# Patent

## Title
Cell growth inhibitor and screening method thereof

## Abstract

An object is to provide a cell growth inhibitor also effective for androgen-independent prostate cancer. The present invention provides a cell growth inhibitor having, as an active ingredient, an expression inhibitor or function inhibitor of an antisense RNA (CTBP1-AS) expressed in the vicinity of an androgen receptor (AR) binding site of a C-terminal binding protein (CTBP1) gene.

## Background
A computer readable text file, entitled “SequenceListing.txt,” created on or about Sep. 24, 2012 with a file size about 74 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
The present invention relates to a growth inhibitor of cells, such as prostate cancer cells, in which expression of a predetermined antisense RNA of a CTBP1 gene has been enhanced, a method of screening a cell growth inhibitor, and the like.
BACKGROUND ART
Prostate cancer is a cancer which manifests in men most frequently in Europe and United States. Also in Japan, with westernization of dietary habits and aging of population, the number of patients suffering from prostate cancer is increasing drastically. Methods used widely for the treatment of prostate cancer are surgical therapy including prostatectomy, chemotherapy with an anticancer agent, and radiation therapy. Surgical therapy is the first-line treatment, but when cancer is diagnosed as being in an advanced stage or when surgery cannot be selected because it is a recurrent cancer after surgery, a therapeutic method other than surgery is selected.
In general, proliferation of prostate cancer is stimulated by androgen. Androgen is a steroid hormone having functions such as sex differentiation into male, function maintenance of reproductive organs, secondary sexual development, spermatogenesis, and promotion of anabolic action in skeletal muscles and the like. Two androgens (testosterone and dihydrotestosterone (DHT) are mainly involved in masculinization of humans. Testosterone is, after synthesis in testicular interstitial cells, transported to target cells such as prostate cells via the blood stream. In the cells, testosterone is converted into DHT by 5-α-hydrogenase and the resulting DHT binds to an androgen receptor (AR) in the cytoplasm. AR bound to DHT becomes an active type, is transported into the nucleus, and binds to an androgen responsive sequence on the target gene to function as a transcription factor activating expression of the target gene.
As therapeutic methods of prostate cancer other than surgical treatment, hormone therapy for inhibiting production and function of androgen is often employed and in most cases, it produces considerably good effects. Within several years after this hormone therapy, however, androgen-independent prostate cancer sometimes occurs. It therefore becomes an important object to control androgen-independent cancer.
Detailed molecular mechanism how androgen-dependent cancer progresses to androgen-independent cancer has not yet been elucidated, but involvement of AR in it has been suggested. More specifically, it has been suggested that in androgen independent cancer, AR which has undergone mutation or amplification shows sensitivity to ultra-low-concentration androgen or another steroid hormone (refer to, for example, Non-patent Documents 1 to 3). A method of treating prostate cancer by inhibiting binding of AR to ligand or inhibiting expression or function of AR by RNAi (RNA interference) method has been studied (refer to, for example, Non-patent Documents 4 to 6).
There is however a limitation in the method of inhibiting the function or expression of AR particularly for the treatment of prostate cancer which has recurred and a clinically sufficient method has not yet been developed.
PRIOR ART DOCUMENTS
Non-Patent Documents


    Non-patent Document 1: Suzuki H, et al. Endocr. Relat. Cancer 10, 2003, 209-216
    Non-patent Document 2: Chen C D, et al. Nat Med. 10, 2004, 33-39
    Non-patent Document 3: Debes J D, et al. N Eng J Med 351, 2004, 1488-1490
    Non-patent Document 4: Sun A, et al. Int J Cancer. 2009 Jul. 29. [Epub ahead of print] PMID: 19642108
    Non-patent Document 5: Compagno D, et al. PLoS One. 2007 Oct. 10; 2(10): e1006.PMID: 17925854
    Non-patent Document 6: Snoek R, et al. Clin Cancer Res. 2009 Jan. 1; 15(1): 39-47


DISCLOSURE OF THE INVENTION
Problem to be Solved by the Invention
An object of the present invention is to provide a cell growth inhibitor also effective for androgen-independent prostate cancer.
Means for Solving the Problem
With a view to overcoming the above-mentioned problem, the present inventors thought that since inhibition of AR was an important therapeutic method of prostate cancer and in androgen-independent cancer for which endocrine treatment was ineffective, AR signals were activated by mutation or amplification of AR, signals downstream of AR were useful as a therapeutic target. Based on this thought, an AR binding sites and a histone H3 acetylation sites were identified by using the chromatin immunoprecipitation method and a human whole genome tiling array in combination (ChIP-chip method; refer to, for example, Cawley S, et al. Cell 116, 2002, 499-509, Kaneshiro K, et al. Genomics. 89, 2007, 178-188) and moreover, by cap analysis gene expression (CAGE method, refer to, for example, Shiraki T, et al. Proc Natl Acad Sci USA. 100, 2003, 15776-81, FANTOM Consortium, Nat Genet. 41, 2009, 553-562), transcriptional start sites were genome-widely identified and the promoter usage was profiled.
As a result, it has been found that a CTBP1 protein known to function as a corepressor in the nucleus functions as a transcription inhibitor of AR. It has been found during the study on CTBP1 that a transcript of a sense DNA in the vicinity of the AR binding site of the CTBP1 gene, that is, an antisense RNA in the vicinity of the AR binding site of the CTBP1 gene is induced strongly by androgen.
It has also been confirmed that suppression of the function of the antisense RNA by the RNAi enhances CTBP1 expression at both mRNA level and protein level, while it suppresses transcriptional activity of AR and suppresses proliferation of prostate cancer cells.
It has also been confirmed that the expression amount of the antisense RNA is significantly high in prostate cancer and metastatic cancer cells thereof compared with that of normal prostate and the expression amount of CTBP1 is significantly low in prostate cancer and metastatic cancer cells thereof compared with that of normal prostate.
It has further been confirmed that in a nude-mouse prostate cancer cell transplant model, the antisense RNA controls CTBP1 negatively and administration of a double-stranded RNA having an RNAi effect against the antisense RNA suppresses proliferation of prostate cancer.
Moreover, it has been confirmed that with regard to a mechanism of suppressing CTBP1-AS and thereby suppressing the growth of prostate cancer, PSF which is an RNA-binding transcriptional repressor binds to CTBP1-AS and this complex suppresses transcription of a cell cycle-inhibiting gene to promote cell growth; the complex suppresses the expression of CTBP1, resulting in promotion of transcription activation by AR; and accordingly, suppression of the expression of CTBP1-AS leads to inhibition of cell proliferation and suppression of activation of AR, leading to the completion of the present invention.
The present invention therefore relates to:
[1] a cell growth inhibitor, which inhibits the growth of cells in which any of the following antisense RNAs (which will hereinafter be called “CTBP1-AS”):
(i) an antisense RNA of a CTBP1 gene containing a partial sequence of the base sequence represented by SEQ ID NO:1;
(ii) an antisense RNA of a CTBP1 gene containing the base sequence represented by SEQ ID NO:5; and
(iii) a mutant or variant of the antisense RNA described above in (i) or (ii) or an antisense RNA in which one or several bases have been deleted, added, or substituted in the antisense RNA described above in (i) or (ii)
has been expressed, containing a CTBP1-AS expression inhibitor or function inhibitor as an active ingredient;
[2] the cell growth inhibitor as described in Claim 1, wherein the CTBP1-AS is an antisense RNA having a base sequence described in any of SEQ ID NOS: 17 to 20;
[3] the cell growth inhibitor as described above in [1] or [2], wherein the expression inhibitor or function inhibitor is a compound selected from the group consisting of the following (a) to (d);
(a) antisense nucleic acids against CTBP1-AS or a portion thereof,
(b) nucleic acids having ribozyme activity to specifically cleave CTBP1-AS;
(c) double-stranded nucleic acids having an RNAi effect against CTBP1-AS; and
(d) nucleic acids encoding the nucleic acids described in any of (a) to (c);
[4] the cell growth inhibitor as described above in [3], wherein the expression inhibitor or function inhibitor is a double-stranded RNA having an RNAi effect against CTBP1-AS and one strand of the double-stranded RNA has a sequence complementary to consecutive 19 to 30 bases in CTBP1-AS;
[5] the cell growth inhibitor as described above in [3], wherein the expression inhibitor or function inhibitor is a double-stranded RNA having an RNAi effect against CTBP1-AS and one strand of the double-stranded RNA has a base sequence complementary to the base sequence represented by any of SEQ ID NOS:3 to 6 and 24 to 52;
[6] the cell growth inhibitor as described above in any one of [1] to [5], wherein the cells in which CTBP1-AS has been expressed are prostate cancer cells and/or metastatic cancer cells thereof;
[7] a pharmaceutical composition containing the cell growth inhibitor as described above in any one of [1] to [6];
[8] a method of preventing or treating prostate cancer, which includes administering a therapeutically effective amount of the cell growth inhibitor as described above in any one of [1] to [6];
[9] a method of screening growth inhibitors of cells in which CTBP1-AS has been expressed, including a step of bringing cells in which CTBP1-AS has been expressed or a cell extract thereof into contact with test compounds in the presence of androgen; a step of measuring an expression level of CTBP1-AS; and a step of selecting, from the test compounds, test compounds decreasing the expression level of CTBP1-AS compared with an expression level measured in the absence of the test compounds;
[10] a method of screening proliferation inhibitors of cells in which CTBP1-AS has been expressed, including a step of bringing cells which have expressed a CTBP1 gene or a cell extract thereof into contact with test compounds in the presence of androgen; a step of measuring an expression level of the CTBP1 gene; and a step of selecting, from the test compounds, test compounds increasing the expression level of the CTBP1 gene compared with an expression level measured in the absence of the test compounds;
[11] the method as described above in [9] or [10], wherein the cells in which CTBP1-AS has been expressed are prostate cancer cells and/or metastatic cancer cells thereof;
[12] a testing method for judging the prognosis of prostate cancer, including a step of measuring the expression level of CTBP1-AS in cells sampled from the prostate of a patient and a step of comparing the expression level with an expression level in normal prostate cells; and
[13] a testing method for judging the prognosis of prostate cancer, including a step of measuring the expression level of a CTBP1 gene in cells sampled from the prostate of a patient and a step of comparing the expression level with an expression level in normal prostate cells.
Effect of the Invention
The cell growth inhibitor of the present invention can efficiently inhibits the growth of cells in which expression of CTBP1-AS has been enhanced such as prostate cancer cells by inhibiting the expression or function of CTBP1-AS.
The cell growth inhibitor of the present invention suppresses transcriptional activity of signals downstream of AR by inhibiting expression or function of CTBP1-AS so that it is presumed to be effective against androgen-independent cancer in which AR signals have been activated by AR variation or amplification.
Using a double-stranded nucleic acid having an RNAi effect against CTBP1-AS as a cell growth inhibitor of the present invention enables high target specificity and high safety, because it makes use of a mechanism which cell originally have.
In addition, the present invention provides a screening method capable of efficiently selecting, from test compounds, a growth inhibitor of cells in which expression of CTBP1-AS has been enhanced; a method of preventing or treating prostate cancer; and a testing method for evaluating the prognosis of prostate cancer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows measurement results of the expression amount of CTBP1-AS when siRNA against CTBP1-AS or a control double-stranded RNA was administered to LNCaP cells. The CTBP1-AS expression was induced by androgen (R1881) and suppressed by the administration of siRNA against CTBP1-AS.
FIG. 2 shows measurement results of the expression amount of CTBP1 (mRNA level) when siRNA against CTBP1-AS or a control double-stranded RNA was administered to LNCaP cells. The CTBP1 expression was suppressed by androgen and the suppressive effect by androgen was decreased by the administration of siRNA against CTBP1-AS.
FIG. 3 shows measurement results of the expression amount of CTBP1 (protein level) when siRNA against CTBP1-AS or a control double-stranded RNA was administered to LNCaP cells. The CTBP1-AS expression was suppressed by androgen and the suppressive effect by androgen was decreased by the administration of siRNA against CTBP1-AS.
FIG. 4A shows the results of experiments to confirm the influence of CTBP1-AS on the transcriptional activity of AR obtained by introducing a PSA luciferase vector into LNCaP cells and administering siRNA against CTBP1-AS or a control double-stranded RNA to the resulting cells. Luciferase activity, that is, transcriptional activity of AR was increased by androgen and suppressed by the administration of siRNA against CTBP1-AS. FIGS. 4B and 4C are studying results of the expression of an androgen responsive gene in the same cells. The expression amount of the androgen responsive gene was increased by androgen, but suppressed by the administration of siRNA.
FIG. 5 shows the results of experiments to investigate the influence of function suppression of CTBP1-AS on the proliferation of LNCaP cells by using MTS assay. Administration of siRNA against CTBP1-AS suppressed cell proliferation.
FIG. 6 shows the results of experiments to confirm the influence of function suppression of CTBP1-AS on a tumor volume in a mouse tumor model. Proliferation of tumor cells subcutaneously transplanted to mice was markedly suppressed by the administration of siRNA against CTBP1-AS.
FIG. 7 shows the results of experiments to confirm the influence of function suppression of CTBP1-AS on a tumor weight in a mouse tumor model. Proliferation of tumor cells subcutaneously transplanted to mice was markedly suppressed by the administration of siRNA against CTBP1-AS.
FIG. 8 shows the measurement results of the RNA level expression amounts of CTBP1-AS and CTBP1 in mouse tumor model. Administration of siRNA against CTBP1-AS decreased the expression amount of CTBP1-AS in tumor but increased the expression amount of CTBP1.
FIG. 9 shows the measurement results of the protein level expression amounts of CTBP1 in mouse tumor model. Administration of siRNA against CTBP1-AS increased the expression amount of the CTBP1 protein in tumor.
FIG. 10 shows the comparison results of the expression amount among normal, cancer, and metastatic cancer samples determined by extracting the signal intensity of a probe specific to AX747592 based on the published expression profile data. The expression amount of CTBP1-AS in cancer and metastasis cancer tissues was significantly higher than that in normal prostate.
FIG. 11A shows the results of experiments to assess the CTBP1 expression in prostate cancer cells by using immunostaining method. The CTBP1 expression amount was decreased in prostate cancer cells compared with that in normal cells. FIG. 11B shows the comparison results of a survival rate between CTBP1 high expression group and low expression group. The survival rate was significantly higher in the CTBP1 high expression group than that in the low expression group. FIG. 11C shows images of typical sections of normal prostate and prostate cancer obtained by immunostaining method.
FIG. 12 is a schematic view showing sequences of CTBP1-ASa, CTBP1-ASb, and CTBP1-ASd whose existence has been confirmed as a result of searching for CTBP1-ASs other than CTBP1-ASd by using the 3′ RACE PCR method.
FIG. 13 shows the northern blotting results by which expression of CTBP1-AS was confirmed using LNCaP-cell-derived RNA. An about 5-kb long transcription product is detected, suggesting that CTBP1-ASb is expressed mainly.
FIG. 14 shows the results of analyzing localized CTBP1-AS by northern blotting using nuclear RNA and cytoplasmic RNA (FIG. 14a) and the results of measuring the expression amount of cDNA synthesized from RNA by using qRT-PCR (FIG. 14b, corrected by GAPDH).
FIGS. 15a and 15b show the analysis results of the influence of androgen stimulation on the expression of CTBP1 and CTBP1-AS in LNCaP cells and LTAD cells which will be an androgen depletion resistant model by western blotting and northern blotting. FIGS. 15c and 15d show comparison results of cell proliferation ability compared by suppressing CTBP1-AS expression by RNAi method with or without androgen stimulation in LNCaP cells or LTAD cells.
FIG. 16 shows the measurement results of a tumor volume (b), mRNA-level CTBP1-AS expression amount (c), and protein-level CTBP1 expression amount (d), after transplanting LTAD cells of an androgen-depletion resistant model to nude mice and locally injecting siRNA against CTBP1-AS.
FIG. 17 shows the results of experiments to confirm the involvement of PSF, an RNA-binding transcriptional repressorr, in expression suppression of CTBP1. When transcription of PSF was suppressed by RNAi, the suppression degree of CTBP1 expression by androgen stimulation decreased.
FIG. 18 shows immunoprecipitation results showing androgen-dependent binding of PSF to CTBP1-AS.
FIG. 19 shows the results of analyzing localization of PSF and CTBP1-AS by RNA-FISH. Localization of PSF almost coincided with that of CTBP1-AS.
FIG. 20 shows the results of analyzing the relationship between PSF and cell cycle.
FIG. 21 shows the results of analyzing PSF-mediated control of SMAD3 and p53 by using siRNA against PSF through qRT-PCR (a) and western blotting (b).
FIG. 22 shows the results of studying the control of a cell cycle-related factor by CTBP1-AS by using siRNA against CTBP1-AS.
FIG. 23 is an explanatory view showing a gene expression control mechanism by CTBP1-AS and PSF.
FIG. 24 shows the results of studying the CTBP1-AS expression suppression effect of 26 siRNAs.



MODE FOR CARRYING OUT THE INVENTION
<Cell Growth Inhibitor>
The cell growth inhibitor according to the present invention is characterized in that it suppresses proliferation of cells in which CTBP1-AS has been expressed and that it contains a CTBP1-AS expression inhibitor or function inhibitor as an active ingredient.
The term “CTBP1” as used herein means a C-terminal binding protein 1 and the term “CTBP1 gene” means a gene encoding a CTBP1 protein. CTBP1 is known to function as a corepressor in the nucleus (Kim J H, et al. Nat Struct Mol Biol. 12, 2005, 423-428, Senyuk V, et al. Arch Biochem Biophys. 441, 2005, 168-173). For example, involvement of CTBP1 in transcription suppression of a ZEB1 gene and the like has been reported and generally, it is presumed to negatively control the transcription (Chinnadurai G, Int J Biochem Cell Biol. 39, 2007, 1593-1607).
As described later in Examples, the present inventors have found that in prostate cancer cells, expression of CTBP1 is significantly reduced and it is controlled by androgen in both mRNA level and protein level expression.
The term “CTBP1-AS” as used herein means any one of antisense RNAs (i) to (iii) of a CTBP1 gene expressed in the vicinity of the AR binding site of the CTBP1 gene.
(i) An antisense RNA of a CTBP1 gene containing a partial sequence of the base sequence represented by SEQ ID NO:1,
(ii) an antisense RNA of the CTBP1 gene containing the base sequence represented by SEQ ID NO:5, and
(iii) a mutant or variant of the antisense RNA described above in (i) or (ii) or an antisense RNA in which one or several bases have been deleted, added, or substituted in the antisense RNA described above in (i) or (ii).
The term “antisense RNA” means a transcription product formed with a sense chain (sense DNA) of a double-stranded DNA of a gene as a template.
CTBP1-AS is a molecule which will be a target of the cell growth inhibitor according to the present invention. In the living body, there is a plurality of antisense RNAs capable of satisfying any one of the above (i) to (iii) and all of them will become CTBP1-AS. The antisense RNAs (i) to (iii) will next be described respectively in detail.
(i) Antisense RNA of a CTBP1 gene containing a partial sequence of the base sequence represented by SEQ ID NO:1
The antisense RNA of a CTBP1 gene having a base sequence represented by SEQ ID NO:1 is a transcription product of a sense DNA in the vicinity of the AR binding site of the CTBP1 gene and its cDNA is known to have Accession Number: AX747592 (SEQ ID NO:2).
Examples of the antisense RNA of a CTBP1 gene containing a partial sequence of the base sequence represented by SEQ ID NO:1 include:
an antisense RNA consisting of only a partial base sequence of the base sequence represented by SEQ ID NO:1;
an antisense RNA comprising a partial sequence containing the 5′-end of the base sequence represented by SEQ ID NO:1 and further having one or more bases bound to the 5′-end; and
an antisense RNA comprising a partial sequence containing the 3′ end of the base sequence represented by SEQ ID NO:1 and further having one or more bases bound to the 3′ end.
The antisense RNAs expressed in the living body correspond to the antisense RNA of (i) insofar as it contains a partial sequence of the base sequence represented by SEQ ID NO:1. As a target of the cell growth inhibitor of the present invention, the antisense RNA consisting of a partial sequence containing the 3′ end of the base sequence represented by SEQ ID NO:1 and the antisense RNA containing a partial sequence containing the 3′ end of the base sequence represented by SEQ ID NO:1 and having one or more bases bound to the 3′ end are particularly preferred. Examples of the “partial sequence containing the 3′ end of the base sequence represented by SEQ ID NO:1” include, but not limited to, a partial sequence from position 1300 to the 3′ end of the base sequence described in SEQ ID NO:1, a partial sequence from position 1200 to the 3′ end, and a partial sequence from position 800 to the 3′ end.
(ii) Antisense RNA of the CTBP1 gene containing the base sequence represented by SEQ ID NO: 5.
Examples of the antisense RNA of the CTBP1 gene containing the base sequence represented by SEQ ID NO:5 include an antisense RNA consisting only of the base sequence represented by SEQ ID NO:5 and an antisense RNA having one or more bases bound to the 3′ end and/or 5′ end of the base sequence described in SEQ ID NO:5.
The base sequence represented by SEQ ID NO:5 corresponds to from position 2348 to position 2372 of the base sequence described in SEQ ID NO:1. The number of bases bound to the 3′ end and/or 5′ end is not particularly limited. The present inventors have confirmed that as shown later in Examples, administration of siRNA to this region as a target inhibits transcriptional activity of AR and also inhibits proliferation of prostate cancer cells. Accordingly, it is obvious that an expression inhibitor or function inhibitor of CTBP1-AS containing the base sequence represented by SEQ ID NO:5 is suited as an active ingredient of the cell growth inhibitor of the present invention.
As an example of the antisense RNA (ii), an antisense RNA containing the base sequence described in SEQ ID NO:23 is also preferred.
(iii) Mutant or variant of the antisense RNA as described above in (i) or (ii) or an antisense RNA in which one or several bases have been deleted, added, or substituted in the antisense RNA described in (i) or (ii).
Examples of the mutant or variant of the antisense RNA described in (i) or (ii) include various variants including splicing variants such as 5′ alternative splicing and 3′ alternative splicing, polymorphisms such as single nucleotide polymorphism (SNP) and copy number variation (CNV), and mutants (for example, cancer-derived mutants).
Also these mutants or variants can be expressed as a transcription product of a sense DNA in the vicinity of the AR binding site of a CTBP1 gene and will be a target of the cell growth inhibitor according to the present invention.
Although no particular limitation is imposed on the number of bases deleted or the like from the antisense RNA described in (i) or (ii) in which one or several bases have been deleted, added, or substituted insofar as it is a transcription product of a sense DNA in the vicinity of the AR binding site of the CTBP1 gene. The number is from 1 to 10, preferably from 1 to 5, more preferably from about 1 to 3 or corresponds to within 10%, preferably within 5%, more preferably within 1% of the entire length. Also the position of the bases to be deleted or the like is not particularly limited and it may be the base(s) at the 5′ end or 3′ end of the antisense RNA or the base(s) other than that at the ends. Alternatively, bases may be deleted, added, and/or substituted at a plurality of positions.
Such antisense RNAs may be expressed as a transcription product of the sense DNA at the AR binding site of CTBP1 gene and will be a target of the cell growth inhibitor according to the present invention.
Specific examples of the CTBP1-AS include RNAs consisting of the base sequence described in SEQ ID NOS:17 to 21.
The RNA having the base sequence described in SEQ ID NO:17 is an RNA of 3710 bases in total length which contains from position 698 to the 3′ end of the base sequence described in SEQ ID NO:1 and further has an RNA of 522 bases bound to the 3′ end (which will hereinafter be called “CIBP1-ASc”).
The RNA having the base sequence described in SEQ ID NO:18 is an RNA of 5041 bases in total length which contains from position 698 to the 3′ end of the base sequence described in SEQ ID NO:1 and further has an RNA of 1853 bases bound to the 3′ end (which will hereinafter be called “CTBP1-ASb”).
The RNA having the base sequence described in SEQ ID NO:19 is an RNA of 15756 bases in total length which contains from position 698 to the 3′ end of the base sequence described in SEQ ID NO:1 and further has an RNA of 12568 bases bound to the 3′ end (which will hereinafter be called “CTBP1-ASa”).
The RNA having the base sequence described in SEQ ID NO:20 is an RNA of 3189 bases in total length corresponding to from position 697 to the 3′ end of the base sequence described in SEQ ID NO:1 (which will hereinafter be called “CTBP1-ASd”).
It has already been confirmed that any of these antisense RNAs has been expressed as a transcription product of a sense DNA in the vicinity of the AR binding site of a CTBP1 gene so that they will be a target of the cell growth inhibitor according to the present invention.
The present inventors have recently found newly by analysis using prostate cancer cells LNCaP that expression of CTBP1-AS is strongly induced by androgen. CTBP1-AS is a non-coding RNA which does not encode a protein motif. CTBP1-AS has an enhanced expression level in prostate cancer cells. Inhibition of CTBP1-AS by the RNAi method leads to suppression of transcriptional activity of AR and remarkable inhibition of cancer cell growth.
As will be described later in Examples, when CTBP1-AS binds to PSF, which is an RNA-binding transcriptional repressor, to form a complex, the resulting complex suppresses transcription of a cell-cycle inhibiting gene (for example, SMAD3 and p53). As a result, cell proliferation is accelerated.
On the other hand, the complex between CTBP1-AS and PSF also suppresses transcription of CTBP1 in cooperation with a histone deacetylation enzyme. Since CTBP1 originally has a function of suppressing transcription of an androgen receptor, suppression of transcription of CTBP1 enhances transcription activation by the androgen receptor. This leads to cell proliferation.
Accordingly, when the expression of CTBP1-AS is suppressed, as will be shown later in Examples, the proliferation of androgen-resistant cancer cells can also be suppressed.
The term “inhibition of cell growth” means that the proliferation of cells can be terminated or retarded.
The cell growth inhibitor according to the present invention is effective for all the cells in which CTBP1-AS has been expressed, but it is particularly useful for suppressing proliferation of cells showing enhanced CTBP1-AS expression. The term “cells showing enhanced CTBP1-AS expression” as used herein means cells having a significantly increased CTBP1-AS expression level compared with normal non-dividing cells. Examples of the cells having enhanced CTBP1-AS expression include, but not limited to, prostate cancer cells and metastatic cancer cells thereof. The term “metastatic cancer cells of prostate cancer” means cancer cells found in metastasis of prostate cancer.
The term “CTBP1-AS expression inhibitor” as used herein means a substance completely inhibiting transcription of CTBP1-AS, that is, transcription for production of CTBP1-AS using the DNA of a CTBP1 gene as a template or a substance significantly reducing such transcription. The term “expression” as used herein embraces transcription for production of a complementary RNA by using a DNA as a template and a transcriptase, synthesis of a protein based on genetic information which a DNA has, and translation for synthesis of a protein based on genetic information which mRNA has.
The term “CTBP1-AS function inhibitor” as used herein means a substance acting on CTBP1-AS itself and completely inhibiting its function or a substance significantly reducing the function. Examples include, but not limited to, substances promoting degradation of CTBP1-AS and substances hybridized with CTBP1-AS. The term “function of CTBP1-AS” as used herein means a function of suppressing expression of CTBP1 and activating AR signals downstream. Inhibition of the function of CTBP1-AS therefore means enhancing CTBP1 expression and suppressing activation of AR signals downstream.
The CTBP1-AS expression inhibitor or function inhibitor to be used in the present invention may be any substance insofar as it has the above-described function. Examples include low molecular compounds, high molecular compounds, proteins including antibodies and enzymes, and nucleic acids including single-stranded DNAs, double-stranded DNAs, single-stranded RNAs, double-stranded RNAs, chimeric nucleic acids containing a single-stranded or double-stranded DNA and a single-stranded or double-stranded RNA, and artificial nucleic acids (peptide nucleic acids (PNA) and locked nucleic acids (LNA)).
As the CTBP1-AS expression inhibitor or function inhibitor, the following ones are preferably used:
(a) antisense nucleic acids against CTBP1-AS or a portion thereof,
(b) nucleic acids having ribozyme activity to specifically cleave CTBP1-AS,
(c) double-stranded nucleic acids having an RNAi effect against CTBP1-AS, and
(d) nucleic acids capable of expressing the nucleic acid described in any one of (a) to (c).
(a) Antisense nucleic acids against CTBP1-AS or a portion thereof.
The antisense nucleic acid method is well known as an expression inhibiting method. It is a method of introducing a single-stranded nucleic acid (antisense nucleic acid) having a base sequence complementary to a target gene (basically, mRNA which is a transcription product), hybridizing it with the target gene, and thereby inhibiting gene expression.
The antisense nucleic acid to be used in the present invention has preferably a sequence complementary to a portion of CTBP1-AS but it is not necessary that the sequence is completely complementary. For example, 90% or more, preferably 95% or more, more preferably 98% or more of the antisense nucleic acid is complementary to a portion of CTBP1-AS. No particular limitation is imposed on the length of the antisense nucleic acid insofar as it can produce an effect of inhibiting the function of CTBP1-AS. It has usually a length of from 10 bases to 100 bases, preferably a length of from 15 to 30 bases.
The antisense nucleic acid can be designed and synthesized as needed based on the base sequence of CTBP1-AS by those skilled in the art by using known software or the like. The antisense nucleic acid may be any of DNA, RNA, and chimeric nucleic acids containing DNA and RNA. They may be modified. Examples of the modified nucleic acid include those obtained by substituting a phosphate group with a thiophosphate group or a methyl phosphate group.
(b) Nucleic acids having ribozyme activity to specifically cleave CTBP1-AS
Examples of the CTBP1-AS function inhibitor also include nucleic acids having ribozyme activity to specifically cleave CTBP1-AS. Ribozyme is a nucleic acid molecule catalytically hydrolyzing a target RNA and is composed of an antisense region having a sequence complementary to a target RNA and a catalytic center region involved in cleavage reaction (for example, Ribozyme: Biochemistry and Biotechnology (Krupp, G. & Gaur, R. K. eds: Eaton Publishing, MA, 2000). Ribozyme which specifically cleaves CTBP1-AS can also be designed as needed in a manner known by those skilled in the art. Ribozyme is usually an RNA molecule but a chimeric DNA-RNA molecule can also be used.
(c) Double-stranded nucleic acids having an RNAi effect against CTBP1-AS
As the CTBP1-AS function inhibitor, double-stranded nucleic acids having an RNAi effect against CTBP1-AS are also preferred. RNAi is a sequence-specific gene expression suppressing mechanism induced by a double-stranded nucleic acid. It has very high target specificity and is highly safe because it utilizes a gene expression suppressing mechanism originally present in the living body.
When it is used for mammal cells, a small double-stranded RNA (small interference RNA; siRNA) usually with from about 19 to 30 bases, preferably from about 21 to 25 bases is used. A longer double-stranded RNA which will be cleaved in the cell by an enzyme (Dicer) and become siRNA may also be used.
Cleavage of a target RNA molecule by RNAi is conducted as follows. First, siRNA is single-stranded by one-side chain cleavage by a protein called Argonaute or by rewinding by RNA helicase and then it binds to Argonaute to form a RISC(RNA induced silencing complex) which is an effector complex. In the RISC, siRNA serves as a guide molecule searching for the target RNA, while Argonaute functions as a ribonuclease (Slicer) cutting the target RNA.
A siRNA has typically a completely complementary double-stranded region of 19 bases and has two protruding bases (overhangs) at the 3′ end of each of the sense strand and antisense strand but it may be a blunt end type having no overhangs. For example, a blunt end RNA with 25 bases is advantageous because it minimizes the activation of an interferon responsive gene, prevents an off-target effect derived from the sense strand, and has considerably high stability in the serum so that it is suited for use also in vivo.
Judging from the sequence of CTBP1-AS, the siRNA to be used in the present invention can be designed, by a known method for obtaining siRNA with high activity, by selecting a target region not having a sequence similar to that of another gene, containing a consensus sequence of siRNA with high activity, having a periodicity of three bases in its sequence, and having a GC content of from about 35 to 45%. For designing of siRNA, a known activity prediction algorithm can also be used. As such an activity prediction algorithm, siExplorer, siDirect, BIOPREDsi, and the like have been made public.
The double-stranded nucleic acid having an RNAi effect may be a double-stranded RNA or a chimeric DNA-RNA double-stranded nucleic acid, or it may contain an artificial nucleic acid. The chimeric type is obtained by substituting a portion of the double-stranded RNA having an RNAi effect with DNA so that it is known to have high stability in the serum and a low immunoresponse induction property.
The above-described double-stranded nucleic acid can have improved resistance to nuclease or be made more stable by modification of the 2′-OH group, phosphorothioate backbone substitution, modification with a boranophosphate group, or introduction of LAN (locked nucleic acid) having ribose bridged between the 2 and 4 positions thereof. Such a modified double-stranded nucleic acid is also embraced in the present invention.
(d) Nucleic acids capable of expressing the nucleic acid described above in any one of (a) to (c)
The nucleic acids described above in (a) to (c) can be administered to the living body after synthesis. It is also possible to introduce a nucleic acid encoding any of these nucleic acids (a) to (c) into the living body and express the nucleic acid in cells.
For example, when a vector containing a DNA capable of expressing an antisense nucleic acid by transcription or a DNA capable of expressing a nucleic acid having ribozyme activity by transcription introduced into cells, RNA expressed in the cells can inhibit the function of CTBP1-AS.
Further, a vector containing DNAs encoding the respective strands of a double-stranded RNA having an RNAi effect against CTBP1-AS may be introduced into cells. These two strands are expressed respectively and hybridized in the cells and as a result, produce an RNAi effect against CTBP1-AS. Alternatively, in order to express a double-stranded RNA having an RNAi effect, a vector containing a DNA encoding a single-stranded RNA obtained by binding respective strands of the double-stranded RNA via a loop can also be used. The single-stranded RNA thus expressed is hybridized in the molecule, constitutes a short hairpin RNA, is cleaved with Dicer at the loop structure portion thereof, and becomes an siRNA.
A construct capable of expressing a desired nucleic acid in cells can be designed as needed by those skilled in the art and introduced into the cells.
Examples of the double-stranded nucleic acids (c) having an RNAi effect against CTBP1-AS include those that target regions, in the base sequence of CTBP1-AS represented by SEQ ID NO:1, from position 1804 to position 1828 (SEQ ID NO:3), from positions 1894 to position 1918 (SEQ ID NO:4), from position 2348 to position 2372 (SEQ ID NO:5), and from position 3063 to position 3087 (SEQ ID NO:6).
Examples of the double-stranded nucleic acids targeting them include those having, as one of two strand, an RNA having a base sequence complementary to these targets and, as the other strand, an RNA having a base sequence complementary to the former one. Such double-stranded nucleic acids are shown in the following table.









TABLE 1





Abbreviation
Target
siRNA







siRNA[1]
1804-1828
UGACCAGUCCGUUUGACACUGAGUG (SEQ ID NO. 7)




CACUCAGUGUCAAACGGACUGGUCA (SEQ ID NO. 8)


 


siRNA[2]
1894-1918
UUGAGAUGCCGGAAACAUUGAUGGG (SEQ ID NO. 8)




CCCAUCAAUGUUUCCGGCAUCUCAA (SEQ ID NO. 10)


 


siRNA[3]
2346-2372
UUAUGUCUCCAGCAAGCUUGGDCUU (SEQ ID NO. 11)




AAGACCAAGCUUGCUGGAGACAUAA (SEQ ID NO. 12)


 


siRNA[4]
3063-3087
GAGACAGGAGGAUGUAGUUUCUAAU (SEQ ID NO. 13)




AUUAGAAACUACAUCCUCCUGUCUC (SEQ ID NO. 14)









The double-stranded nucleic acids of the present invention include the double-stranded nucleic acids having the above-described four regions as a target consisting of a strand having mismatches with the target RNA at from one to three bases and the other strand having a base sequence complementary to it, insofar as they produce an RNAi effect.
As the CTBP1-AS function inhibitor or expression inhibitor, an siRNA (for example, siRNA [3]) targeting a region represented by SEQ ID NO:5 is particularly preferred.
Examples of the double-stranded nucleic acids (c) having an RNAi effect against CTBP1-AS include those that target the sequence of CTBP1-AS represented by SEQ ID NOS:24 to 52. Such double-stranded nucleic acids include double-stranded nucleic acids having an RNA composed of a base sequence represented by SEQ ID NOS:53 to 78 and an RNA complementary thereto. Of these, nucleic acids Nos. 3, 4, 6, 10, 12, 18, and 24 are highly effective for suppressing the expression of CTBP1-AS.
<Pharmaceutical Composition>
The pharmaceutical composition of the present invention contains the above-mentioned cell growth inhibitor according to the present invention. The pharmaceutical composition of the present invention administered to cells in which CTBP1-AS expression has been enhanced therefore produces a proliferation suppressive effect against these cells so that it is particularly useful as a preventive or remedy of diseases in which AR-dependent cell proliferation is involved. Examples of the diseases in which AR-dependent cell proliferation is involved include AR-dependent cancers (typically, prostate cancer) and benign prostatic hyperplasia.
The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier or additive. Examples of such a carrier include, but not limited to, water, saline, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, carboxymethyl cellulose sodium, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, surfactants, excipients, flavoring agents, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity accelerators, taste corrigents, and the like.
The pharmaceutical composition of the present invention can be formulated into various forms, for example, liquids (such as injections), dispersants, suspensions, tablets, pills, powders, and suppositories. A preferred mode of the pharmaceutical composition is an injection and it is preferably administered parenterally (for example, intravenously, transdermally, intraperitoneally, or intramuscularly).
When the pharmaceutical composition of the present invention contains, as a cell proliferation inhibitor, a nucleic acid such as that described above in (a) to (c), preparations can be obtained by enclosing the nucleic acid with a carrier such as liposome, high-molecular micelle, or cationic carrier. A nucleic acid carrier such as protamine may be used. An affected part is preferably targeted by an antibody or the like bound to such a carrier. In addition, the retention in the blood can be improved by binding cholesterol or the like to the nucleic acid.
When a nucleic acid encoding the nucleic acid described above in any of (a) to (c) is contained as the cell growth inhibitor, the nucleic acid inserted in a virus vector such as retrovirus, adenovirus, or Sendai virus or in a non-virus vector such as liposome may be administered to cells.
<Therapeutic Method>
The method of preventing or treating prostate cancer according to the present invention is characterized by that a therapeutically effective amount of the cell growth inhibitor of the present invention is administered.
The term “therapeutically effective amount” as used herein means an amount of an acting substance that ameliorates, to a certain extent, one or a plurality of symptoms to be treated. In prostate cancer, therefore, it means the amount that can achieve at least one of reduction in tumor size, inhibition (retardation or termination) of metastasis of tumor, inhibition (retardation or termination) of tumor growth, and relaxation of one or more symptoms related to cancer. The cell growth inhibitor can be administered as the above-mentioned pharmaceutical composition.
<Screening Method>
In a first mode, a method of screening cell growth inhibitors according to the present invention includes a step of bringing cells expressing CTBP1-AS or a cell extract thereof into contact with test compounds in the presence of androgen, a step of measuring the expression level of CTBP1-AS, and a step of selecting, from the test compounds, test compounds decreasing the expression level of CTBP1-AS compared with an expression level measured in the absence of the test compounds
The cells in which CTBP1-AS has been expressed are not particularly limited insofar as they are cells in which CTBP1-AS has been expressed. For example, a variety of human cancer cells can be used. Of these, prostate cancer cells (LnCap cells and the like) showing high expression of CTBP1-AS and metastatic cancer cells thereof are preferred. Uterine cervix cancer cell strain HeLa cells, cell line 293 cells derived from kidney, mammary tumor cell line MCF7 cells, and the like can also be used. Cells in which CTBP1-AS has not been expressed sufficiently can be used as the cells in which CTBP1-AS has been expressed after introduction of an expression vector containing a DNA encoding CTBP1-AS.
In the screening method of the present invention, not cells themselves but a cell extract can be used. The cell extract can be prepared in a known manner by those skilled in the art.
Although test compounds to be used in the screening method of the present invention are not particularly limited, examples include organic compounds, inorganic compounds, nucleic acids (antisense nucleic acids, ribozymes, siRNAs, RNA adaptors, and the like), peptides, proteins (antibodies), and saccharides.
The test compounds can be brought into contact with cells in which CTBP1-AS has been expressed or a cell extract thereof, for example, by adding the test compounds to a medium of the cells or the cell extract.
When a nucleic acid is used as the test compound, the test compound can be introduced into cells by using cationic liposome method, electroporation method, microinjection method or the 11. When siRNA is used as the test compound, a commercially available transfection kit may be used according to its instruction.
By bringing the test compounds into contact with cells or a cell extract in the presence of androgen, CTBP1-AS expression can be enhanced stably, making it possible to confirm the effect of the test compounds. For example, it is recommended to add androgen to the medium of cells or a cell extract three days after bringing the test compounds into contact with the medium or cell extract.
The step of measuring the expression level of CTBP1-AS can be performed by any method capable of measuring the expression amount of mRNA. For example, RNA collected from cells can be analyzed using Real-time PCR or northern hybridization method. It is to be noted that CTBP1-AS is a non-coding RNA so that the term “expression level” means an expression level of RNA.
The term “measured in the absence of the test compounds” means that the expression level of CTBP1-AS is measured by adding only androgen, without adding the test compounds, to similar cells or cell extract to those or that with which the test compounds are brought into contact. When compared with the value measured in this case, the value is significantly lower when measured by bringing the test compounds into contact with the cell or cell extract, the test compounds are selected as a cell growth inhibitor for cells showing enhanced CTBP1-AS expression.
In a second mode, a method of screening cell growth inhibitors according to the present invention includes a step of bringing cells in which a CTBP1 gene has been expressed into contact with test compounds in the presence of androgen, a step of measuring the expression level of the CTBP1 gene, and a step of selecting, from the test compounds, test compounds increasing the expression level of the CTBP1 gene compared with an expression level measured in the absence of the test compounds.
From the first mode, the second mode is different in using cells in which a CTBP1 gene has been expressed and selecting the test compounds increasing the expression level of the CTBP1 gene.
As the cells in which CTBP1 gene has been expressed, for example, prostate cancer cells (LNCap cells, etc.) or metastatic cancer cells thereof can be used. The cells in which CTBP1 has been expressed can also be prepared y introducing, into cells in which the CTBP1 gene has not been expressed, an expression vector containing a DNA encoding the CTBP1 gene. When the expression vector is introduced, a portion of the CTBP1 gene may be expressed not only by using a DNA encoding the full length of the CTBP1 gene but also by using a DNA encoding a portion of it. The CTBP1 gene encodes a CTBP1 protein so that the term “expression” embraces both transcription and translation.
In the expression level of the CTB1 gene, either the mRNA level expression or the protein level expression can be measured by those skilled in the art in a known manner. The mRNA level expression can be measured using Real-time PCR or northern hybridization similar to the measurement of CTBP1-AS. The protein level expression can also be measured by using electrophoresis such as SDS-PAGE or western blotting using an antibody against CTBP1 protein.
As described above, inhibition of the expression or function of CTBP1-AS leads to an increase in expression of CTBP1 protein, suppression of transcriptional activity of AR, and suppression of proliferation of cells showing enhanced CTBP1-AS expression. It is therefore possible to obtain proliferation inhibitors for cells having enhanced CTBP1-AS expression by selecting test compounds capable of increasing the expression level of the CTBP1 gene compared with the expression level measured in the absence of the test compounds.
The screening methods of the present invention may further include a step of administering the test compounds to cells having enhanced CTBP1-AS expression such as prostate cancer cells and performing cell proliferation assay (MIS assay) or a step of administering the test compounds to cancer-carrying animals to confirm the effect of them against the proliferation of cancer cells. By these steps, the effect of the test compounds can be examined further.
<Test Method>
The test method for evaluating the prognosis of prostate cancer according to the present invention includes a step of measuring the expression level of CTBP1-AS or a CTBP1 gene in cells collected from the prostate gland of a patient and a step of comparing the expression level with the expression level in normal prostate cells.
The present inventors have confirmed that the expression amount of CTBP1-AS in the prostate cancer cells of a patient and metastatic cancer cell thereof is significantly greater than that in normal cells and the expression amount of a CTBP1-gene is significantly smaller than that in normal cells. In addition, they have found that an increase in the expression amount of CTBP1-AS or a decrease in the expression amount of the CTBP1 gene has a correlation with the prognosis of a prostate cancer patient.
Accordingly, when as a result of the test using the test method of the present invention, expression of CTBP1-AS is significantly higher than that in normal cells, there is a high possibility of poor prognosis, while when the expression of CTB1 is significantly smaller than that in normal cells, there is a high possibility of good prognosis.
EXAMPLES
The present invention will hereinafter be described in detail based on Examples, but it should be noted that the invention is not limited by them.
Methods employed in the following Examples will next be described.
[RNA Fish and Fluorescent Immunostaining]
An antisense CTBP1-AS probe for detecting CTBP1-AS was prepared by using an RNA labeling kit (Roche) according to its manual. For colocalization study, LNCaP cells were cultured on a cover glass in a 24-well culture vessel. Twenty four hours after treatment with androgen, the cells were washed with PBS, allowed to stand in 3.6% formaldehyde and 10% acetic acid (in PBS) at room temperature for 20 minutes to fix them, and allowed to stand in 0.5% Triton X-100 at room temperature for 5 minutes for permeabilization. After washing with PBS, the cells were hybridized with the probe (overnight at 42° C. in a moist chamber) and detected using a donkey anti-DIG antibody bound to Alexa555. Endogenous PSF was reacted overnight with a rabbit anti-PSF antibody at 4° C. and detected using an anti-rabbit IgG antibody bound to Alexa488, followed by imaging using a confocal microscope.
[RNA Immunoprecipitation (RIP)]
Confluent LNCaP cells were collected on a 15-cm dish and re-suspended in 1 ml NP40 lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, and 1% NP40). An antibody to Sin3A, PSF, or MONO or IgG (Santa cruz Biotechnology, Sigma, BD, or Sigma, respectively) was added to the supernatant and the resulting mixture was rotated overnight at 4° C. Protein G beads (30 μL) were added and the mixture was incubated at 4° C. for 2 hours while mildly rotating it. The beads were washed three times with a lysis buffer and then re-suspended in 1 ml ISOGEN. The RNA co-precipitated was isolated and the mRNA level of CTBP1-AS was analyzed using qRT-PCR.
[Northern Blotting]
Preparation of an antisense CTBP1-AS probe and subsequent analysis by northern blotting were conducted by using a Northern blot starter kit (Roche) according to its manual. Total RNA (1 μg) was modified and then loaded on a formaldehyde-containing agarose gel (in 1×MOPS buffer). RNA was transferred on a Hybond-XL membrane (Roche) and detected using an RNA probe labeled with DIG and extending across the center portion of CTBP1-AS.
[Microarray]
The total RNA of LNCaP cells was collected using an ISOGEN reagent (Nippon Gene). As an expression analysis microarray, Gene chip human exon 1.0 ST array (Affymetrix) was used according to its manual. Data were analyzed using Affymetrix Microarray Suite software. With regard to a comparison array, data from all probe sets were standardized.
[ChIP and Quantitative Real Time PCR]
ChIP was performed in a conventional manner (Takayama K, et al. Oncogene 26, 4453-63 (2007)). After treatment for 5 minutes with 1% formaldehyde to crosslink protein and DNA, the cells were collected and a protein-DNA extract was prepared. The extract was subjected to sonication. After reaction overnight with a specific antibody and a non-specific IgG, the protein-DNA complex was collected using a protein A/G agarose. The complex was then washed and reacted overnight at 65° C. to de-crosslink it. DNA was collected by ethanol precipitation. Fold enrichment relative to IgG control was quantified by real time PCR using SYBR green PCR master mix (Applied Biosystems) and ABI Prism 7000 system (product of Applied Biosystems) based on SYBR green I fluorescence. PCR products thus obtained respectively were relatively quantified by the Comparative cycle threshold method (Ct method). GAPDH was used as an external standard. The sequences of the primers used are shown in the following table.









TABLE 2





Gene
Primer
SEQ ID NO.







CTBP1
F: GGACGCCTGTATGGAAGCA
105


(intron 1)
R: TCCGCAGACGCCTTTTG
106


 


CTBP1-ARBS
F: GCACTGTGTGGCATAAAAAGAAAA
107



R: TGGAACGTGCCCCAGAA
108









[Real Time RT-PCR]

The total RNA was isolated using an ISOGEN reagent. The first cDNA strand was produced using a Primescript RT reagent kit (Takara) and mRNA was quantified using real time PCR.
The primers used are shown in the following table.









TABLE 3





Gene
Primer
SEQ ID NO.







CTBP1
F: TGGCCACTGTGGCCTTCT
109



R: CGTTCAGGACCTTCTCATGGA
110


 


CTBP1-AS
F: AACCTGGCAGCACGGAAGT
111



R: GAGCACAACCACCACCTCATC
112


 


TMPRSS2
F: TCAACCCCTCTAACTGGTGTGA
113



R: AGGCGAACACACCGATTCTC
114


 


SMAD3
F: CCCCAGAGCAATATTCCAGA
115



R: GGCTCGCAGTAGGTAACTGG
116


 


p53
F: CCCCTCTGAGTCAGGAAACA
117



R: TCATCTGGACCTGGGTCTTC
118









[siRNA]

A Stealth RNAi system targeting CTBP1-AS, CTBP1 (H55102437), NONO (H55143135), PSF (H55109643), and Sin3A (H55177954) and a negative control were purchased from Invitrogen. Transfection of cells was conducted using a Lipofectamine RNAi MAX reagent (Invitrogen) from 48 to 72 hours before the test. The sequence of siRNA against CTBP1-AS is siRNA 5′-UUAUGUCUCCAGCAAGCUUGGUCUU.
[In Vivo Tumor Formation Assay]
LNCaP cells (3×106 cells) or LTAD cells (1×107 cells) were transdermally injected to both sides of 20 male, 5-week-old nude mice. From the mice transplanted with the LTAD cells, the testicle was removed by surgery at the time when the tumor volume reached 100 mm3. Three times a week, 5 μg of siRNA against CTBP1-AS or control siRNA was transfected into the tumor by using Lipofectamine RNAi MAX transfection Reagent. The tumor volume was determined according to the following formula: 0.5×r1×r2×r3 (r1<r2<r3).
[Cell Proliferation Assay]
Cells were cultured on a 96 well plate at 3×103 cells/well. Stable cell lines of pcDNA3 CTBP1-AS were inoculated onto a 1%-FBS-containing PRMI medium. For RNAi test, the cells were transfected with stealth RNA 24 hours after transfer to the plate. MTS assay was performed using a cell titer reagent (Promega) according to its manual. The test was repeated five times for each.
[Cell Culture and Reagent]
LNCaP cells (human prostate cancer cells) were cultured on a RPMI medium containing 10% FBS, 50 units/ml of penicillin, and 50 μg/ml of streptomycin. Prior to treatment with androgen, the cells were cultured for from 48 hours to 72 hours on a phenol red free medium containing 5% dextran charcoal stripped FBS. The antibodies used were Sin3A (AK-11), p53 (Pab-240), p21 (F-5), CyclinD1 (C-20), CyclinA (H-432), CyclinB1 (H-433) (Santa cruz Biotechnology), CTBP1 (#612042), NONO (#611278) (BD bioscience), ACH3K9 (#07-352) (Upstate), PSF (#61045) (Novus Biologicals), and SMAD3 (#04-1035) (Millipore). Antibodies against AR, ACH3, and β-actin are described in the report of Takayama, et al. (Takayama K, et al. Oncogene 26, 4453-63 (2007); Cancer Res. 69(1):137-42 (2009); Oncogene 30(5):619-30 (2011)). Dihydrotestosterone and Bicaltamide were purchased from Wako Pure Chemical Industries.
[Western Blotting, Immunoprecipitation]
Conducted in a conventional manner (Takayama K, et al. Oncogene 26, 4453-63 (2007)). The cells were collected in an NP40 buffer and a protein extract was prepared. AN adequate amount of the extract was boiled for 5 minutes in a Laemmli sample buffer and separated using SDS-PAGE. After transcription to a PVDF membrane, it was blocked with 5% non-fat dry milk and reacted overnight with a primary antibody at 4° C. The reaction product was then reacted for one hour with a secondary antibody against rabbit or mouse IgG, followed by color development and photographing.
[Luciferase Assay]
Prior to transfection, LNCaP cells were incubated for 24 hours on a phenol red free medium containing 5% charcoal stripped FBS. Then, with a FuGENE6 reagent (Roche Diagnostics), the cells were transfected with an ARBS-containing pGL3 vector and tk-PRL. Twenty four hours after transfection, luciferase activity was measured in a conventional manner. In a luciferase assay using RNAi in combination, the transfection of RNA was conducted 72 hours before stimulation with androgen.
[Analysis of Cell Cycle]
After transfection of control siRNA or siRNA against PSF, incubation was conducted for 96 hours and cells were collected. The cells were centrifuged and washed with PBS. While agitating mildly, 3 ml of ice-cooled 70% ethanol was added slowly to fix them. Until use, they were stored at 4° C. On the cell cycle analysis day, the cells were centrifuged, washed with PBS, re-suspended in PBS containing 100 μg/ml RNaseA (Takara) at 106 cells/ml, and incubated at 37° C. for 30 minutes. In order to measure the DNA content, 30,000 cells were analyzed by FACS Calibur flow cytometry using Cell Quest software (BD Biosciences).
[Analysis of Microarray Data]
Microarray analyses of siRNA against CTBP1-AS and siRNA against PSF were conducted, independently. As a result of a test using siRNA against PSF, 700 genes are presumed to be suppressed by PSF. The function of these target genes were identified using DAVID and for pathway analysis, REACTOME was used. In each analysis, 300 genes obtained from the control sample were identified as genes suppressed by androgen.
<Discovery of CTBP1-AS>
Considering that inhibition of signals downstream of AR is effective as a target of prostate cancer treatment, the present inventors have established a method of finding a gene directly targeted by AR by using chromatin immunoprecipitation and tiling array in combination (ChIP-chip method) (Takayama K. et al. Oncogene 26, 2007, 4453-4463, Takayama K. et al. Cancer Res. 69, 2009, 137-142). Moreover, to elucidate the transcription network by androgen, ChIP-chip analysis was conducted on the whole human genome level by using, in addition to AR, an antibody against acetylation at histone H3K9/K14 to identify the AR binding site (ARBS) and the histone H3 acetylation site (ACH3).
In addition, Cap Analysis gene expression (CAGE) was conducted using LNCaP cells. CAGE is a method of sequencing concatemers of DNA tags derived from 20 bases from the 5′ end of mRNA, carrying out genome-wide identification of a starting point of transcription, and profiling the promoter usage and is therefore a high throughput method. A region where CAGE tags were mapped to the human genome and aggregated as a result of CAGE analysis was defined as a tag cluster (TC). Of the TCs, those undergoing a significant change in the distribution of tags were identified.
Based on the recent report (Katayama S. et al. Science 309, 2005, 1564-1566) that an antisense transcription product controls the expression of genes in the vicinity of the product and the finding that transcription control of AR is an important signal of the onset of prostate cancer, the relationship between the transcriptional activity of AR and an antisense transcription product was considered to be an important factor for describing the advance of cancer. As a result of searching for an androgen responsive gene controlled by an antisense, CTBP1 was found.
In addition, in the vicinity of ARES of CTBP1, an antisense TC to be activated by androgen was found. According to GENBANK, it was found to have ex3 of AX747506 as a starting point of transcription. The antisense RNA was therefore named CTBP1-ASd (SEQ ID NO:20).
CTBP1-ASd contains a sequence encoding the sequence of 330 amino acids. It has been found from the analysis by the present inventors by using Pfam that this amino acid sequence does not encode a protein motif. Analysis using Netstart has revealed that the first methionine is unlikely to function as a starting point of protein translation. Furthermore, as a result of a test by the IVT method (in vitro transcription method), protein synthesis from CTBP1-ASd was not recognized (data not shown). These results mean that CTBP1-ASd is a non-coding RNA not encoding a protein motif.
<Preparation of siRNA Having RNAi Effect Against CTBP1-ASd>
As siRNA, stealth RNAi (Invitrogen) designed based on the base sequence of CTBP1-Asd was used. It is a blunt end type double-stranded RNA having a length of 25 bases longer than the typical siRNA and is characterized by that it can minimize the activation of an interferon responsive gene, can prevent an off-target effect derived from the sense strand, and has very high stability in the serum. It also has actual using results in vivo.
The optimum site of the siRNA sequence was determined using the RNAi Designer on invitrogen's website. The sequence of siRNA thus prepared is as described below.









TABLE 4





Abbreviation
Target
siRNA







siRNA[1]
1804-1828
UGACCAGUCCGUUUGACACUGAGUG (SEQ ID NO. 7)




CACUCAGUGUCAAACGGACUGGUCA (SEQ ID NO. 8)


 


siRNA[2]
1894-1918
UUGAGAUGCCGGAAACAUUGAUGGG (SEQ ID NO. 9)




CCCAUCAAUGUUUCCGGCAUCUCAA (SEQ ID NO. 10)


 


siRNA[3]
2348-2372
UUAUSUCUCCAGCAAGCUUGGUCUU (SEQ ID NO. 11)




AAGACCAAGCUUGCUGGAGACAUAA (SEQ ID NO. 12)


 


siRNA[4]
3063-3087
GAGACAGGAGGAUGUAGUUUCUAAU (SEQ ID NO. 13)




AUUAGAAACUACAUCCUCCUGUCUC (SEQ ID NO. 14)









In Examples described below, the above-mentioned siRNA [3] was administered to a siRNA administration group. As a control in the siRNA test (except in vivo test), a double-stranded RNA (StealthTM RNAi Negative Control Medium GC Double-stranded, Invitrogen 12935-300) not showing a RNAi effect against CTBP1-ASd was administered.
<Androgen-Dependent Expression of CTBP1-AS and Effect of SiRNA Administration on Androgen-Dependent Expression Suppression of CTBP1 Gene>
In the test, LNCaP cells of an AR-positive human prostate cell line were used. For cell culture, RPMI1640 (Sigma) containing 10% fatal bovine serum (FBS, Sigma), 100 μg/ml streptomycin, and 100 U/ml penicillin (Invitrogen) was used as a cell broth. The cells were cultured at 37° C. in an incubator containing 5% carbon dioxide gas in the air.
On a 6-well plate, 1×105 LNCaP cells were cultured. On the following day, 20 nM and 50 nM siRNAs were introduced into the cells by using Hiperfect Transfection Reagent (Quiagen) and OPTI-MEM (Invitrogen). Forty eight hours later, the resulting cells were stimulated with 10 nM androgen R1881 or Vehicle. After culturing for further 24 hours, RNA was collected using ISOGEN (Nippon gene).
Expression analysis of mRNA was conducted using Real-time PCR (Prism7000 system; Applied biosystems). From an amplification curve available by the Real-time PCR method, an intracellular expression amount of a transcription product was determined by the ΔΔCt method. The expression amount was corrected based on the expression amount of a GADPH gene.
Measurement results of the CTBP1-AS expression are shown in FIG. 1. In Control, expression of CTBP1-AS was accelerated androgen-dependently, but in the cells of the siRNA administration group, CTBP1-AS expression was suppressed in a siRNA-concentration dependent manner. In the cells administered with 50 nM siRNA, expression induction by androgen was suppressed by from 60 to 80%.
The measurement results of the expression of mRNA of CTBP1 are shown in FIG. 2. In Control, the expression of mRNA of CTBP1 was suppressed by androgen. On the other hand, in the siRNA administration group, the expression suppression effect of CTBP1 by androgen disappeared in a siRNA-concentration dependent manner.
In addition, 24 hours after treatment with 10 nM R1881, a protein was collected and the protein-level expression amount of CTBP1 was measured by western blotting. The results are shown in FIG. 3. The protein-level expression was also suppressed by androgen administration, but due to the administration of siRNA, the expression suppression effect decreased in a siRNA-concentration dependent manner.
<Luciferase Assay>
Luciferase assay was conducted to confirm the influence of CTBP1-AS on transcriptional activity of AR.
A luciferase vector was prepared by inserting, in the promoter region of pGL3-basic (Promega), the promoter and enhancer of PSA (Prostate specific antigen), that is, a typical androgen responsive gene (PSA-LUC). PSA-LUC has an AR binding site and the promoter is activated androgen-responsively.
LNCaP cells were sprayed and cultured on a 24-well plate at 3×104 cells/well. After culturing for 2 days on a phenol red-free medium containing 2.5% charcoal serum, PSA-LUC was introduced. On the following day, the resulting cells were stimulated with 10 nM R1881 or Vehicle. The cells were collected after 24 hours. Luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega).
Two days before introduction of the vector into the cells, siRNA and control double-stranded RNA were administered by the above-described method.
The results are shown in FIG. 4A. It has been demonstrated in Control that luciferase activity showed a marked increase and administration of androgen enhanced AR transcriptional activity, which activated the PSA promoter and enhancer. In the siRNA administration group, on the other hand, luciferase activity was markedly suppressed.
The results of studying the expression of an androgen responsive gene in the same cells are shown in FIGS. 4B and 4C. In Control, the expression amounts of FKBP5 and TMPRSS2, that is, typical androgen responsive genes, increased, while in the siRNA administration group, an increase in the expression amount was suppressed in a concentration dependent manner.
It has been confirmed from the above-mentioned results that suppression of CTBP1-AS leads to suppression of AR transcriptional activity.
<Influence of Function Suppression of CTBP1-AS on Proliferation of LNCaP Cells>
The cell-level influence of CTBP1-AS on LNCaP cell was analyzed using MIS assay.
On a 96-well plate, LNCaP cells were continuously cultured at 3×103 cells/well. Similar to luciferase assay, siRNA was administered on the following day. Forty eight hours, 72 hours, and 96 hours after administration, the resulting cells were reacted for one hour by using Cell titer 96 (Promega).
MTS assay is a method of measuring a viable cell count based on a reduction reaction for converting, via PES (phenazine ethosulfate), a tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MIS) into a formazan product which is a chromogenic substance. The cell proliferation ability was measured at an absorbance of 490 nm by using a microplate reader.
The results are shown in FIG. 5. It has been found that in the siRNA administration group, compared with Control, cell proliferation was significantly suppressed and CTBP1-AS promoted the proliferation of LNCaP cells.
<Influence of Function Suppression of CTBP1-AS on Tumor Proliferation In Vivo>
Influence of CTBP1-AS and function suppression of CTBP1-AS on the tumor proliferation in vivo was analyzed.
To subcutaneously transplant LNCaP cells to mice, they were mixed with PBS and matrigel (BD bioscience). The mixture for 20 mice was prepared at a time so that the cells, PBS, and matrigel per mouse be 1×107 cells/mice, 100 μl, and 100 μl, respectively.
The mixture was injected subcutaneously to 6-week-old male BALB/c nude mice (CLEA Japan) by using a 25 G injection needle.
Injection of siRNA into tumor was conducted using Gene silencer (Genlantis). A mixture obtained by mixing 5 μg of siRNA with 5 μl of Gene silencer in OPTI-MEM was locally injected into the mouse tumor. In control group, a double-stranded RNA having the following base sequence was similarly administered.









(SEQ ID NO: 15)



AAGACGAUCGUUCGGGACAACAUAA



 



(SEQ ID NO: 16)



UUAUGUUGUCCCGAACGAUCGUCUU






The siRNA injection was started two weeks after the cancer cells transplanted into mice seemed to be subcutaneously engrafted and it was conducted twice a week.
The size of the mouse tumor was measured once a week. The major axis (r1) and the minor axes (r2, r3) were measured at two positions and the size of the tumor was determined based on the formula: (r1×r2×r3)/2.
Ten weeks after transplantation, mice were sacrificed and their tumor was excised from under the skin. After the tumor was weighed, a portion of it was provided for RNA extraction using ISOGEN. After cDNA synthesis, RNA level expression amounts of CTBP1-AS and CTBP1 were measured using Real time-PCR. Another portion was dissolved in an SDS lysis buffer (10 mM Tris-HCl, pH7.5, 2% SDS, 10% mercaptoethanol). The resulting solution was provided for protein extraction and a CTBP1 expression amount was measured using western blotting.
The results are shown in from FIG. 6 to FIG. 9.
As shown in FIG. 6, in the siRNA administration group, compared with Control, proliferation of the tumor was significantly suppressed (p<0.01). As shown in FIG. 7, it has been observed that in the siRNA administration group, an increase in the tumor weight was suppressed markedly (p<0.001).
FIG. 8 shows the measurement results of the RNA level expression amount of CTBP1-AS and CTBP1 in the tumor by using Real time-PCR. Compared with Control group, the expression level of CTBP1-AS in the tumor of four mice was reduced by administration of RNAi against CTBP1-AS. On the other hand, the expression amount of CTBP1 was increased by the administration of RNAi against CTBP1-AS compared with Control group. These results have revealed that also in vivo, CTBP1 is under negative control of CTBP1-AS.
FIG. 9 shows the measurement results of the protein-level expression amount of CTBP1 in tumor by western blotting. In the RNAi administration group, compared with Control group, the protein level expression amount of CTBP1 was also increased.
It has been confirmed from the above results that a mechanism of suppressing CTBP1 by CTBP1-AS also functions in in vivo tumor proliferation in the tumor model and suppression of tumor proliferation is promoted by the suppression targeting CTBP1-AS.
<Expression of CTBP1-AS in Actual Tumor>
Expression profile data (GSE3325) of prostate and prostate cancer cases using Human Genome U133 Plus 2.0 array of Affymetrix made public in GEO Datasets on NCBI (http://www.ncbi.nlm.nih.gov) were downloaded.
Signal intensity was extracted while paying attention to the probe (1563571_at) specific to CTBP1-AS (AX747592) of normal, cancer, and metastasis samples. Mann-Whitney U test was conducted and the expression amount was compared among these groups.
The results are shown in FIG. 10. The expression amount of CTBP1-AS in cancer and the cancer tissue of distant metastasis were significantly higher than that of normal prostate (p<0.05, Mann-Whitney U test). It has been presumed from this result that CTBP1-AS is involved in the onset and development of cancer.
<Expression of CTBP1 in Actual Cancer>
Expression of CTBP1 in the actual prostate cancer was assessed by immunostaining. Immunostaining was performed according to the method of Suzuki, et al. (Suzuki T. et al. Clin. Cancer Res. 2005 Sep. 1; 11(17): 6148-54).
Cases were selected from 104 cases with a surgical specimen of prostate cancer excised from 1987 to 2001 in the Urology/the University of Tokyo Hospital. After 99 sections corresponding to the normal site and 101 sections corresponding to the prostate cancer were fixed in formalin, paraffin sections were prepared. As a primary antibody, CTBP1-specific mouse monoclonal antibody (BD Bioscience) was used. With Histofine Kit (Nichirei) for immunohistochemical staining, staining was conducted with a 3,3′-diaminobenzidine solution [1 mmol/L 3,3′-diaminobenzidine, 50 mmol/L Tris-HCl buffer (PH7.6), and 0.006% H2O2]. Images of sections representative of normal prostate and prostate cancer are shown in FIG. 11C.
A labeling index (LI) in the nucleus of CTBP1 in each section was measured. The expression of CTBP1 protein in the normal tissue and cancer tissue was quantified and compared.
The results are show in FIG. 11A. The expression amount of CTBP1 decreased in prostate cancer cells compared with that in normal cells.
With regards to 101 prostate cancer cases, a survival rate was compared between a CTBP1 high expression group and a CTBP1 low expression group based on the data obtained by tracking the progress on an outpatient basis every three months after surgery for five years and the results were analyzed using the Kaplan-Meier method with the Long-rank test. The classification into the high expression group and the low expression group was conducted based on the median of Labeling Indices showing the expression of CTBP1.
The results are shown in FIG. 11B. The survival rate was significantly high in the CTBP1 high expression group compared with that in the low expression group.
It has been confirmed from the above results that a suppressing mechanism of CTBP1 by CTBP1-AS also functions in proliferation of actual tumor.
<Identification of CTBP1-AS Except CTBP1-ASd>
CTBP1-ASs except CTBP1-ASd expressed in prostate cancer cells were searched in the following manner.
Twenty four hours after stimulation of LNCaP cells with 10 nM R1881, RNA was collected. RNA (1 μg) was treated with 1 μl of DNase (Roche, 10 U/μl) and a reverse transcription reaction was then conducted using a RACE Kit (Roche). Next, a PCR reaction was conducted using primers obtained by the CAGE method, one of which was in the vicinity of a starting point of transcription and the other one had an adapter-specific sequence at the terminal. The PCR product thus obtained was inserted into a plasmid by using a TA Cloning Kit (Invitrogen). The plasmid was sequenced and the sequence of the transcription product obtained by the PCR reaction was analyzed. The sequence thus obtained was mapped to a human genome sequence and a range of the transcription product was determined.
The following are the 3′ RACE primer and PCR adaptor primer used.









3′ RACE primer:



(SEQ ID NO: 21)



5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3′



 



PCR adaptor primer:



(SEQ ID NO: 22)



5′-GACCACGCGTATCGATGTCGAC-3′






The results show that there are at least four kinds of CTBP1-AS as shown in FIG. 12. One of them is the above-described CTBP1-ASd. The base sequence of CTBP1-ASa is shown in SEQ ID NO:19, the base sequence of CTBP1-ASb is shown in SEQ ID NO:18, and the base sequence of CTBP1-ASc is shown in SEQ ID NO:17.
These CTBP1-Ass each contain the target sequence of siRNA described in the above table. It is therefore presumed that in the expression suppression test using siRNA in the above Example, expression of these CTBP1-AS was also suppressed.
A probe for detecting, as a sequence which CTBP1-ASa to CTBP1-ASd have in common, a region (SEQ ID NO:23) from position 1286 to position 3515 of the RNA sequence described in SEQ ID NO:1 was prepared and northern blotting was conducted. The probe was prepared following the protocol by using DIG Northern Starter Kit (Roche).
LNCaP cells were stimulated with 10 nM R1881 and 0, 6, 12, 18, 24, and 48 hours later, RNAs were collected. RNAs (each, 1 μg) were electrophoresed on an agarose gel containing formaldehyde and transferred to a membrane. The membrane was hybridized with the above probe (100 ng/ml) and a DIG-specific antibody was used for detection.
In order to measure the amount of RNA used in the test, a probe (Roche) against β actin was purchased at the same time and the same membrane was used for detection.
The results are shown in FIG. 13. A transcription product having a length of about 5 kb was detected, suggesting that among the above-mentioned four kinds of CTBP1-ASs, CTBP1-ASb was expressed mainly.
<Localization of CTBP1-AS>
LNCaP cells were treated for 24 hours with 10 nM R1881 or Vehicle. From the cells collected, intranuclear RNA and cytoplasmic RNA were extracted using a PARIS kit (Ambion). From respective samples of them, 1 μg of RNA was electrophoresed and an expression amount was analyzed using northern blotting. As Loading Control, a probe against β-actin was hybridized and an expression amount was measured. The expression amount of each of the genes was corrected by GAPDH.
The results are shown in FIG. 14. Intranuclear localization of CTBP1-AS was confirmed.
<Involvement of CTBP1-AS in Androgen-Depletion Resistant Cancer Cell Proliferation-In Vitro>
Lysates of LTAD cells and LNCaP cells (2.5% charcoal serum, cultured for 3 days, stimulated with R1381, R1881+Bicaltamide added) and western blotting was conducted using a CTBP1 antibody. A probe against β-actin was used as a Roding Control.
In order to compare the expression amount of CTBP1-AS between LTAD cells and LNCaP cells, Total RNA of LTAD cells and LNCaP cells (2.5% charcoal serum, cultured for 3 days, stimulated with R1881, R1881+Bicaltamide added) were collected. The RNA (1 μg) was electrophoresed, transferred, and subjected to northern blotting with a probe against CTBP1-AS.
The results are shown in FIGS. 15a and 15b. The LTAD cells were cultured under an androgen-depleted state to be a hormone therapy resistant model. When the LNCaP cells were stimulated with androgen, expression of CTBP1 was decreased to a similar level to that of the LTAD cells. In the LTAD cells, expression of CTBP1-AS was increased remarkably.
Next, an influence of CTBP1-AS on hormone-dependent or hormone-depleted proliferation of prostate cancer cells was analyzed.
LNCaP cells were sprayed on a phenol red-free medium containing 2.5% charcoal serum at 3×103 cells/well. On the following day, RNAi against CTBP1-AS was transfected. Two days later, the cells were stimulated with Et or 81881 and on Day 1, 3, and 5, MTS assay was performed to evaluate proliferation ability.
Separately, the LTAD cells were sprayed on a phenol red-free medium containing 2.5% charcoal serum at 3×103 cells/well. On the following day, RNAi against CTBP1-AS was transfected. Two days later, the cells were stimulated with Et or R1881 and on Day 1, 3, and 5, MTS assay was performed to evaluate proliferation ability.
The results are shown in FIG. 15c. Cancer cells proliferate even without stimulating the androgen depletion resistant LTAD cells with androgen. Either androgen-dependent proliferation or androgen-independent proliferation was suppressed by knockdown of CTBP1-AS.
<Involvement of CTBP1-AS in Androgen-Depletion Resistant Cancer Cell Proliferation-In Vitro>
The role of CTBP1-AS in proliferation of hormone therapy resistant cells was analyzed in vivo.
LTAD cells were subcutaneously transplanted to nude mice at 1×107 cells/mouse. The testicle was removed by surgery at the time when the tumor volume reached 100 mm3. Control siRNA or siRNA against CTBP1-AS was locally injected (5 μg/mouse, twice/week).
The results are shown in FIG. 16. Even in vivo, by suppressing expression of CTBP1-AS, an increase in the volume of the androgen-depletion resistant tumor was suppressed (FIGS. 16a and 16b). As a result of measurement of the expression amount of CTBP1-AS in tumor by qRT-PCR, decrease in the mRNA level expression has been confirmed (FIG. 16c). On the other hand, CTBP1 in tumor was measured by western blotting. Due to knock down of CTBP1-AS, the expression amount of CTBP1 increased (FIG. 16d).
<Involvement of RNA-Binding Protein PSF in Expression Suppression of CTBP1>
Expression of three genes, that is, PSF, NONO, and Sin3A was suppressed by RNAi. LNCaP cells were transfected with siRNAs against PSF, NONO, and Sin3A. Two days later, lysates were collected and the expression amount was examined by western blotting. It has been confirmed that any of the siRNAs specifically suppressed the target expression.
LNCaP cells were treated with ethanol or R1881 for 24 hours and two days before stimulation, the resulting cells were transfected with control siRNA or siRNA against PSF. The RNA was collected, cDNA was synthesized, and an expression amount of CTBP1 was analyzed by RT-PCR.
The results are shown in FIG. 17b. When the expression of PSF was suppressed, the suppression degree of CTBP1 expression by stimulation with androgen decreased.
In addition, ChIP was conducted using an ACH3 antibody. A histone acetylation level of a promoter region of CTBP1 and CTBP1-AS was measured using the real time PCR, followed by correction with GAPDH. The myoglobin exon region was used as a negative control.
The results are shown in FIG. 17c. When the expression of PSF was suppressed, the suppression degree of CTBP1 expression by stimulation with androgen decreased. This has suggested a promoter-level involvement of PSF in CTBP1 expression.
<Androgen-Dependent Binding of PSF to CTBP1-AS>
PSF is a RNA-binding transcriptional repressor. The presence or absence of binding between PSF and CTBP1-AS was analyzed.
LNCaP cells were treated with ethanol or R1881 for 24 24 hours and two days before stimulation, the resulting cells were transfected with control siRNA or siRNA against PSF. After a nuclear compartment was extracted and a lysate was collected, immunoprecipitation was conducted using an antibody specific to Norman IgG, PSF and NONO. Immunoprecipitation was conducted using Protein G and after washing, RNA was extracted using Isogen. A reverse transcription reaction was performed using total RNA and expression amounts of GAPDH, Myoglobin (MG), and CTBP1-AS were measured using the real time RT-PCR.
The results are shown in FIG. 18. It has been confirmed that CTBP1-AS binds to PSF in an androgen-dependent manner.
<Analysis of CTBP1-AS Localization by RNA-FISH>
Intranuclear localization of PSF and CTBP1-AS was analyzed. Ethanol or R1881 was added to LNCaP cells, followed by reaction for 24 hours. Then, RNA FISH of CTBP1-AS was performed. After fixed with formalin and methanol, the cells were reacted overnight at 42° C. for binding by using a probe against DIG-labeled CTBP1-AS. On the following day, a fluorescence-labeled antibody against DIG was bound and after washing, immunostaining with an anti-PSF antibody was performed. A second antibody against a mouse antibody was reacted for detection. The nucleus was stained with DAPI.
The results are shown in FIG. 19. PSF and CTBP1-AS showed almost the same localization, suggesting that they have been bound to each other.
<Control of Cell Cycle by PSF>
Next, another gene whose transcription was suppressed by PSF was searched.
Cells were transfected with control siRNA or siRNA against PSF. Twenty four hours after stimulation with ethanol or R1881, RNA was collected. The RNA was amplified and gene expression was analyzed using a microarray. From an expression-suppressed gene group and an expression-enhanced gene group, a gene whose change was suppressed by androgen was extracted. A change rate of gene expression caused by androgen was Log 2 transformed and a Heat map was created using Cluster and Tree view (FIG. 20a). Suppressed transcription is shown with a lighter color.
70% of the gene suppressed by androgen was released from the suppression by siPSF. Moreover, induction of most of the androgen-mediated genes was suppressed by siPSF. This suggests that PSF widely mediates the expression suppression by androgen.
The gene group in which PSF was engaged in androgen action was subjected to pathway analysis. It has been found as a result of analysis of the gene group whose change was suppressed by PSF suppression, among the gene groups suppressed by androgen, that genes involved in cell cycle such as p53 and SMAD3 were concentrated (in the drawing, UGT2B15 is a negative control whose expression is suppressed by androgen but is not influenced by PSF).
Next, an influence of suppression by PSF on the cell cycle was analyzed using FACS. LNCaP cells, LTAD cells, and BicR cells were transfected with control siRNA or siRNA against PSF and 96 hours later, the cells were collected. The cell cycle was analyzed using FACS. A change in the percentage of S-phase cell fraction is shown in FIG. 20b. When PSF was knocked down, the tendency to decrease in the percentage of the DNA synthesis S-phase and retardation of the cell cycle were found.
A similar test to that in FIG. 20a was conducted except that CTBP1-AS, instead of PSF, was knocked down and kinds of genes to be released from the androgen-mediated suppression were compared. The results are shown in FIG. 20c. About 50% of the cells to be released from the androgen-mediated suppression by knockdown of PSF showed a tendency of being released from the androgen-mediated suppression even by knockdown of CTBP1-AS. This suggests that CTBP1-AS and PSF are cooperatively involved in androgen-mediated suppression of gene expression.
<Analysis of p53 and SMAD3, Target Genes of PSF>
Influence of PSF on the expression of p53 and SMAD3 was analyzed.
LNCaP cells were transfected with control siRNA or siRNA against PSF and 48 hours after the transfection, they were stimulated with ethanol or R1881. Twenty four hours later, RNA and protein were collected. Measurement results of the expression amount of p53 and SMAD3 by qRT-PCR are shown in FIG. 21a (corrected by GAPDH). The results of western blotting are shown in FIG. 21b actin was used as Loading control).
Stimulation with androgen decreased both the mRNA level expression and protein level expression of SMAD3 and p53. When expression of PSF was suppressed, the androgen-mediated suppression of expression tended to be released. This suggests that SMAD3 or p53 is hardly influenced by androgen without PSF, in other words, suppression of SMAD3 or p53 expression by androgen is mediated by PSF.
<Androgen-Dependent Expression Suppression of Cell-Cycle-Related Factors by CTBP1-AS>
The influence of CTBP1-AS on the expression of p53 and SMAD3 was analyzed.
LNCaP cells were transfected with control siRNA or siRNA against CTBP1-AS and 48 hours later, they were stimulated with ethanol or androgen. Forty eight hours later, RNA and protein were collected and the expression amounts of p53, p21, and SMAD3 were analyzed by western blotting. The results are shown in FIG. 22a. 
Expression of each of p53, p21, and SMAD3 was decreased by stimulation with androgen, but a decreasing degree was reduced by suppressing the expression of CTBP1-AS.
LTAD cells, that is, hormone-depletion resistant prostate cancer cells were transplanted to nude mice. After formation of a tumor, their testis was excised and locally injected with control siRNA or siRNA against CTBP1-AS. Three weeks later, the tumor was excised, protein was collected, and the expression amount of a cell cycle-related gene product was analyzed by western blotting. The results are shown in FIG. 22b (each, n=2). Suppression of the expression of CTBP1-AS led to release of androgen-dependent expression suppression of the cell cycle-related gene product.
FIG. 23 is a schematic view showing a gene expression control mechanism by CTBP1-AS and PSF which is suggested by the above results.
First, when androgen binds to an androgen receptor AR, transcription of CTBP1-AS increases.
As a result, in a global genome region (within the left framework in the drawing), CTBP1-AS binds to PSF to form a complex and this complex suppresses expression of SMAD3 or p53. SMAD3 or p53 is a cell cycle inhibiting gene so that when its expression is suppressed, cell proliferation is promoted.
On the other hand, locally (within the right framework in the drawing), when transcription of CTBP1-AS increases, a complex of CTBP1-AS and PSF binds to a histone deacetylation enzyme (HDAC) and suppresses transcription of CTBP1. Since CTBP1 is a transcription suppressor of an androgen receptor, suppression of the transcription of CTBP1 increases the transcription of the androgen receptor and activates the receptor.
Thus, either globally or locally, an increase in the transcription of CTBP1-AS serves to cause proliferation of cancer cells. This mechanism has proved that suppression of CTBP1-AS or PSF leads to suppression of the proliferation of cancer cells.
<Expression Suppression of CTBP1-AS by siRNA>
After designing and synthesizing siRNAs against CTBP1-AS shown in the following table, their effect on the expression suppression of CTBP1-AS was measured.













TABLE 5






Target sequence







Oligo
(target sequence for
SEQ ID

SEQ ID

SEQ ID


name
designing siRNA)
NO.
>sense_strand
NO.
>anitsense_strand
NO.





















No. 1
accaacaggaaacgtcctactta
24
CAACAGGAAACGUCCUACUUA
53
AGUAGGACGUUUCCUGUUGGU
79


 


No. 2
ctcaacatcagaccaattaatta
25
CAACAUCAGACCAAUUAAUUA
54
AUUAAUUGGUCUGAUGUUGAG
80


 


No. 3
accaactgtcaagaaacaattag
26
CAACUGUCAAGAAACAAUUAG
55
AAUUGUUUCUUGACAGUUGGU
81


 


No. 4
ctcactgctgctgatgattgtag
27
CACUGCUGCUGAUGAUUGUAG
56
ACAAUCAUCAGCAGCAGUGAG
82


 


No. 5
gacataccacacaaacactgatc
28
CAUACCACACAAACACUGAUC
57
UCAGUGUUUGUGUGGUAUGUC
83


 


No. 6
gaccaattaattagaccacaaaa
29
CCAAUUAAUUAGACCACAAAA
58
UUGUGGUCUAAUUAAUUGGUC
84


 


No. 7
tcccacatcaacacggtaacacc
30
CCACAUCAACACGGUAACACC
59
UGUUACCGUGUUGAUGUGGGA
85


 


No. 8
cacccggcctaacatggagaatt
31
CCCGGCCUAACAUGGAGAAUU
60
UUCUCCAUGUUAGGCCGGGUG
86


 


No. 9
acccggcctaacatggagaattt
32
CCGGCCUAACAUGGAGAAUUU
61
AUUCUCCAUGUUAGGCCGGGU
87


 


No. 10
ggcctcataacgcactaggataa
33
CCUCAUAACGCACUAGGAUAA
62
AUCCUAGUGCGUUAUGAGGCC
88


 


No. 11
aacgctgtgggatatgaggttgg
34
CGCUGUGGGAUAUGAGGUUGG
63
AACCUCAUAUCCCACAGCGUU
89


 


No. 12
cccggcctaacatggagaattta
35
CGGCCUAACAUGGAGAAUUUA
64
AAUUCUCCAUGUUAGGCCGGG
90


 


No. 13
gcctaacatggagaatttagaac
36
CUAACAUGGAGAAUUUAGAAC
65
UCUAAAUUCUCCAUGUUAGGC
91


 


No. 14
tcctacttaatggtgaaatgtca
37
CUACUUAAUGGUGAAAUGUCA
66
ACAUUUCACCAUUAAGUAGGA
92


 


No. 15
ctctatactattaataacagaca
38
CUAUACUAUUAAUAACAGACA
67
UCUGUUAUUAAUAGUAUAGAG
93


 


No. 16
gcctcataacgcactaggataac
39
CUCAUAACGCACUAGGAUAAC
68
UAUCCUAGUGCGUUAUGAGGC
94


 


No. 17
ttctcccacatcaacacggtaac
40
CUCCCACAUCAACACGGUAAC
69
UACCGUGUUGAUGUGGGAGAA
95


 


No. 18
gcctcggcctcataacgcactag
41
CUCGGCCUCAUAACGCACUAG
70
AGUGCGUUAUGAGGCCGAGGC
96


 


No. 19
tgctgctgatgattgtagctaat
42
CUGCUGAUGAUUGUAGCUAAU
71
UAGCUACAAUCAUCAGCAGCA
97


 


No. 20
aggaactaactctatactattaa
43
GAACUAACUCUAUACUAUUAA
72
AAUAGUAUAGAGUUAGUUCCU
98


 


No. 21
cactaagcagagtataggttaaa
44
CUAAGCAGAGUAUAGGUUAAA
73
UAACCUAUACUCUGCUUAGUG
99


 


No. 22
ttaacacctgattaatataaaag
45
GACACCUGAUUAAUAUAAAAG
74
UUUAUAUUAAUCAGGUGUCAA
100



(ttgacacctgattaatataaaag)
(46)






 


No. 23
tgaacactcaagaggcaaagact
47
GACACUCAAGAGGCAAAGACU
75
UCUUUGCCUCUUGAGUGUCCA
101



(tggacactcaagaggcaaagact)
(48)






 


No. 24
gaccgtataaacgcactacatcc
49
CCGUCUAAACGCACUACAUCC
76
AUGUAGUGCGUUUAGACGGUC
102


 


No. 25
cccgcgaacgtggtgtctcctgc
50
CGCGAACGUGGUGUCUCCUGC
77
AGGAGACACCACGUUCGCGGG
103


 


No. 26
tgacaacaagtgtggatgtaaac
51
GCAACAAGUGUGGAUGUAAAC
78
UUACAUCCACACUUGUUGCCA
104



(tggcaacaagtgtggatgtaaac)
(52)









The results are shown in FIG. 24. In this drawing, NC and NC2 are negative controls. It has been confirmed that compared with the negative controls, siRNAs of Nos. 3, 4, 6, 10, 12, 18, and 24 suppress the transcription of CTBP1-AS.
[Sequence Listing Free Text]
SEQ ID NO:1 is a base sequence in one mode of CTBP1-AS.
SEQ ID NO:2 is a base sequence of AX747592.
SEQ ID NO:3 is a base sequence of a target region of siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:4 is a base sequence of a target region of siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:5 is a base sequence of a target region of siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:6 is a base sequence of a target region of siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:7 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:8 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:9 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:10 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:11 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:12 is a base sequence of TNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:13 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:14 is a base sequence of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NO:15 is a base sequence of RNA constituting siRNA used as a control in siRNA in Examples.
SEQ ID NO:16 is a base sequence of RNA constituting siRNA used as a control in siRNA in Examples.
SEQ ID NO:17 is a base sequence of CTBP1-ASc.
SEQ ID NO:18 is a base sequence of CTBP1-ASb.
SEQ ID NO:19 is a base sequence of CTBP1-ASa.
SEQ ID NO:20 is a base sequence of CTBP1-ASd.
SEQ ID NO:21 is a base sequence of 3′ Race primer used for 3′ RACE PCR.
SEQ ID NO:22 is a base sequence of a PCR adaptor primer used for 3′ RACE PCR.
SEQ ID NO:23 is a base sequence of a probe used in northern blotting of RNA derived from LNCaP cells.
SEQ ID NOS:24 to 52 are base sequences of a target region of siRNA inhibiting the function of CTBP1-AS.
SEQ ID NOS:53 to 104 are base sequences of RNA constituting siRNA inhibiting the function of CTBP1-AS.
SEQ ID NOS:105 to 108 are base sequences of a primer used in real time PCR.
SEQ ID NO:109 to 118 are base sequences of a primer used in real time RT-PCR.

## Claims

1. A cell growth inhibitor
comprising a CTBP1-AS (C-terminal binding protein 1-antisense) expression inhibitor or function inhibitor as an active ingredient, wherein
the CTBP1-AS expression inhibitor or function inhibitor is a double-stranded RNA having an RNAi effect against CTBP1-AS and
one strand of the double-stranded RNA comprises a base sequence complementary to the base sequence of any of SEQ ID NO:3 to 6 and 24 to 52.

2. A method of treating prostate cancer, comprising administering a therapeutically effective amount of the cell growth inhibitor of claim 1.

3. A method of screening growth inhibitors of cells in which CTBP1-AS has been expressed, comprising:
bringing cells in which CTBP1-AS has been expressed or a cell extract thereof into contact with test compounds in the presence of androgen;
measuring an expression level of CTBP1-AS; and
selecting, from the test compounds, test compounds decreasing the expression level of CTBP1-AS compared with an expression level measured in the absence of the test compounds.

4. A method of screening proliferation inhibitors of cells in which CTBP1-AS has been expressed, comprising:
bringing cells in which a CTBP1 gene has been expressed or a cell extract thereof into contact with test compounds in the presence of androgen;
measuring an expression level of the CTBP1 gene; and
selecting, from the test compounds, test compounds increasing the expression level of the CTBP1 gene compared with an expression level measured in the absence of the test compounds.

5. The method according to claim 3 or 4, wherein the cells in which CTBP1-AS has been expressed are prostate cancer cells and/or metastatic cancer cells thereof.

6. A method of treating prostate cancer, comprising: measuring the expression level of CTBP1-AS in cells sampled from the prostate of a patient; comparing the expression level with an expression level in normal prostate cells; determining the prognosis of prostate cancer for the patient, wherein an increased CTBP1-AS expression level in the patient indicates a poor prognosis; treating the patient with the poor prognosis by administering a therapeutically effective amount of the cell growth inhibitor of claim 1 to the subject.

7. The cell growth inhibitor according to claim 1, wherein one strand of the double-stranded RNA consists of a base sequence complementary to the base sequence of any of SEQ ID NO:3 to 6 and 24 to 52.

8. A method of treating prostate cancer, comprising administering a therapeutically effective amount of the cell growth inhibitor of claim 7.

9. The method according to claim 2, wherein the administering comprises contacting the cell growth inhibitor to a cell expressing:
(i) an antisense RNA to a CTBP1 gene comprising from position 2348 to position 2372 of the base sequence represented by SEQ ID NO:1; or
(ii) a mutant or variant of the antisense RNA to a CTBP1 gene comprising a sequence that is 90% identical to from position 2348 to position 2372 of the base sequence represented by SEQ ID NO:1.

10. The method according to claim 2, wherein the administering comprises contacting the cell growth inhibitor to a prostate cancer cell and/or a metastatic cancer cell thereof.