, the length of the amino acid sequence may range from 23 to 70 residues, for example, from 23 to 50, preferably from 23 to 30, more specifically from 23 to 25 amino acid residues.

Furthermore, the present invention relates to variants of the polypeptide having the amino acid sequence of SEQ ID NO: 43. In the present invention, the variants can be those which contain any mutations selected from addition, deletion, substitution and insertion of one, two or several amino acid residues and are functionally equivalent to the polypeptide having the amino acid sequence of SEQ ID NO: 43. The phrase "functionally equivalent to the polypeptide having the amino acid sequence of SEQ ID NO: 43" refers to having the function to inhibit an activity of the RASEF polypeptide. In general, the modifications of one, two or several amino acids in a polypeptide will not influence the function of the polypeptide, and in some cases will even enhance the desired function of the original polypeptide (See, "Genes and Polypeptides"). Thus, the polypeptide of the present invention encompass polypeptides that have an amino acid sequence in which one, two or several amino acids are substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43 and have the function to inhibit an activity of the RASEF polypeptide. In preferred embodiments, the modifications in the amino acid sequence of SEQ ID NO: 43 can be conservative amino acid substitutions (See, "Genes and Polypeptides"). However, the polypeptides of the present invention are not restricted thereto and may include non-conservative modifications, so long as the modified polypeptide retains the function to inhibit an activity of the RASEF polypeptide. Furthermore, modified polypeptides should not exclude inhibitory polypeptides of polymorphic variants, interspecies homologues, and alleles of RASEF. To retain the function to inhibit the binding between the RASEF polypeptide and ERK1/2 protein, one can modify (insert, add, delete and/or substitute) a small number (for example, 1, 2 or several) or a small percentage of amino acids. Herein, the term "several" means 5 or fewer amino acids, for example, 4 or 3 or fewer. The percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less or 1 to 5%.

Alternatively, the number of amino acids that may be mutated is not particularly restricted, so long as the modified polypeptide retains the function to inhibit an activity of the RASEF polypeptide. Likewise, the site of mutation is not particularly restricted, so long as the mutation does not result in the disruption of the function to inhibit an activity of the RASEF polypeptide.
As demonstrated in Examples, the amino acid sequence of SEQ ID NO: 43 corresponds to a binding domain to ERK1/2 protein of the RASEF polypeptide (Fig. 11). Therefore, it is considered that the polypeptide having the amino acid sequence of SEQ ID NO: 43 competitively inhibits the binding of the RASEF polypeptide to ERK1/2 protein and consequently, inhibits cell proliferation promoting activity of the RASEF polypeptide and inhibits ERK1/2 dependent phosphorylation of RASEF. Accordingly, in preferred embodiments, the activities of the RASEF polypeptide to be inhibited by the polypeptide of the present invention include cell proliferation promoting activity and binding activity to the ERK1/2 protein. Detection of cell proliferation promoting activity and binding activity to the ERK1/2 protein can carried out by methods known in the art, for example, methods described above section "7.(II) Screening Method, (iii) Screening for Substances that Suppresses the Biological Activity of RASEF: and (v) Screening for a Substance that Inhibits the Binding between RASEF and ERK1/2 protein:".

The polypeptides of the present invention can be chemically synthesized. Methods used in the ordinary peptide chemistry can be used for the method of synthesizing polypeptides (See, "Genes and Polypeptides"). Alternatively, the polypeptides of the present invention can be also prepared by known genetic engineering techniques (See, "Genes and Polypeptides").
When genetic engineering techniques are used, the polypeptide of the present invention can be expressed as a fused protein with a peptide having a different amino acid sequence. A vector expressing a desired fusion protein can be obtained by linking a polynucleotide encoding the polypeptide of the present invention to a polynucleotide encoding a different peptide so that they are in the same reading frame, and then introducing the resulting nucleotide into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides to be used in forming fusion proteins include the following peptides: FLAG (Hopp et al., (1988) BioTechnology 6, 1204-10), 6xHis consisting of six His (histidine) residues, 10xHis, Influenza hemagglutinin (HA), Human c-myc fragment,
VSV-GP fragment, p18 HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), HA (Influenza hemagglutinin), Immunoglobulin constant region, beta-galactosidase, and MBP (maltose-binding protein).

The polypeptide of the present invention can be obtained by treating the fusion protein thus produced with an appropriate protease, and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein is captured in advance with affinity chromatography that binds with the fusion protein, and then the captured fusion protein can be treated with a protease. With the protease treatment, the desired polypeptide is separated from affinity chromatography, and the desired polypeptide with high purity is recovered.
Furthermore, the polypeptides of the present invention may be modified by being linked to other substances. The other substances include organic compounds such as peptides, lipids, saccharides, and various naturally-occurring or synthetic polymers. The polypeptides of the present invention may be linked to any other substances so long as the polypeptides retain the function to inhibit an activity of the RASEF polypeptide. Modifications can also confer additive functions on the polypeptides of the present invention. Examples of the additive functions include, but are not limited to, targetability, deliverability, permeability and stability.

Preferred examples of modifications in the present invention include, for example, the introduction of a cell-membrane permeable substance. Usually, the intracellular structure is cut off from the outside by the cell membrane. Therefore, it is difficult to efficiently introduce an extracellular substance into cells. Cell membrane permeability can be conferred on the polypeptides of the present invention by modifying the polypeptides with a cell-membrane permeable substance. As a result, by contacting the polypeptide of the present invention with a cell, the polypeptide can be delivered into the cell to act thereon.

As used herein, the phrase "cell-membrane permeable substance" refers to a substance capable of penetrating the mammalian cell membrane to enter the cytoplasm. For example, a certain liposome fuses with the cell membrane to release the content into the cell. Meanwhile, a certain type of polypeptide penetrates the cytoplasmic membrane of mammalian cell to enter the inside of the cell. In a preferred embodiment, the polypeptide of the present invention has the following general formula:
[R]-[D];
wherein,
[R] represents a cell-membrane permeable substance; [D] represents a polypeptide comprising the amino acid sequence of (a) or (b) below:
(a) the amino acid sequence of SEQ ID NO: 43;
(b) the amino acid sequence in which one, two or several amino acid is substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43.

In the above-described general formula, [R] and [D] can be linked directly or indirectly through a linker. Peptides, compounds having multiple functional groups, or such can be used as a linker. Specifically, amino acid sequences containing -G- can be used as a linker. Alternatively, a cell-membrane permeable substance and the polypeptide can be bound to the surface of a minute particle. [R] can be linked to any positions of [D]. Specifically, [R] can be linked to the N terminal or C terminal of [D], or to a side chain of amino acids constituting [D]. Furthermore, more than one [R] molecule can be linked to one molecule of [D]. The [R] molecules can be introduced to different positions on the [D] molecule. Alternatively, [D] can be modified with a number of [R]s linked together.

There have been reported a variety of naturally-occurring or artificially synthesized polypeptides having cell-membrane permeability (Joliot A. & Prochiantz A., Nat Cell Biol. 2004; 6: 189-96). All of these known cell-membrane permeable substances can be used for the polypeptides in the present invention. Examples of cell-membrane permeable substances include, for example:
poly-arginine; Matsushita et al., (2003) J. Neurosci.; 21, 6000-7.
Tat / RKKRRQRRR (SEQ ID NO: 26) (Frankel et al., (1988) Cell 55,1189-93.,Green & Loewenstein (1988) Cell 55, 1179-88.);
Penetratin / RQIKIWFQNRRMKWKK (SEQ ID NO: 27)(Derossi et al., (1994) J. Biol. Chem. 269, 10444-50.);
Buforin II / TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 28)(Park et al., (2000) Proc. Natl Acad. Sci. USA 97, 8245-50.);
Transportan / GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 29)(Pooga et al., (1998) FASEB J. 12, 67-77.);
MAP (model amphipathic peptide) / KLALKLALKALKAALKLA (SEQ ID NO: 30)(Oehlke et al., (1998) Biochim. Biophys. Acta. 1414, 127-39.);
K-FGF / AAVALLPAVLLALLAP (SEQ ID NO: 31)(Lin et al., (1995) J. Biol. Chem. 270, 14255-8.);
Ku70 / VPMLK (SEQ ID NO: 32)(Sawada et al., (2003) Nature Cell Biol. 5, 352-7.);
Ku70 / PMLKE (SEQ ID NO: 33)(Sawada et al., (2003) Nature Cell Biol. 5, 352-7.);
Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 34)(Lundberg et al., (2002) Biochem. Biophys. Res. Commun. 299, 85-90.);
pVEC / LLIILRRRIRKQAHAHSK (SEQ ID NO: 35)(Elmquist et al., (2001) Exp. Cell Res. 269, 237-44.);
Pep-1 / KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 36)(Morris et al., (2001) Nature Biotechnol. 19, 1173-6.);
SynB1 / RGGRLSYSRRRFSTSTGR (SEQ ID NO: 37)(Rousselle et al., (2000) Mol. Pharmacol. 57, 679-86.);
Pep-7 / SDLWEMMMVSLACQY (SEQ ID NO: 38)(Gao et al., (2002) Bioorg. Med. Chem. 10, 4057-65.); and
HN-1 / TSPLNIHNGQKL (SEQ ID NO: 39)(Hong & Clayman (2000) Cancer Res. 60, 6551-6.).

The poly-arginine, which is listed above as an example of cell-membrane permeable substances, may be constituted by any number of arginine residues. Specifically, for example, it may be constituted by consecutive 5-20 arginine residues. The preferable number of arginine residues is 11 (SEQ ID NO: 40).

Compositions and Methods for treating cancer using dominant negative polypeptides
The polypeptides of the present invention inhibit proliferation of cancer cells. Therefore, the present invention provides pharmaceutical compositions for treating and/or preventing cancer which contains as an active ingredient the polypeptide of the present invention; or a polynucleotide encoding the same. Alternatively, the present invention relates to methods for treating and/or preventing cancer including the step of administering the polypeptide of the present invention to a subject. Furthermore, the present invention relates to the use of the polypeptides of the present invention in manufacturing pharmaceutical compositions for treating and/or preventing cancer. Cancers to be treated or prevented by the present invention are not limited, so long as expression of RASEF is up-regulated in the cancer cells. For example, the polypeptides of the present invention are useful for treating lung cancer.

Alternatively, the polypeptides of the present invention can be used to suppress growth of cancer cells. Therefore, the present invention provides compositions for suppressing cancer cell growth, which contain as an active ingredient the polypeptide of the present invention; or a polynucleotide encoding the same. Alternatively, the present invention relates to methods for suppressing cancer cell growth which include the step of administering the polypeptides of the present invention. Furthermore, the present invention relates to the use of polypeptides of the present invention in manufacturing pharmaceutical compositions for suppressing cancer cell growth.

When the polypeptides of the present invention are administered, as a prepared pharmaceutical, to human and other mammals such as mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, cattle, monkey, baboon and chimpanzee for treating and/or preventing cancer, the polypeptides of the present invention can be administered directly, or formulated into an appropriate dosage form using known methods for preparing pharmaceuticals. For example, if necessary, the pharmaceuticals can be orally administered as a sugar-coated tablet, capsule, elixir, and microcapsule, or alternatively parenterally administered in the injection form that is a sterilized solution or suspension with water or any other pharmaceutically acceptable liquid. For example, the polypeptides of the present invention can be mixed with pharmacologically acceptable carriers or media, specifically sterilized water, physiological saline, plant oil, emulsifier, suspending agent, surfactant, stabilizer, corrigent, excipient, vehicle, preservative, and binder, in a unit dosage form necessary for producing a generally accepted pharmaceutical. Depending on the amount of active ingredient in these formulations, a suitable dose within the specified range can be determined.

Examples of additives that can be mixed in tablets and capsules are binders such as gelatin, corn starch, tragacanth gum, and gum arabic; media such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharine; and corrigents such as peppermint, wintergreen oil and cherry. When the unit dosage from is capsule, liquid carriers such as oil can be further included in the above-described ingredients. Sterilized mixture for injection can be formulated using media such as distilled water for injection according to the realization of usual pharmaceuticals.

Physiological saline, glucose, and other isotonic solutions containing adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. They can be used in combination with a suitable solubilizer, for example, alcohol, specifically ethanol and polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants such as Polysorbate 80TM and HCO-50.
Sesame oil or soybean oil can be used as an oleaginous liquid, and also used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Furthermore, they can be further formulated with buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants. Injections thus prepared can be loaded into appropriate ampoules.

Methods well-known to those skilled in the art can be used for administering pharmaceutical compositions of the present invention to subjects, for example, by intraarterial, intravenous, or subcutaneous injection, and similarly, by intranasal, transtracheal, intramuscular, or oral administration. Doses and administration methods are varied depending on the body weight and age of patients as well as administration methods. However, those skilled in the art can routinely select them. DNA encoding the polypeptide of the present invention can be inserted into a vector for the gene therapy, and the vector can be administered for treatment. Although doses and administration methods are varied depending on the body weight, age, and symptoms of patients, those skilled in the art can appropriately select them. For example, a dose of the polypeptide of the present invention is, when orally administered to a normal adult (body weight 60 kg), about 0.1 mg to about 100 mg/day, preferably about 1.0 mg to about 50 mg/day, more preferably about 1.0 mg to about 20 mg/day, although it is slightly varied depending on symptoms.

When the polypeptide of the present invention is parenterally administered to a normal adult (body weight 60 kg) in the injection form, it is convenient to intravenously inject a dose of about 0.01 mg to about 30 mg/day, preferably about 0.1 mg to about 20 mg/day, more preferably about 0.1 mg to about 10 mg/day, although it is slightly varied depending on patients, target organs, symptoms, and administration methods. Similarly, the polypeptide of the present invention can be administered to other animals in an amount converted from the dose for the body weight of 60 kg.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

Materials and Methods
Cell lines and tissue samples. Lung cancer cell lines and human bronchial epitherial cells (BEAS-2B) used in this study were listed in Table 1. All cells were grown in monolayer in appropriate medium supplemented with 10% FCS and maintained at 37degrees C in humidified air with 5% CO₂. Primary lung cancer samples had been obtained earlier as previously described (6 Kikuchi T, et al., Oncogene 2003;22:2192-205.; Taniwaki M, et al., Int J Oncol 2006;29:567-75.). All tumors were staged on the basis of the pTNM pathological classification of the UICC (International Union Against Cancer) (Sobin L, et al., 6th ed. New York: Wiley-Liss; 2002). A total of 341 formalin-fixed samples of primary NSCLCs (100 female and 241 male patients; median age of 65 with a range of 35-85 years; 93 never smoke cases and 248 ex- or current smokers; 205 ADCs, 105 SCCs, 20 LCCs, 11 ASCs; 141 pT1, 157 pT2 and 43 pT3 cases; 223 pN0, 42 pN1, and 76 pN2 cases: see Table 2) had been obtained earlier along with clinicopathological data from patients undergoing surgery at Saitama Cancer Center (Saitama, Japan). These patients received resection of their primary cancers, and among them only patients with positive lymph node metastasis were treated with cisplatin-based adjuvant chemotherapies after their surgery. This study and the use of all clinical materials mentioned were approved by individual institutional Ethical Committees.

Semiquantitative reverse transcription-PCR. A total of 3 microgram aliquot of mRNA from each sample was reversely 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 to RASEF or beta-actin (ACTB) specific primers as an internal control: RASEF, 5'-ATCTAACCGGGACCAATTCC-3'(SEQ ID NO: 1) and 5'-CAAAGTTCCAGAGGGACCTG-3' (SEQ ID NO: 2); ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3'(SEQ ID NO: 3) and 5'-CAAGTCAGTGTACAGGTAAGC-3'(SEQ ID NO: 4). PCRs 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 covering 16 tissues (BD Biosciences) were hybridized with an [alpha-³²P]-dCTP-labeled, 421-bp PCR product of RASEF that was prepared as a probe using primers 5'-GGCTGACATTCGTGACACTG-3'(SEQ ID NO: 5) and 5'-CAAAGTTCCAGAGGGACCTG-3' (SEQ ID NO: 6). Prehybridization, hybridization, and washing were done following the manufacturer's specifications. The blots were autoradiographed with intensifying screens at -80 degerees C for 10 days.

Western blot analysis. Cell lysates from lung cancer cell line or normal airway epitherial cells were subjected to western blotting. In brief, cells were incubated in 1 mL lysis buffer (0.5% NP-40, 50mmol/L Tris-HCl, 150mmol NaCl) in the presense of protease inhibitor (Protease Inhibitor Cocktail Set III; Calbiochem). Western blotting were done using an ECL western-blotting analysis system (GE Healthcare Bio-sciences), as previously described (Yamabuki T, et al. Cancer Res 2007;67:2517-25.). A rabbit polyclonal anti-human RASEF antibody (Proteintech Group, Inc.) and the specific antibodies indicated later were used as the primary antibody and donkey anti-rabbit and -mouse IgG-HRP antibody (GE Healthcare Bio-sciences) were served as the secondary antibodies for the experiments.

Immunocytochemical analysis. Cells were plated on glass coverslips (Becton Dickinson Labware), fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 3 min at room temperature. Nonspecific binding was blocked by Casblock (ZYMED) for 10 min at room temperature. Cells were then incubated for overnight at 4 degerees C with a rabbit polyclonal anti-human RASEF antibody diluted in PBS containing 1% BSA. After being washed with PBS, the cells were stained by Alexa488-conjugated secondary antibody (Invitrogen) for 60 min at room temperature. After another wash with PBS, each specimen was mounted with Vectashield (Vector Laboratories, Inc.) containing 4',6-diamidino-2-phenylindole (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS; Leica Microsystems).

Immunohistochemistry and tissue microarray. To investigate the significance of RASEF overexpression in clinical lung cancers, tissue sections were stained using ENVISION+ Kit/HRP (DakoCytomation). Anti-RASEF antibody (Proteintech Inc.) 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. Tumor tissue microarrays were constructed with formalin-fixed 341 primary lung cancers, each of which had been obtained by a single institutional group (please see above) with an identical protocol to collect, fix, and preserve the tissues after resection (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.). The tissue area for sampling was selected based on visual alignment with the corresponding H and E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments). A core of normal tissue was punched from each case, and 5-micrometer sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed RASEF positivity without prior knowledge of clinicopathologic data. The intensity of RASEF staining was evaluated using the following criteria: strong positive (scored as 2+), brown staining in > 50% of tumor cells completely obscuring cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell cytoplasm; and absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted as strongly positive only if the three reviewers independently defined them as such.

Statistical analysis. Statistical analyses were done using the StatView statistical program (SAS). Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for RASEF expression; differences in survival times among patient subgroups were analyzed using the log-rank test. The expected sample numbers assigned in this analysis was calculated as follows; a difference in survival rate after following up for 2000 days was supposed as more than 15% with an 80% power for a two-sided significance level at 5%. 345 assessable patients were expected to be required. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, it was analyzed associations between death and possible prognostic factors, including age, gender, pathologic tumor classification, and pathologic node classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong RASEF expression into the model, along with any and all variables that satisfied an entry level of a P value of < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.

RNA interference assay. Previously, a vector-based RNA interference system, psiH1BX3.0 that was designed to synthesize small interfering RNAs (siRNA) in mammalian cells was established (Suzuki C, et al. Cancer Res 2003;63:7038-41.). Ten micrograms of siRNA expression vector were transfected using 30 micro-L of LipofectAMINE 2000 (Invitrogen) into lung cancer cell lines LC319 and A549. The transfected cells were cultured for 7 days in the presence of appropriate concentrations of geneticin (G418); 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 assay at 7 days after the treatment. Briefly, cell counting kit-8 solution (Dojindo) was added to each dish at a concentration of 1/10 volume, and the plates were incubated at 37 degerees C for additional 1 hour. Absorbance was then measured at 490 nm, and at 630 nm as a reference, with a Microplate Reader 550 (Bio-Rad). To confirm suppression of RASEF mRNA expression, semiquantitative RT-PCR experiments were carried out with the following synthesized RASEF-specific primers according to the standard protocol. The target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (EGFP: enhanced green fluorescent protein gene, a mutant of Aequorea victoria green fluorescent protein), 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 7); control 2 (luciferase/LUC: Photinus pyralis luciferase gene), 5'-CGTACGCGGAATACTTCGA-3'(SEQ ID NO: 8); siRNA-RASEF-1, 5'-GTTAGTACCTTGTACCAAA-3'(SEQ ID NO: 9); siRNA-RASEF-2, 5'-CTTCATCCGTGAGATCAGA-3'(SEQ ID NO: 10).
To evaluate the biological functions of RASEF in lung cancer cells, used small interfering RNA (siRNA) duplexes against the target genes (Sigma-Aldrich) were used.
siRNAs were transfected into NCI-H2170 and NCI-H1373, using 30 microlitter of Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Cell numbers and viability were measured by Giemsa staining and triplicate MTT assays (cell counting kit-8 solution; Dojindo Laboratories) at 5 days after the transfection. Expression of endogenous RASEF protein was detected by Western blotting.

Construction of RASEF-expressing vector. The entire coding region of RASEF was amplified by RT-PCR using the primer sets (5'-GGAATTCCGCCAGGCGGGATGGAGGCGGATG-3' (SEQ ID NO: 11) and 5'-ATTTGCGGCCGCTTGCCATTGCAACAATTCTTCATC-3'(SEQ ID NO: 12)). The product was digested with EcoRI and XhoI or EcoRI and Not I , and cloned into appropriate sites of a pcDNA3.1-myc/His A (+) vector (Invitrogen) that contained c-myc-His epitope sequences (LDEESILKQEHHHHHH) (SEQ ID NO: 13) at the COOH-terminal of the RASEF protein. Additionally, expression vector was constructed using pCAGGSn-3FH vector, which contained 3 X Flag epitope sequences (DYKDHDGDYKDHDIDYKDDDDK) (SEQ ID NO: 14) at the NH₂-terminal of the RASEF protein.

Cell growth assays. COS-7 and DMS114 cells transfected either with plasmids expressing RASEF or with mock plasmids were seeded onto six-well plates (5 X 10⁴ cells/well), and maintained in medium containing 10% FBS and 0.4 mg/ml geneticin. After 120 hours cell proliferation was evaluated by the MTT assay using Cell Counting Kits (Wako).

In vitro kinase assay. To evaluate whether RASEF was phosphorylated by extracellular signal-related kinase (ERK), in vitro kinase assay using active recombinant ERK2 (Upstate Biotechnology) was done. Briefly, 1 microgramERK2 protein was incubated in 20 microliter kinase assay buffer[50 mM tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 1 mM NaF, and 0.1microliter protease inhibitor] and then supplemented with 1 mM ATP containing 3 microCi [gamma-³²P] ATP (GE Healthcare Bio-sciences). For the substrates, 4 microliter or 8 microliter full length RASEF recombinant proteins (see below) were added in the reaction solutions. After 20-minute incubation at 30 degrees C, the reactions were terminated by addition of SDS sample buffer. After boiling, the protein samples were electrophoresed on 10% to 20% gradient gel (Bio-Rad), and transferred to nitrocellulose membranes. It was analyzed by autoradiography. To prepare RASEF protein as a substrate, the cell lysates from DMS114 transfected with Flag-tagged RASEF expression vector were immunoprecipitated with anti-Flag M2 agarose antibody (Sigma-Aldrich) at 4 degrees C for 2 hour. The immunoprecipitates were washed six times with lysis buffer, and then eluted with 3 X Flag peptide (Sigma-Aldrich). Briefly, 0.5 microgram of RASEF immunoprecipitant was incubated in 20 microlitter kinase assay buffer [50 mM tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 1 mM NaF, and 0.1 micro-L protease inhibitor] supplemented with 1 mM ATP containing 3 micro Ci [gamma-³²P] ATP (GE Healthcare) and 10 ng ofglutathione S-transferase (GST)-tagged active recombinant ERK2 (Upstate Biotechnology) for 20 minutes at 30 degrees C. The reactions were terminated by addition of SDS sample buffer. After boiling, the protein samples were electrophoresed on 10% to 20% gradient gel (Bio-Rad Laboratories), and transferred to nitrocellulose membrane which was analyzed by autoradiography. MBP was used as a positive control.

Preparation of RASEF recombinant protein. To use the full length RASEF recombinant protein as a substrate for in vitro kinase assay, Flag-tagged RASEF expression vectors was transfected into DMS114 cells. The cell lysates were immunoprecipitated with Flag-conjugated agarose M2 gel (Sigma-Aldrich) at 4 degerees C for 1 hour. These immunoprecipitates were washed six times with lysis buffer containing 50 mM Tris-HCL (pH 7.5), 150 mM NaCL and 1% NP-40. Aliquots (20 microliter) of immunoprecipitates were subjected to immunoblotting using rabbit anti-Flag antibody (Sigma-Aldrich) to check the success of the immunoprecipitation experiments.

Synthesized dominant-negative peptide
The three different kinds of 23-amino acid polypeptides covering the ERK1/2-binding site on RASEF 520-575 with a membrane-permeable 11 residues of arginine (11R) at its N-terminus (11R-RASEF 520-542, RRRRRRRRRRR-GGG-SALSPQTDLVDDNAKSFSSQKAY(SEQ ID NO 41); 11R-RASEF 536-558, RRRRRRRRRRR-GGG-FSSQKAYKIVLAGDAAVGKSSFL(SEQ ID NO42); 11R-RASEF 553-575, RRRRRRRRRRR-GGG-GKSSFLMRLCKNEFRENISATLG(SEQ ID NO43)) were synthesized as previously described (Hayama S, et al., Cancer Res 2006;66:10339-48.; Yamabuki T, et al., Cancer Res 2007;67:2517-25.). Scramble peptides (SCR) derived from the most effective 11R-RASEF 553-575 peptides were synthesized as a control (RRRRRRRRRRR-GGG-RSENKMSLFRGSEFTLLKGCINA(SEQ ID NO44)). Peptides were purified by preparative reverse-phase high-performance liquid chromatography and were >95% purity.
NCI-H12170, NCI-H1373 and BEAS-2B cells were incubated with the 11R peptides at the concentration of 5, 10, or 15 micro-M for 5 days. The medium was exchanged at every 48 hours at the appropriate concentrations of each peptide, and the viability of cells was evaluated by MTT assay.

Results
RASEF expression in lung cancers and normal tissues. To identify novel molecules that can be applicable to develop novel biomarkers and treatments based on the biological characteristics of cancer cells, genome-wide expression profile analysis of 120 lung carcinomas was performed using a cDNA microarray (Kikuchi T, et al. Oncogene 2003;22:2192-205.; Kakiuchi S, et al. Mol Cancer Res 2003;1:485-99.; Kakiuchi S, et al. Hum Mol Genet 2004;13:3029-43.; Kikuchi T, et al. Int J Oncol 2006; 28:799-805.; Taniwaki M, et al. Int J Oncol 2006;29:567-75.). Among 32,256 genes screened, an elevated expression (5-fold or higher) of RASEF transcript was identified in cancer cells in the great majority of the lung cancer samples examined. Its overexpression was confirmed by means of semiquantitative RT-PCR experiments in 12 of 15 lung cancer tissues, and in 14 of 22 lung cancer cell lines (Fig. 1A). Also, its expression levels in clinical lung cancers and adjacent normal lung were confirmed, and confirmed its overexpression (Fig. 1B). The expression levels of RASEF protein in lung cancer cells were evaluated by western blotting (Fig. 1C). Signals on about 100kD were detected in NCI-H1373 and NCI-H2170 cells which were confirmed its expression by semi-quantitative RT-PCR, whereas no signals were detected in NCI-H226 cells as well as bronchial epithelia derived BEAS-2B cells whose expressions of RASEF gene were negative (Fig.1A, lower panel). The upper weak signals represented phosphorylated RASEF protein, which was confirmed by western blotting after phosphatase treatment (Fig. 1D). Immunofluorescence analysis was done to examine the subcellular localization of endogenous RASEF in lung cancer cells. RASEF was detected mainly at cytoplasm of tumor cells at a high level in NCI-H2170, NCI-H1373 and A549 cells in which RASEF transcript was detected by semiquantitative RT-PCR experiments, but not in NCI-H226 cells and BEAS-2B cells (Fig. 1E, F).

Northern blot analysis using a RASEF cDNA fragment as a probe identified a transcript of 5.8kb only in Prostate and testis, but not in any other normal tissues (Fig. 2A). Also, expression of RASEF protein was examined with polyclonal antibody specific to RASEF on five normal tissues (liver, heart, kidney, lung, and testis) and lung cancer tissues (ADC, SCC, and SCLC). RASEF staining was mainly observed in cytoplasm of lung tumor cells, moderately in prostate and weakly in testis, but not detected in other four normal tissues (Fig. 2B). Additionally, RASEF staining in NSCLC and adjacent normal tissues from patients who underwent surgery were evaluated, and found that most NSCLC tissues were stained but adjacent normal lung tissues were scarcely stained (Fig. 2C).

Association of RASEF expression with poor prognosis for NSCLC patients. To investigate the biological and clinicopathological significance of RASEF in pulmonary carcinogenesis, it was carried out immunohistochemical staining on tissue microarray containing tissue sections from 341 NSCLC cases that underwent curative surgical resection. RASEF staining detected with polyclonal antibody specific to RASEF was mainly observed at cytoplasm of tumor cells but was not in normal lung cells. A pattern of RASEF expression on the tissue array was classified ranging from absent (scored as 0) to weak/strong positive (scored as 1+ to 2+; the examples of staining were on Fig. 3A). Of the 341 NSCLCs, RASEF was strongly stained in 126 (37.0%) cases (score 2+), weakly stained in 150 (44.0%) cases (score 1+), and not stained in 65 (19.1%) cases (score 0) (Table 3A). Then, a correlation of RASEF expression (strong positive vs weak positive/absent) was examined with various clinicopathologic parameters such as age, gender pathologic tumor stage (tumor size; T1 vs T2-3), pathologic node stage (node status; N0 vs N1-2), and histology (ADC vs other histology types), and found that strong RASEF expression was significantly correlated with T factors (P=0.0006, Table 3A). The median survival time of NSCLC patients was significantly shorter in accordance with the higher expression levels of RASEF (P < 0.0001 log-rank test; Fig. 3B). Univariate analysis was applied to evaluate associations between patient prognosis and several factors including age, gender pathologic tumor stage, pathologic node stage, histology, and RASEF status (

score

0, 1+ vs score 2+). All those variables were significantly associated with poor prognosis. Multivariate analysis using a Cox proportional hazard model determined that RASEF (P = 0.0035) as well as other three factors (age, tumor size, and lymph node metastasis) were independent prognostic factors for surgically treated NSCLC patients (Table 3B).

Growth promoting effect of RASEF protein. To disclose the role of RASEF in progression of lung cancer, evaluated the growth promoting/ survival ability of RASEF was evaluated by inhibiting endogenous RASEF expression using siRNA, along with two/three different control siRNAs (siRNAs for LUC, SCR and EGFP). Treatment of effective siRNA on lung cancer cells, A549 and LC319 reduced the expression of RASEF (Fig. 4A), and resulted in significant inhibition of cell viability and colony numbers measured by MTT and colony formation assays (Fig. 4B and Fig. 4C; statistical analysis of colony formation assay is in Fig. 4D). Treatment of siRNA on lung cancer cells, NCI-H2170 and NCI-H1373, reduced the expression of RASEF protein (Fig. 4E), and resulted in significant inhibition of cell viability and colony numbers measured by MTT (Fig. 4F) and colony formation assays (Fig. 4G). These results suggested that RASEF is indispensable for cell growth or survival of lung cancer cells. Furthermore, the role of RASEF in cell growth was evaluated by introducing plasmids expressing RASEF or mock plasmids into COS-7 cells and DMS114 cells, which does not express RASEF. The expression of RASEF protein was confirmed by western blotting (Fig. 5A), and revealed that significant cell proliferation was seen in exogenous RASEF expressing cells (Fig. 5B). In accordance with the result of siRNA assays and with the fact that strong RASEF expression is associated with tumor size, these data strongly suggest that RASEF associates the progression of lung cancer by promoting cell proliferation.

RASEF is a substrate for ERK. The sequence of RASEF contains several consensus sites, serine or threonine followed by a proline (x-x-S/T-P) known as minimal consensus sequence for phosphorylation by ERK kinase, present inventors first focused on the possibility that RASEF was one of the substrate of ERK1/2 kinase. To demonstrate this hypothesis that RASEF is an ERK substrate, in vitro kinase assay of RASEF and ERK was carried out using full length RASEF immunoprecipitatant and active recombinant ERK2 protein. The phosphorylation of RASEF was increased in the presence of ERK2 protein (Fig. 6A, 6B), indicating that RASEF is could be phosphorylated by ERK2.

Furthermore, phosphoproteome analysis was performed using Mass spectrometry to identify ERK-dependent phosphorylation sites of RASEF. COS-7 cells that were transfected with Flag-taged RASEF or co-transfected with Flag-taged RASEF and myc-taged ERK2 were lysed 8 minutes after 100 ng/ml EGF (Sigma-Aldrich) stimulation. The cell lysates were immunoprecipitated with Flag as described above, and the proteins were electrophoresed on SDS-PAGE gel. The gels were stained with CBB (Fig. 6C). The RASEF bands were excised selectively to serve for analysis by matrix-assistedlaser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS; AXIMA-CFRplus, SHIMADZU BIOTECH, Kyoto, Japan). As the result of direct amino acid sequencing of RASEF (Table 4), the sequence coverage of both samples was higher than 80%. The RASEF protein treated with ERK2 transfection showed additional phosphorylated serine sites (Serine-377, Serine-386, Serine-402, Serine-406, Serine-523 and Serine-719), compared with untreated RASEF protein. It was determined that these serine-sites could be phosphorylated by ERK inside mammalian cells, and be important for cell proliferation.

Identification of ERK1/2-dependent phosphorylation site of RASEF
Another phosphoproteome analysis using Mass spectrometry was performed to identify ERK1/2-dependent in vivo phosphorylation site on RASEF as previously described (Nguyen MH, et al. Cancer Res 2010;70:5337-47.). COS-7 cells transfected with Flag-taged RASEF expression vector and myc-taged ERK2 expression vector were cultured in FCS-free medium for 20 hours. Then, cells were stimulated by 50 ng/mL of epidermal growth factor (EGF, Sigma-Aldrich) for 10 minutes with or without 20 micro-M MEK inhibitor U0126 (Cell Signaling Technology). The colloidal Coomassie brilliant blue (CBB) staining was performed after immunoprecipitation assay using anti-Flag M2 agarose antibody and electrophoresis on SDS-PAGE gel (Fig. 7A). The three bands corresponding to immunoprecipitated RASEF from the cells that were treated with or without EGF, or treated with both EGF and U0126 were excised selectively to serve for subsequent Mass spectrometric analysis.As the result of direct amino acid sequencing of RASEF, the sequence coverage of each sample was higher than 60%. Although several phosphorylated serine-sites (Ser-377, Ser-393 and Ser-523) were common to all samples, phosphorylated Ser-520 was shown in only the RASEF from the cells stimulated by EGF without U0126. Therefore, it was determined that Ser-520 was ERK1/2-dependently phosphorylated in mammalian cells. Then the expression vector of phospho-defective mutant RASEF whose Ser-520 was replaced with alanine (RASEF-S520A) was constracted, and it was confirmed that ERK1/2-dependent phosphorylation was decreased in RASEF-S520A compared with wild type RASEF by in vitro kinase assay (Fig. 7B).

The expression vector of phospho-mimicking RASEF whose Ser-520 was replaced with glutamic acid (RASEF-S520E) was also constructed. Growth assay of DMS114 cells transfected either with wild type RASEF or with RASEF-S520E or with RASEF-S520A expression vectors or with mock vector, revealed that phospho-mimicking RASEF-S520E enhanced growth promoting effect of RASEF, whereas phospho-defective RASEF-S520A negated that effect (Fig. 7C). This result suggests that ERK1/2-dependent phosphorylation of RASEF at Ser-520 has stimulatory influence on lung cancer cell growth.

RASEF is directly interacting with ERK. Subsequently the interaction between exogenous RASEF and endogenous ERK in mammalian cells was confirmed by immunoprecipitation (Fig. 8A). The lysates from COS-7 cells transfected Flag-taged RASEF were prepared and performed immunoprecipitation. The immunoprecipitates using rabbit polyclonal antibody to ERK (Santa-cruz) were subjected to Western blot analysis to detect exogenous RASEF and the immunoprecipitates using mouse monoclonal antibody to Flag were subjected to Western blot analysis to detect ERK. The direct interaction between exogenous RASEF and endogenous ERK was demonstrated as shown in Fig. 8A. Additionally, the interaction between exogenous RASEF and exogenous ERK2 was confirmed using COS-7 co-transfected with RASEF and ERK2 (Fig. 8B). Furthermore, the direct interaction between endogenous RASEF and ERK1/2 was confirmed by immunoprecipitation assay using extracts from lung cancer cell line NCI-H2170. The direct binding between endogenous RASEF and ERK1/2 was demonstrated as shown in Fig. 8C.

ERK1/2-dependent phosphorylation is important for sufficient RASEF-ERK1/2 binding
The significance of phosphorylation at Ser-520 of RASEF on the molecular biological function of RASEF protein was investigated. The present inventors first examined whether phosphorylation at Ser-520 could influence to subcellular localization and/or protein stability of RASEF, but there were no differences between wild type RASEF and RASEF-S520A (data not shown). Subsequently, to examine the effect of phosphorylation at Ser-520 on RASEF-ERK1/2 binding, the immunoprecipitation assay was performed with anti-ERK1/2 antibody or anti-Flag M2 agarose antibody using DMS114 cells transfected either with wild type RASEF or with RASEF-S520A expression vector. Western blotting with anti-ERK1/2 antibody or anti-Flag antibody revealed that RASEF-ERK1/2 binding was significantly decreased in RASEF-S520A compared with wild type RASEF (Fig. 8D), suggesting that ERK1/2-dependent phosphorylation at Ser-520 of RASEF is important for sufficient RASEF-ERK1/2 binding.

RASEF-ERK1/2 interaction promotes ERK1/2 activity
Because it has been reported that some of Rab proteins were involved in regulation of signal molecules (He D, et al. J Mol Med 2011;89:137-50.; Li L, et al. J Biol Chem 2010;285:19705-9.), the present inventors examined whether RASEF influences kinase activity of ERK1/2. A phosphorylation status of ERK1/2 was investigated through Western blot analysis using RASEF-overexpressing or -knockdown lung cancer cells. RASEF-overexpressing DMS114 cells showed enhanced phosphorylation of ERK1/2 compared with controls (Fig 9A), whereas NCI-H2170 treated with siRNA for RASEF showed significant decrease of ERK1/2 phosphorylation (Fig 9B). Overexpression and knockdown of RASEF showed no effect on phosphorylation status of both MEK1/2 and c-Raf which are known as upstream molecules of ERK1/2, strongly suggesting that RASEF directly mediates ERK1/2 kinase activity in lung cancer cells. The present inventors also made a comparison of ERK1/2 activity promoting effect between wild type RASEF and phospho-defective RASEF-S520A which weakly binds to ERK1/2, and confirmed that the effect was decreased in RASEF-S520A induced DMS114 cells (Fig. 9C).

Identification of the ERK1/2-binding site of RASEF
To further investigate the biological importance of the interaction of these two proteins, either of 3 partial constructs of RASEF with Flag sequence at its NH₂-terminal (RASEF1-240, RASEF170-520, and RASEF455-750; Fig. 10A) was transfected into COS-7 cells and DMS114 cells. Immunoprecipitation with polyclonal anti-ERK1/2 antibody, anti-ERK1/2 antibody or anti-Flag M2 agarose antibody indicated that the COOH-term side construct (RASEF455-750) including RAB domain was able to interact with endogenous ERK1/2 (Fig. 10B).

To further define the minimal and high-affinity ERK1/2-binding site in RASEF520-740, either of 4 additional constructs of RASEF was transfected into DMS114 cells (RASEF520-575, RASEF575-630, RASEF630-685 and RASEF685-740; Fig. 10C) and the immunoprecipitation assay was perfprmed with anti-Flag M2 agarose antibody. Western blotting with anti-ERK1/2 antibody revealed that RASEF520-575 was able to bind with ERK1/2, but other constructs were not (Fig. 10E). These experiments determined that the 55-amino acid polypeptide in RASEF should be important for the binding with ERK1/2.

Growth inhibition of lung cancer cells by dominant-negative peptides
To examine whether the interaction between RASEF and ERK1/2 could be target of lung cancer treatment, three different kinds of 23-amino acid polypeptides covering the ERK1/2-binding site on RASEF 520-575 (11R-RASEF 520-542, 11R-RASEF 536-558, and 11R-RASEF 553-575) was synthesized as shown in Fig. 11A. Initially, the inhibition of binding between RASEF and ERK1/2 was elevated by these cell-permeable peptides through immunoprecipitation assay. It was found that 11R-RASEF 553-575 reduced the binding between exogenous RASEF and endogenous ERK1/2 in COS-7 cells treated with each of three cell-permeable peptides (Fig. 11B). It was also confirmed that the endogenous RASEF-ERK1/2 binding was reduced by 11R-RASEF 553-575, but not by control scrambled peptide in NCI-H2170 (Fig. 11C). Furthermore, in vitro kinase assay revealed that 11R-RASEF 553-575 could inhibit ERK1/2-dependent phosphorylation of RASEF (Fig. 11D).

To evaluate the dominant negative effect of these peptides in lung cancer cell growth/survival, NCI-H2170, NCI-H1373 and BEAS-2B cells were incubated with each of these peptides at the concentration of 5, 10, or 15 microM. As a results of MTT assay, 11R-RASEF 553-575 showed concentration-dependent growth suppressive effect in NCI-H2170 and NCI-H1373, which were RASEF-positive cells, but not in BEAS-2B, which was RASEF-negative cells (Fig. 11E and F).

Discussion
Recent accumulation of knowledge in cancer genomics and molecular biochemistry provides new strategies for treatment of cancer, such as molecular target drugs / therapy (Daigo Y, et al., Gen Thorac Cardiovasc Surg 2008;56:43-53.). Molecular targeted drugs are highly specific to malignant cells, with minimal adverse effects due to their well-defined mechanisms of action. To find such molecules, a powerful screening system was established to identify proteins that were activated specifically in lung cancer cells. The strategy was as follows: (a) identification of up-regulated genes in 120 lung cancer samples through the genome-wide cDNA microarray system, containing more than 32,256 genes, coupled with laser microdissection (Daigo Y, et al., Gen Thorac Cardiovasc Surg 2008;56:43-53.; Kikuchi T, et al. Oncogene 2003;22:2192-205.; Kakiuchi S, et al. Mol Cancer Res 2003;1:485-99.; Kakiuchi S, et al. Hum Mol Genet 2004;13:3029-43.; Kikuchi T, et al. Int J Oncol 2006; 28:799-805.; Taniwaki M, et al. Int J Oncol 2006;29:567-75.); (b) verification of very low or absent expression of such genes in normal organs by cDNA microarray analysis and multiple-tissue Northern blot analysis; (c) confirmation of the clinicopathologic significance of their overexpression using tissue microarray consisting of hundreds of NSCLC tissue samples ( Suzuki C, et al. Cancer Res 2003;63:7038-41. ; Ishikawa N, et al. Clin Cancer Res 2004;10:8363-70. ;Kato T, et al. Cancer Res 2005;65:5638-46.; Furukawa C, et al. Cancer Res 2005;65:7102-10.; Ishikawa N, et al. Cancer Res 2005;65:9176-84. ; Suzuki C, et al. Cancer Res 2005;65:11314-25. ; Ishikawa N, et al. Cancer Sci 2006;97:737-45. ; Takahashi K, et al. Cancer Res 2006;66:9408-19.; Hayama S, et al. Cancer Res 2006;66:10339-48. ; Kato T, et al. Clin Cancer Res 2007;13:434-42. ; Suzuki C, et al. Mol Cancer Ther 2007;6:542-51.;Yamabuki T, et al. Cancer Res 2007;67:2517-25.; Hayama S, et al. Cancer Res 2007; 67:4113-22.; Taniwaki M, et al. Clin Cancer Res 2007;13:6624-31. ; Ishikawa N, et al. Cancer Res 2007;67:11601-11.; Mano Y, et al. Cancer Sci 2007;98:1902-13.); and (d) verification of the targeted genes whether they are essential for the survival or growth of lung cancer cells by siRNA (Suzuki C, et al. Cancer Res 2003;63:7038-41. ; Ishikawa N, et al. Clin Cancer Res 2004;10:8363-70. ;Kato T, et al. Cancer Res 2005;65:5638-46. ;Furukawa C, et al. Cancer Res 2005;65:7102-10.; Suzuki C, et al. Cancer Res 2005;65:11314-25. ; Ishikawa N, et al. Cancer Sci 2006;97:737-45. ; Takahashi K, et al. Cancer Res 2006;66:9408-19.; Hayama S, et al. Cancer Res 2006;66:10339-48. ; Kato T, et al. Clin Cancer Res 2007;13:434-42. ; Suzuki C, et al. Mol Cancer Ther 2007;6:542-51.;Yamabuki T, et al. Cancer Res 2007;67:2517-25.; Hayama S, et al. Cancer Res 2007; 67:4113-22.;Taniwaki M, et al. Clin Cancer Res 2007;13:6624-31. ; Ishikawa N, et al. Cancer Res 2007;67:11601-11.; Mano Y, et al. Cancer Sci 2007;98:1902-13. ; Kato T, et al. Clin Cancer Res 2008;14:2363-70.; Hirata D, et al. Clin Cancer Res 2009,15:256-66.). Through this screening system, several genes encoding oncoantigens were identified that good targets for the development of novel diagnostic markers, therapeutic drugs, and/or immunotherapy. In this study, one of these genes, RASEF, has been selected and shown to be frequently overexpressed in clinical lung cancer samples and cell lines, and that its gene product plays important roles in the growth of lung cancer cells.

RASEF is a member of Rab GTPase family which generally plays an important role in vesicle trafficking. Among Rab GTPase proteins only RASEF has the unique structure that consists of small GTPase domain on the COOH-terminus and a EF-hand domain which enables the protein to bind calcium ions on the NH₂-terminus (Shintani M, et al., Biochem Biophys Res Commun. 2007; 357: 661-7.). The function of this 100kD protein was not known , except for some suggestions that it is a tumor suppessor gene (Jonsson G, et al., J Natl Cancer Inst. 2005; 97: 1377-82.; Maat W, et al., Invest Ophthalmol Vis Sci. 2008; 49: 1291-8.). Several studies have reported on the implication of RASEF in malignant diseases, but the results of the studies have been contradictory; RASEF was overexpressed in the cDNA microarray using early esophageal squamous cell carcinoma samples (Zhang C, et al. Clin Chem 2010;56:1871-9.), whereas it was down-regulated in cutaneous malignant melanoma ( Jonsson G, et al. J Natl Cancer Inst 2005; 97:1377-82.). Until now, the relationship between RASEF and lung cancer has been unknown, and molecular biological function of RASEF in carcinogenesis and cancer progression has been not closely examined.

However,in this study, it was demonstrated that frequent overexpression of RASEF was seen in clinical lung cancer tissues and little or no expression of RASEF gene and protein was seen in normal organs including lung, heart, liver, and kidney. By analyzing tissue microarray, it was revealed that strong RASEF expression was associated with poorer clinical outcome for surgically treated NSCLC patients and was an independent prognostic factor by multivariate regression analyses. Furthermore it was also confirmed that inhibition of endogenous expression of RASEF by siRNA resulted in marked reduction of cell viability of lung cancer cells and exogenous expression of RASEF promoted cell growth in mammalian cells.

The results presented here show that RASEF plays a stimulatory role in lung cancer cells and is a prognostic biomarker and novel molecule target for this disease.

The mechanism of growth promotion effect of RASEF was described at the molecular level. The results of in vitro kinase assay, phosphoproteome analysis and immunoprecipitation assay suggest that RASEF interacts with ERK directly and is phosphorylated by ERK. Activation of ERK signaling frequently occurs in various human cancers, and has been recognized as an important target in the diagnosis and treatment of cancer (Roberts PJ et al., Oncogene. 2007: 26:3291-310.). Without wishing to be bound by theory, this study has revealed that RASEF positively mediates ERK1/2 activity through the direct binding with ERK1/2. Furthermore, it is interesting that ERK1/2-dependent phosphorylation at Ser-520 of RASEF is required for sufficient RASEF-ERK1/2 binding. We estimate that the regulation of RASEF-ERK1/2 binding is similar to that of ERK1/2-c-Fos interaction. The c-Fos protein known as a nuclear substrate of ERK1/2 is initially phosphorylated at C-terminal site of c-Fos without binding (Murphy LO,et al. Nat Cell Biol 2002;4:556-64.). This initial phosphorylation contributes exposure of ERK1/2-binding domain of c-Fos and allows the binding of ERK1/2 and facilitation of the secondary phosphorylation.

The present invention shows that RASEF plays an important role for pulmonary carcinogenesis and tumor progression as one of the downstream molecules of the ERK pathway.

It is suggested that ERK1/2 is able to be effectively and strongly cancer progression through the interaction with RASEF in RASEF-positive lung cancer cells. In other words, RASEF is thought to contribute lung carcinogenesis and tumor progression as an amplification mechanism of mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade including ERK is well defined as an important intracellular signaling pathway that regulates cell proliferation, cell cycle, cell survival, angiogenesis, and cell migration in human cancer, thus it has been the subject of intense research for discovery of new anticancer drugs. As an example, Selumetinib (AZD6244, ARRY-142886), a novel, selective inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), was reported to be effective for certain proportions of cancer patients in clinical trials ( Friday BB, et al. Clin Cancer Res 2008;14:342-6.).

Thus, the selective inhibition of functional interaction between ERK and RASEF using inhibitory peptide is an effective therapeutic strategy.
In this study, we demonstrated that inhibition of RASEF-ERK1/2 interaction using cell permeable peptide covering ERK1/2-binding site of RASEF resulted in marked suppression of RASEF-positive lung cancer cell growth.
In conclusion, RASEF is frequently expressed in lung cancers and RASEF overexpression in resected specimens is a useful index for application of adjuvant therapy for early stage lung cancer patients. Moreover, RASEF plays an important role in lung carcinogenesis and tumor progression through the interaction with ERK1/2 kinase. Inhibition of RASEF itself as well as its functional interaction with ERK1/2 kinase is therefore a useful therapeutic strategy for the lung cancer treatment.

The gene-expression analysis of cancers described herein, using the combination of laser-capture dissection and genome-wide cDNA microarray, has identified a specific gene as a target for cancer prevention and therapy. Based on the expression of this differentially expressed gene, i.e., RASEF, the present invention provides a novel molecular diagnostic marker for identifying and detecting cancers as well as assessing the prognosis. Further, ERK1 and/or ERK2, identified as the gene that its translation product was interacted with RASEF,. Therefore, the present invention also provides a novel diagnostic strategy using RASEF.
Furthermore, as described herein, RASEF is involved in cancer cell survival. Therefore, the present invention also provides a novel molecular target for treating and preventing cancer. This target is useful for developing novel therapeutic drugs and preventative agents without adverse effects.

The methods described herein are also useful for the identification of additional molecular targets for prevention, diagnosis, and treatment of cancers. The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, 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 of detecting or diagnosing lung cancer in a subject, comprising determining a expression level of RASEF in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing lung cancer, or the presence of lung cancer in said subject, wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of a RASEF gene,
(b) detecting a protein encoded by the RASEF gene, and
(c) detecting biological activity of a protein encoded by the RASEF gene.
The method of claim 1, wherein said increase is at least 10% greater than said normal control level.
The method of claim 1, wherein the subject-derived biological sample is a biopsy sample.
A kit for use in diagnosis or detection of lung cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the RASEF gene.
A method for assessing prognosis of a subject with lung cancer, wherein the method comprises steps of:
(a) detecting an expression level of RASEF in a subject-derived biological sample;
(b) comparing the detected expression level to a control level; and
(c) determining prognosis of the patient based on the comparison of (b).
The method of claim 5, wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis.
The method of claim 6, wherein the increase is at least 10% greater than said control level.
The method of claim 5, wherein said expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of a RASEF gene;
(b) detecting a protein encoded by the RASEF gene; and
(c) detecting a biological activity of the protein encoded by the RASEF gene.
A kit for assessing a lung cancer prognosis, wherein the kit comprises any one component selected from the group consisting of:
(a) a reagent for detecting the presence of an mRNA encoding the amino acid sequence of SEQ ID NO: 16.
(b) a reagent for detecting the presence of a protein comprising the amino acid sequence of SEQ ID NO: 16 , and
(c) a reagent for detecting the biological activity of a protein comprising the amino acid sequence of SEQ ID NO: 16.
The method of claims 5 to 8, and the kit of claim 9, wherein the lung cancer is NSCLC.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a RASEF polypeptide, or fragment thereof;
(b) detecting binding activity between the polypeptide or fragment thereof, and the test substance; and
(c) selecting the test substance that binds to the polypeptide or fragment thereof.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a cell expressing a RASEF gene;
(b) detecting an expression level of the RASEF gene; and
(c) selecting the test substance that reduces the expression level of the RASEF gene in comparison with the expression level detected in absence of the test substance.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a RASEF polypeptide or fragment thereof;
(b) detecting a biological activity of the polypeptide or fragment thereof of step (a); and
(c) selecting the test substance that suppresses a biological activity of the polypeptide or fragment thereof in comparison with a biological activity detected in the absence of the test substance.
The method of claim 13, wherein the biological activity is cell proliferation enhancing activity.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
(a) contacting a test substance with a cell into which a vector comprising a transcriptional regulatory region of a RASEF gene and a reporter gene that is expressed under control of transcriptional regulatory region has been introduced,
(b) measuring expression or activity of said reporter gene; and
(c) selecting the test substance that reduces an expression or activity level of said reporter gene, in comparison with the level detected in the absence of the test substance.
A method of screening for a candidate substance for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising the steps of:
(a) contacting a RASEF polypeptide or functional equivalent thereof with an ERK1 polypeptide or functional equivalent thereof and/or an ERK2 polypeptide or functional equivalent thereof in the presence of a test substance ;
(b) detecting a binding 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 or inhibits the binding level in comparison with the level detected in the absence of the test substance .
The method of claim 16, wherein the functional equivalent of the RASEF polypeptide comprises an ERK1 and/or ERK2-binding domain.
The method of claim 17 wherein the ERK1 and/or ERK2-binding domain is comprised in residues 553-575 of SEQ ID NO: 16.
The method of claim 17 wherein the ERK1 and/or ERK2-binding domain is comprised in residues 520-575 of SEQ ID NO: 16.
The method of claim 17 wherein the ERK1 and/or ERK2-binding domain is comprised in residues 455-740 of SEQ ID NO: 16.
The method of claim 18, 19 or 20, wherein the functional equivalent of the ERK1 polyeptide and/or the ERK2 polypeptide comprises a RASEF -binding domain.
A method of screening for a candidate substance for treating or preventing a disease associated with overexpression of a RASEF gene, or inhibiting proliferation of a cell expressing the RASEF gene, said method comprising the steps of:
(a) contacting a RASEF polypeptide or a fragment thereof with an ERK1 polypeptide or functional equivalent thereof and/or an ERK2 polypeptide or a fragment thereof in the presence of a test substance under a suitable condition for phosphorylation;
(b) detecting the phosphorylation level of the RASEF polypeptide or fragment thereof;
(c) comparing the phosphorylation level with that detected in the absence of the test substance; and
(d) selecting the test substance that reduces the phosphorylation level of the RASEF polypeptide or fragment thereof as compared to the phosphorylation level detected in the absence of the test substance.
The method of claims 22, the fragment of the RASEF polypeptide comprises the Serine-377, Serine-386, Serine-402, Serine-406, Serine-520, Serine-523, or Serine-719 in the amino acid sequence of SEQ ID NO: 16.
The merhod of claim 22, the fragment of the ERK1 polypeptide and/or the ERK2 polypeptide is a fragment retaining kinase activity.
The method of claims 22, wherein the step (a) comprises incubating a RASEF polypeptide or a fragment thereof and an ERK1 polypeptide or a fragment thereof and/or an ERK2 polypeptide or a fragment thereof in the presence of a phosphate donor in the incubation mixture.
The method of claims 25, wherein the phosphate donor is ATP.
The method of claim 22, the disease associated with overexpression of RASEF is cancer.
A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10, and the antisense strand comprises a nucleotide sequence complementary to said target sequence, wherein said sense strand and said antisense strand hybridize to each other to form the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits the expression of said gene.
The double-stranded molecule of claim 28 wherein the sense strand hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
The double-stranded molecule of claim 28 or 29 wherein said double-stranded molecule is a single polynucleotide comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
The double-stranded molecule of claim 30 wherein said polynucleotide has a general formula
5'-[A]-[B]-[A']-3'
, wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
A vector comprising each or both of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 and 10, and wherein the antisense strand comprises a nucleotide sequence which is complementary to said sense strand, wherein 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 a RASEF gene, inhibits expression of said RASEF gene.
A vector comprising each or both of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9 and 10 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing a RASEF gene, inhibits the cell proliferation.
The vector of claim 32 or 33, wherein the polynucleotide is a polynucleotide of between about 19 and about 25 nucleotides in length.
The vector of claim 32 or 33, wherein said double-stranded molecule is a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
The vector of claim 35, wherein said polynucleotide has a general formula
5'-[A]-[B]-[A']-3'
wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO:10; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
A method of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against a RASEF gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the RASEF gene.
A method of claim 37, wherein the double stranded molecule is that of any one of claims 27 to 30.
A method of claim 38, wherein the vector is that of any one of claims 31 to 35.
A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against a RASEF gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the RASEF gene, inhibits cell proliferation as well as the expression of the RASEF gene, and a pharmaceutically acceptable carrier.
The composition of claim 40, wherein the double stranded molecule is that of any one of claims 28 to 31.
The composition of claim 41, wherein the vector is that of any one of claims 30 to 34.
A polypeptide comprising the amino acid sequence of (a) or (b) below:
(a) the amino acid sequence of SEQ ID NO: 43;
(b) the amino acid sequence in which one, two or several amino acid is substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43;
wherein the polypeptide inhibits a biological activity of the RASEF polypeptide.
The polypeptide of claim 43, wherein the biological activity of the RASEF polypeptide is a binding activity to the ERK1/2 protein.
The polypeptide of claim 43 or 44, which is modified with a cell-membrane permeable substance.
The polypeptide of claim 45, which has the following general formula:
[R]-[D];
wherein [R] represents the cell-membrane permeable substance; and [D] represents a polypeptide comprising the amino acid sequence of (a) or (b) below:
(a) the amino acid sequence of SEQ ID NO: 43;
(b) the amino acid sequence in which one, two or several amino acid is substituted, deleted, inserted and/or added in the amino acid sequence of SEQ ID NO: 43,
wherein [R] and [D] are linked directly or indirectly through a linker.
A composition for treating and/or preventing cancer expressing RASEF gene, wherein the composition comprises the polypeptide of any one of claims 43 to 46 and a pharmaceutically acceptable carrier.
The composition of claim 47, wherein the cancer to be treated is lung cancer.
A method for treating and/or preventing cancer expressing RASEF gene, wherein the method comprises the step of administering the polypeptide of any one of claims 42 to 45 to a subject.
The method of claim 49, wherein the cancer to be treated is lung cancer.