WO2020067499A1 - Method for screening or evaluating therapeutic drug for prostate cancer - Google Patents

Abstract

The present invention addresses the problem of providing a method for screening and evaluating therapeutic drugs for prostate cancer. In the present invention, a candidate compound is added to cells expressing the SF3B2 gene, and the level of expression of the SF3B2 gene in the cells to which the candidate compound has been added is measured. On the basis of that expression level, screening or evaluation of a therapeutic drug for prostate cancer is conducted.

Description

Method for screening or evaluating therapeutic agent for prostate cancer

The present invention relates to a method for screening or evaluating a therapeutic agent for prostate cancer.

The prostate is divided into an external gland (marginal region) and an internal gland (center region and transition region), and prostate cancer is a cancer mainly occurring in the external gland (marginal region). Prostate cancer is a cancer that accounts for the highest number of affected males in Japan and the United States by site, and there are still many patients. In Japan, the number of people with prostate cancer is estimated to exceed 80,000, and about 12,000 of them are expected to die (National Cancer Research Center Cancer Information Service "Cancer Registry and Statistics", 2017 cancer statistics prediction).

Treatment of prostate cancer includes surgical treatment, radiation treatment, chemotherapy, endocrine therapy (hormone therapy) and the like. Among these, endocrine therapy is a treatment for suppressing the progression of prostate cancer by suppressing the secretion of androgen, which plays a role in promoting the progression of prostate cancer, or by inhibiting the function of androgen.
However, as a problem of endocrine therapy, long-term treatment may result in resistance to drugs used in endocrine therapy, and the therapeutic effect of endocrine therapy may be reduced. Prostate cancer whose prostate cancer symptoms have worsened as a result of becoming resistant to endocrine therapy in this way is called castration-resistant prostate cancer.

ア ン ド It is known that the mechanism of castration-resistant prostate cancer involves the androgen receptor (AR) (Non-Patent Document 1). Furthermore, it is known that AR-V7, one of the AR variants, can promote the growth of prostate cancer even in a castration state by being activated as a transcription factor in a ligand-independent manner (non- Patent Documents 2 and 3).

Here, the full-length AR gene (AR-FL) is a structure having eight exons. On the other hand, its variant AR-V7 has only exons 1-3 and cryptic exon CE3, and has no ligand binding site. Therefore, its activity as a transcription factor is independent of ligand. It can have. That is, it is thought that reducing the expression level of AR-V7 leads to treatment of castration-resistant prostate cancer.

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In view of the above problems, an object of the present invention is to provide a method for screening and evaluating a therapeutic agent for prostate cancer.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, completed an invention relating to a method for screening and evaluating a therapeutic agent for prostate cancer using the expression of SF3B2 (splicing factor 3B subunit 2) as an index. .

Specifically, the AR-V7 splicing inhibitory effect of the administered compound on castration-resistant prostate cancer cells could be measured by using the expression of AR-V7 as an index. This drug having a high AR-V7 splicing inhibitory effect was remarkably effective in a mouse model of castration-resistant prostate cancer. Therefore, a cell capable of monitoring AR-V7 with a fluorescent protein was developed.
GFP was added immediately before the stop codon of AR-V7 in the cryptic exon (CE) 3 region, which encodes V7, one of the androgen receptor (AR) variants of castration-resistant prostate cancer cells. By the introduction, cells capable of monitoring the expression of AR-V7 by flow cytometry were developed.
Addition of Pladienolide B or Spliceostatin A to these cells suppressed the splicing of AR-V7 and reduced the expression of GFP. By using these cells, splicing of castration-resistant prostate cancer cells can be suppressed and cytotoxicity can be simultaneously measured.
Further, since AR-V7 splicing can be suppressed by suppressing the expression of SF3B2, cells capable of indirectly monitoring the expression of SF3B2 with a fluorescent protein have been developed.
Furthermore, it was found that SF3b protein complex formation was inhibited by administering Pradienolide B to an assay system containing SF3B2 protein and an SF3b protein complex-forming molecule other than SF3B2. By utilizing this system, it is possible to screen candidate compounds for a therapeutic agent for prostate cancer using inhibition of SF3b protein complex formation as an index.

Increasing SF3B2 expression increases AR-V7 splicing and exacerbates tumors. Conversely, decreasing SF3B2 expression attenuates AR-V7 splicing. From this, using these cells, a compound that regulates the expression of SF3B2 can be screened and cytotoxicity can be simultaneously measured.
Further, by introducing a luciferase gene instead of GFP, it is possible to construct a higher-throughput screening system by luminescence.
Based on the above findings, the present invention has been completed.

That is, the present invention is as follows.
[1] A method for screening or evaluating a therapeutic agent for prostate cancer, comprising a step of adding a candidate compound to cells expressing the SF3B2 gene and a step of measuring the expression level of the SF3B2 gene in the cell to which the candidate compound has been added.
[2] The method according to [1], wherein a compound that reduces the expression level of the SF3B2 gene as compared with a control is selected or evaluated as a candidate for a therapeutic agent for prostate cancer.
[3] The SF3B2 gene is represented by a DNA having a base sequence of SEQ ID NO: 1 or a DNA hybridizing under stringent conditions with a DNA having a complementary sequence to the base sequence of SEQ ID NO: 1, [1] Or the method according to [2].
[4] The method according to any one of [1] to [3], wherein the expression level of the SF3B2 gene is measured by measuring the expression level of a reporter gene linked to a promoter of the SF3B2 gene. .
[5] A method for screening or evaluating a therapeutic agent for prostate cancer, comprising a step of adding a candidate compound to cells expressing the SF3B2 gene and a step of measuring the expression level of the AR-V7 gene in the cell to which the candidate compound has been added.
[6] The method according to [5], wherein a compound that reduces the expression level of the AR-V7 gene as compared to a control is selected or evaluated as a candidate for a therapeutic agent for prostate cancer.
[7] The SF3B2 gene is represented by a DNA having a base sequence of SEQ ID NO: 1 or a DNA having a sequence complementary to the base sequence of SEQ ID NO: 1 and a DNA that hybridizes under stringent conditions, [5] Or the method according to [6].
[8] The expression according to any one of [5] to [7], wherein the expression level of the AR-V7 gene is measured by measuring the expression level of a reporter gene linked to the promoter of the AR-V7 gene. The method described in.
[9] a step of adding a candidate compound to an assay system containing an SF3B2 protein and an SF3b protein complex-forming molecule other than SF3B2, and a step of selecting a compound that inhibits SF3b protein complex formation as a candidate for a therapeutic agent for prostate cancer , A method for screening or evaluating a therapeutic agent for prostate cancer.
[10] A kit for screening or evaluating a therapeutic agent for prostate cancer, comprising a reagent capable of measuring the expression level of the SF3B2 gene.

According to the present invention, it becomes possible to screen and evaluate therapeutic agents for prostate cancer using the SF3B2 gene.
Compounds that intervene in abnormal splicing control of cancer and have low cytotoxicity can be screened. Compounds targeting splicing control are highly likely to be developed as anticancer agents, but few systems can screen for such compounds. The present invention allows one to screen for compounds that intervene in splicing control of cancer. Since the inventors have found that cancer is exacerbated by splicing control, a compound that suppresses splicing may have a significant effect on cancers that are resistant to existing therapies.

FIG. 2 is a schematic diagram of gene editing for producing an AR-V7-GFP gene. FIG. 2 is a Western blot diagram confirming the expression of a reporter protein (GFP) in CWR22Rv1 cells into which an AR-V7-GFP gene has been introduced (a photograph as a substitute for a drawing). FIG. 4 is a flow cytometry diagram in which a change in the expression level of AR-V7 protein was measured by suppressing the function of the SF3B2 gene by CRISPR / Cas9. In vivo, mice were inoculated subcutaneously with prostate cancer cells in which the SF3B2 gene was forcibly expressed, or prostate cancer cells in which the SF3B2 gene was forcibly expressed and the AR-V7 gene was knocked out, and changes in tumor growth were confirmed. is there. In (A), prostate cancer cell CWR22Rv1 (22Rv1) was used, and in (B), prostate cancer cell LNCaP95 was used. (A) Schematic representation of purification of SF3B2 complex and treatment with Pradienolide B (PLA-B). (B) is a western blot diagram confirming the expression of SF3B2 protein in a nuclear extract or cytoplasmic extract of 22Rv1 cells stably expressing SF3B2-TAP (a photograph as a substitute for a drawing). (C) shows the result of silver staining of the purified SF3B2 complex based on the presence or absence of the treatment with 20 mM ΔPLA-B (a photograph substituted for a drawing). The proteins identified by mass spectrometry are described on the left side of the figure. The lower figure is a Western blot of SF3B2 as an internal standard (a photograph substituted for a drawing). (A) shows the results of flow cytometry analysis of AR-V7-GFP negative group in AR-V7-GFP cells treated with Pradienolide B. (B) is a diagram showing a flow cytometry analysis of AR-V7-GFP in AR-V7-GFP cells treated with Pradienolide B. (C) is a diagram confirming the expression level of AR-V7 in the case of using Pradienolide B (PLA-B) in vivo. FIG. 4 shows the therapeutic effect of Pradienolide B on prostate cancer. (A) shows the change in tumor growth in castrated mice having prostate cancer cells (LNCaP95) stably overexpressing SF3B2 by administering PLA-B or vehicle (DMSO) in vivo. It is. (B) is a figure confirming the change in tumor growth of castrated mice having prostate cancer cells (LNCaP95) stably overexpressing SF3B2 or GFP by administration of PLA-B in vivo. .

The expression level of the SF3B2 gene is positively correlated with the expression level of AR-V7, and it is known that the expression level of AR-V7 is involved in the development of prostate cancer. By screening or evaluating a drug using the activity as an index, a substance that can be a therapeutic agent for prostate cancer can be obtained.

Prostate cancer is classified into stages by, for example, judging the degree of malignancy of the cancer by Gleason score or judging the degree of progression of the cancer by TNM classification. The Gleason score can be classified by observing the tissue structure of a cancer cell under a microscope using a pathological image to determine the degree of malignancy of the cancer. In the case of TNM classification, the disease is classified according to the presence and degree of metastasis (lymph node metastasis, distant metastasis). The prostate cancer in the present invention includes any staging determined based on these indices.
Here, the TNM classification is one of the indices used for staging malignant tumors, and includes T factor (the size and degree of invasion of the primary tumor), N factor (the presence and degree of lymph node metastasis), And M factors (presence or absence and extent of distant metastasis).
In the present invention, the prostate cancer includes castration-resistant prostate cancer. Castration-resistant prostate cancer is a condition in which prostate cancer, once suppressed by endocrine therapy for prostate cancer, becomes resistant to endocrine therapy drugs by long-term endocrine therapy. Is prostate cancer that has become worse again.

に お い て In one embodiment of the present invention, the prostate cancer is preferably a castration-resistant prostate cancer.

SF3B2 is a positive splicing regulator of AR-V7. That is, in splicing of AR-V7, SF3B2 controls splicing by removing the intron to which SF3B2 is bound while leaving the exon to which SF3B2 is bound.
As the SF3B2 gene, a gene having the nucleotide sequence shown in SEQ ID NO: 1 is exemplified. Further, as long as the SF3B2 gene encodes an SF3B2 protein having a function of enhancing the expression of AR-V7, a DNA that hybridizes under stringent conditions with a DNA having a complementary sequence of the nucleotide sequence of SEQ ID NO: 1 Is included. Here, examples of stringent conditions include, for example, conditions for washing under conditions of 0.1 × SDS, 0.1 × SSC, and 68 ° C. SF3B2 is a protein that more easily binds to the AR-V7 pre-mRNA sequence than the AR-FL pre-mRNA sequence, and further binds directly to the CE3 region, which is a potential exon of AR-V7.

Although measuring the expression level of the SF3B2 gene is not particularly limited, it means, for example, measuring the expression level of mRNA or protein.
The expression level of the SF3B2 gene can be determined by, for example, RT-PCR, quantitative PCR, microarray, or Northern blot. The expression level of the SF3B2 protein, which is a product of the SF3B2 gene, can be determined by, for example, Western blot, ELISA, or the like.
The base sequence of the coding region of the SF3B2 gene is, for example, the sequence of SEQ ID NO: 1. Using this sequence and the like, primers and probes for gene expression analysis can be designed or obtained. The nucleotide sequence may have 90% or more, preferably 95% or more, more preferably 98% or more identity with the nucleotide sequence of SEQ ID NO: 1. In addition, the above-mentioned primers and probes have 90% or more, preferably 95% or more, more preferably 98% or more identity with the complementary sequence of the above-mentioned base sequence as long as they can specifically bind to the above-mentioned base sequence. May be.
As the antibody, a commercially available antibody can be used, or an antibody prepared using a part of the amino acid sequence of the SF3B2 protein represented by SEQ ID NO: 2 as an antigen can be used. The amino acid sequence has 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of SEQ ID NO: 2 as long as it is an SF3B2 protein having a function of enhancing AR-V7 expression. May be something. In addition, the antibody or the like has 90% or more, preferably 95% or more, more preferably 98% or more identity with a complementary sequence of the amino acid sequence as long as it can specifically bind to the amino acid sequence. Good.

The screening or evaluation method of the present invention includes a step of adding a candidate compound to cells expressing the SF3B2 gene, and a step of measuring the expression level of the SF3B2 gene in the cell to which the candidate compound has been added, comprising: Screening or evaluation method.
For example, when the expression level of the SF3B2 gene in the presence of the candidate compound is reduced by, for example, 20% or more, preferably 50% or more, more preferably 90% or more, as compared with a control to which the candidate compound has not been added. The candidate compound can be selected as a candidate for a therapeutic agent for prostate cancer.

As the cells, any of a biological sample containing cells that express the SF3B2 gene, a cultured cell line that expresses the SF3B2 gene, and a cultured cell line that expresses the SF3B2 gene may be used, but cultured cells that express the SF3B2 gene forcibly. Preferably, a strain is used.
The biological sample includes, for example, cells or tissues of the prostate, cells or tissues around the prostate, cells or tissues of any site where metastasis of prostate cancer can be considered, collected from a test subject or a healthy subject, and the like. In addition, the biological sample may be a body fluid such as blood or lymph that may contain prostate cancer cells.
The cultured cell line that expresses the SF3B2 gene is not particularly limited as long as it is a cell line that expresses the SF3B2 gene, and examples include a cell line derived from prostate cancer cells.
The cultured cell line in which the SF3B2 gene is forcibly expressed is, for example, a cultured cell line in which the SF3B2 gene is incorporated into a plasmid or a viral vector for introducing the gene into a mammalian cell and transfected into the cell by a normal method such as lipofection. And the like. Transfection may be transient or stable.

As another embodiment of the screening or evaluation method of the present invention, for example, a step of adding a candidate compound to a cell expressing a reporter gene linked to a promoter of SF3B2 gene, and a step of adding a reporter gene to the cell to which the candidate compound is added And a method for screening or evaluating a therapeutic agent for prostate cancer, which comprises a step of measuring the expression level of the drug.
For example, in the presence of a candidate compound, the expression level of a reporter gene linked to the promoter of the SF3B2 gene is, for example, 20% or more, preferably 50% or more, more preferably, as compared to a control to which no candidate compound was added. When is decreased by 90% or more, the candidate compound can be selected as a candidate for a therapeutic agent for prostate cancer.

When using a reporter gene linked to the promoter of the SF3B2 gene, the promoter of the SF3B2 gene is preferably a region containing about 1 kbp upstream of the transcription start site, and more preferably a region containing about 2 kbp upstream.
As a reporter gene, a luciferase gene, a GFP gene, a chloramphenicol acetyltransferase gene, or the like can be used, and preferably, a luciferase gene or a GFP gene can be used.

As the cells, for example, these reporter genes are linked to the promoter of the SF3B2 gene, and this is incorporated into a plasmid or a viral vector for introducing the gene into mammalian cells, and the cells are cultured by a known method such as lipofection. A cultured cell line transfected with a reporter gene and forcibly expressing the reporter gene can be used. Transfection may be transient or stable.

In another embodiment of the present invention, the cells are any of a biological sample expressing the AR-V7 gene, a cultured cell line expressing the AR-V7 gene, and a cultured cell line expressing the AR-V7 gene forcibly. , SF3B2 gene, or the expression of a reporter gene linked to the SF3B2 gene promoter.
The biological sample is not particularly limited as long as it is a biological sample containing cells expressing the AR-V7 gene. For example, prostate cells or tissues, cells around the prostate collected from a test subject or a healthy subject Or a tissue or any other cell or tissue at which metastasis of prostate cancer can be considered. In addition, the biological sample may be a body fluid such as blood or lymph that may contain prostate cancer cells.
The cultured cell line expressing the AR-V7 gene is not particularly limited as long as it is a cell line expressing the AR-V7 gene, and examples include a cell line derived from prostate cancer cells.
A cultured cell line in which the AR-V7 gene has been forcibly expressed is, for example, a cell line in which the AR-V7 gene is incorporated into a plasmid or virus vector for introducing the gene into a mammalian cell, and transfected into the cell by a normal method such as lipofection. Cultured cell lines.

In another embodiment of the screening or evaluation method of the present invention, a step of adding a candidate compound to cells expressing the SF3B2 gene, preferably cells in which the SF3B2 gene is forcibly expressed, A method for screening or evaluating a therapeutic agent for prostate cancer, which includes a step of measuring the expression level of the -V7 gene or the reporter gene linked to the AR-V7 gene promoter.
For example, a candidate compound is added to cells expressing the SF3B2 gene, and after culturing for a certain period of time, the expression levels of the AR-V7 gene or reporter gene are measured. The candidate compound can be selected as a candidate for a therapeutic agent for prostate cancer when it decreases, for example, by 20% or more, preferably 50% or more, more preferably 90% or more compared to the control.

When a reporter gene linked to the AR-V7 gene promoter is used, the AR-V7 gene promoter is preferably a region containing about 1 kbp upstream of the transcription start site, and more preferably a region containing about 2 kbp upstream.
As a reporter gene, a luciferase gene, a GFP gene, a chloramphenicol acetyltransferase gene, or the like can be used, and preferably, a luciferase gene or a GFP gene can be used.

For cells, for example, these reporter genes are linked to the promoter of the AR-V7 gene, which is incorporated into a plasmid or a viral vector for introducing the gene into mammalian cells, and used in a conventional method such as lipofection, which is a known method. , A cultured cell line in which a reporter gene is forcibly expressed can be used.

In the present invention, the candidate compound may be either a high molecular compound or a low molecular compound, and is not particularly limited. Examples of the high molecular compound include proteins, antibodies, peptides, non-peptide compounds, nucleic acids, and RNAs. And the like. These compounds may be novel compounds or known compounds, and may be fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. containing these compounds.
By selecting a compound that reduces the expression level of the SF3B2 gene and / or AR-V7 gene as compared to when no candidate compound is added, a substance that can be a therapeutic agent for prostate cancer can be obtained. it can.

濃度 Concentration of the candidate compound The addition concentration is not particularly limited as long as it can be confirmed that the expression level of the SF3B2 gene and / or the AR-V7 gene is reduced. Further, the addition method, reaction time, reaction temperature and the like can be appropriately selected according to the analyte to be used.

に お い て In the present invention, the substance obtained by the screening can be evaluated as a substance having the ability to decrease the expression of SF3B2 gene and / or AR-V7 gene. The therapeutic effect of the substance can be evaluated using a prostate cancer model animal or the like.

In another embodiment of the screening or evaluation method of the present invention, a step of adding a candidate compound to an assay system containing an SF3B2 protein and an SF3b protein complex-forming molecule other than SF3B2, a method of inhibiting a compound that inhibits SF3b protein complex formation from prostate A method for screening or evaluating a therapeutic agent for prostate cancer, which includes a step of selecting as a candidate for a therapeutic agent for cancer, is mentioned.
The complex of the SF3b protein includes SF3B1, SF3B2, SF3B3, SF3B4, SF3A1, and SF3A3 as shown in Example 4 of the present application and FIG. The SF3b protein complex is also described in MOLECULAR AND CELLULAR BIOLOGY, Oct. 1999, Vol. 19, No. 10, p.6796-6802.
Inhibiting the complex formation of SF3b protein means that SF3B2 inhibits complex formation with one or more of SF3B1, SF3B3, SF3B4, SF3A1 and SF3A3, and preferably the SF3B2 protein inhibits SF3B1, SF3B3, SF3B4, SF3A1. And complex formation with all of SF3A3.
For example, after adding a candidate compound to an assay system containing an SF3B2 protein and an SF3b protein complex-forming molecule other than SF3B2 and incubating for a certain period, when immunoprecipitating with an antibody against SF3B2, SF3B3, SF3B4, and SF3A1 co-precipitate with SF3B2. And the amount of one or more of SF3A3 can be evaluated as an index. A compound is a candidate prostate cancer therapeutic when the amount of one or more of SF3B3, SF3B4, SF3A1 and SF3A3 that co-precipitates with SF3B2 when incubated with a compound is reduced as compared to a control. It is possible to select as
In the present invention, inhibiting the formation of a complex refers to dissociating proteins that have already formed a complex, and / or inhibiting the formation of a new complex by associating proteins.
The assay system may be an in vitro system or a cell system.

The present invention also includes a kit for use in a method for screening or evaluating a therapeutic agent for prostate cancer. The contents of the kit are composed of a combination of devices or reagents. If the kit contains a substance that is essentially the same as each component described below, or a substance that is essentially the same as a part thereof, the configuration or form is Even if they are different, they are included in the kit of the present invention.

The reagent includes, for example, a primer capable of specifically amplifying the SF3B2 gene when the expression level of the SF3B2 gene is measured by the PCR method. If necessary, a reverse transcriptase, a polymerase, a buffer, a fluorescent reagent and the like may be included. As the device, for example, a fluorometer, a thermal cycler or the like can be used.

(4) When the expression level of the SF3B2 gene is measured by the microarray method, a support such as glass, plastic, silicon, or a membrane on which the SF3B2 gene fragment is spotted is included. If necessary, a labeling reagent, a hybridization buffer, a fragmentation buffer, and the like may be included.

When the expression level of SF3B2 protein is measured by an immunoassay, an anti-SF3B2 antibody is included. Further, if necessary, a diluent of a biological sample, an antibody-immobilized solid phase, a buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, a reagent for detecting a labeled body, a standard substance, and the like are also included. Examples of the diluent for the biological sample include an aqueous solution containing a protein such as BSA or casein in a surfactant or a buffer.

(4) As the antibody-immobilized solid phase, a material obtained by immobilizing an anti-molecular marker antibody or an antibody fragment thereof on a material obtained by shaping various polymer materials to suit the intended use is used. Tubes, beads, plates, latex and other fine particles, sticks, etc. are used as materials, and polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, etc. Polymer materials, glass, ceramics and metals. As a method for immobilizing the antibody, known methods such as a physical method and a chemical method or a combination method thereof can be used. For example, there may be mentioned a polystyrene 96-well immunoassay microterplate having an antibody or antibody fragment immobilized on a hydrophobic solid phase.

The reaction buffer may be any as long as it provides a solvent environment for the binding reaction between the antibody in the antibody-immobilized solid phase and the antigen in the biological sample, but may be a surfactant, a buffer, a BSA, Reaction buffers containing proteins such as casein, preservatives, stabilizers, reaction accelerators, and the like.

As the labeled secondary antibody or its antibody fragment, the antibody or antibody fragment used in the present invention is labeled with a labeling enzyme such as horseradish peroxidase (HRP), bovine intestinal alkaline phosphatase, β-galactosidase, and a buffer. , A mixture of proteins such as BSA and casein, preservatives and the like.

Depending on the above-mentioned labeling enzyme, for example, horseradish peroxidase may be used as a reagent for detecting a labeled substance, a substrate for absorption measurement such as tetramethylbenzidine or orthophenylenediamine, or a fluorescent substance such as hydroxyphenylpropionic acid or hydroxyphenylacetic acid. When the luminescent substrate such as a substrate or luminol is alkaline phosphatase, examples thereof include a substrate for measuring absorbance such as 4-nitrophenyl phosphate and a fluorescent substrate such as 4-methylumbelliferyl phosphate.

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the scope of the present invention is not limited to the examples.

Example 1 Method for Producing AR-V7-GFP Expressing Cell In order to examine the association between the AR-V7 gene and the SF3B2 gene, the following examination was performed using a cell line expressing the AR-V7 gene. Was.
AR-V7-GFP (Green Fluorescent Protein) gene was prepared and knocked into prostate cancer cells CWR22Rv1 (hereinafter referred to as 22Rv1). As shown in FIG. 1, AR-V7-GFP expression was achieved by incorporating the nucleotide sequences of GFP, T2A, and NeoR immediately before the stop codon contained in the latent exon 3 (CE3) portion on the AR-V7 gene. Cells were made. Knock-in of the gene was performed using the CRISPR / Cas9 system, which is a known genome editing system. The gRNA (guide RNA) used at the time of producing the AR-V7-GFP-expressing cell is a gRNA having the base sequence represented by SEQ ID NO: 3.
Thereafter, it was confirmed by Western blotting that the AR-V7 gene and the GFP gene were fused (FIG. 2), and cells capable of monitoring AR-V7 with the fluorescent protein GFP could be developed.

Example 2 Inhibition of AR-V7 Expression by Inhibition of SF3B2 Expression In order to examine the relationship between the expression levels of SF3B2 and AR-V7, AR-V7-GFP cells were analyzed using the CRISPR / Cas9 system. Gene transfer of SF3B2 gRNA (guide RNA) was performed. The gRNA (guide RNA) used at the time of preparing the SF3B2-expressing cells is a gRNA having the base sequence represented by SEQ ID NO: 4.
As shown in the flow cytometry diagram shown in FIG. 3, AR-V7-GFP cells into which SF3B2 gRNA was introduced and SF3B2 gene expression was suppressed showed that AR-V7 expression was reduced as compared to control cells. confirmed.
That is, since it was confirmed that the splicing of AR-V7 can be suppressed by suppressing the expression of SF3B2, the created AR-V7-GFP cells are cells in which the expression of SF3B2 can be monitored with a fluorescent protein (GFP). That was confirmed.

Example 3 Tumor Growth Inhibition by AR-V7 Knockout In Vivo The relationship between overexpression of SF3B2 and AR-V7-induced tumor growth under in vivo androgen depletion was confirmed.
Overexpression of SF3B2 was performed by transfecting a vector incorporating the SF3B2 gene into target cells by electroporation using a Neon (registered trademark) system (ThermoFisher scientific). The vector used was pX330.
The cells in which AR-V7 was knocked out were subjected to sgRNA (single-guide RNA) having the nucleotide sequence represented by SEQ ID NO: 3 and Cas9 by electroporation using a Neon (registered trademark) system (ThermoFisher scientific). It was obtained by transfecting cells, culturing the cells in a selective medium, and isolating a single colony.
For tumor cell transplantation into mice, male non-obese diabetics / severe combined immunodeficient (NOD / SCID) mice were first castrated surgically at the age of 7 weeks. Cancer cells (2 × 10 ⁶ cells) were suspended in a PBS / Matrigel mixture and injected subcutaneously into castrated 8-week-old NOD / SCID mice under deep anesthesia. Thereafter, the mice were bred in a temperature-controlled sterile room. Tumor volume following formula: was calculated by tumor volume (mm ³⁾ = Length × (tumor width) tumor ^2/2.
As shown in FIG. 4, a change in tumor volume upon subcutaneous injection of the tumor was confirmed. As a result, in both 22Rv1 cells and LNCaP95 cells, it was found that cells overexpressing SF3B2 had significantly increased tumor volume as compared to control (GFP only) cells (P = 0). .0133, 0.0371). On the other hand, it was found that the cells in which SF3B2 was overexpressed and AR-V7 was knocked out had a significantly suppressed increase in tumor volume as compared with the cells in which SF3B2 was overexpressed (P = 0. 0002, P <0.0001).
In other words, it was found that prostate cancer caused by SF3B2 expression was suppressed by knocking out AR-V7. That is, it was suggested that regulating the splicing of AR-V7 is one of the important roles of SF3B2 in castration-resistant prostate cancer.

<Example 4> Involvement of SF3B2 and SF3b in prostate cancer cells Since it has been known that SF3B2 is contained in the SF3b complex, SF3B2 is involved in SF3b components also in prostate cancer cells. Was discussed.
As shown in FIG. 5A, a SF3B2-related protein was purified from nuclear extracts of 22Rv1 cells stably expressing SF3B2-TAP by tandem affinity purification (TAP), which is a known method. SF3B2-TAP was found to be localized in both nucleus and cytoplasm (FIG. 5B). By mass spectrometry, SF3B2 can be identified as SF3B1, SF3B3 (also known as SF3b130 or SAP130), SF3B4 (also known as SF3b49 or SAP49), SF3A1 (also known as SF3a120 or SAP114) and SF3A3 (also known as SF3a60 or SAP61). Is also known) (Fig. 5C). These results suggested that SF3B2 is an important component in the SF3b complex.

<Example 5> Involvement of Pradienolide B in SF3B2 complex It is known that a certain compound inhibits splicing and has an ability as an antitumor agent. One such compound, Pladienolide B (PLA-B), is a commercially available macrolide, has antitumor activity, and splices AR-V7 through its interaction with SF3B3. It has been known so far to interfere. Since SF3B2 is involved in SF3B3 in prostate cancer, it was examined whether Pradienolide B would be a candidate for a therapeutic drug for aggressive cancer having high SF3B2 expression.
Overexpression of SF3B2 or GFP was performed in the same manner as described in Example 3.
Notably, Pladienolide B reduced the interaction of SF3B2-TAP with its related proteins (FIGS. 5A, 5C). Considering the effect of Pradienolide B on SF3B2-related proteins, the effect on AR-V7 splicing was examined (FIGS. 6A and 6B). Pradienolide B reduced AR-V7-GFP expression in AR-V7-GFP cells in a dose-dependent manner. In addition, when LNCaP95 cells were used, it was found that the administration of Pradienolide B could significantly suppress the expression of AR-V7 as compared to the vehicle (FIG. 6C, P = 0.0005). This result suggests that PLA-B reduced the interaction between the constituent proteins of the protein complex SF3b, which is a splicing factor, and as a result, suppressed the splicing of AR-V7. Similar results were obtained when 22Rv1 cells were used (data not shown).

<Example 6> Inhibition of SF3B2-dependent tumor growth of Pradienolide B in vivo It was examined whether the growth of SF3-B2-dependent tumor of Pradienolide B in vivo was suppressed.
Overexpression of SF3B2 or GFP and transplantation of tumor cells into mice were performed in the same manner as described in Example 3.
Further, when the tumor volume in the 22Rv1 cells reached 200 ~ 400 mm ³ in 100 ~ 300 mm ³ or LNCaP95 cells, Pladienolide B derivative (5 mg / kg) or vehicle (DMSO) 0, 2, 4, and 6 days Administered intraperitoneally in the eyes. Relative tumor volume was calculated by the ratio between the tumor volume at time t and the tumor volume at the start of treatment.
In order to confirm the antitumor effect in vivo, Pradienolide B was intraperitoneally administered to the LNCaP95 cells when the size of the castrated mouse reached 200 to 400 mm ³ . As a result, the tumor volume of mice administered with vehicle (DMSO) tended to grow over time, whereas the mice administered PLA-B significantly reduced tumor volume (FIG. 7A, P <0). .01). This result suggests that PLA-B inhibited the function of SF3B2 and suppressed splicing of AR-V7 by SF3B2, resulting in suppression of tumor growth.
Further, in castrated mice having prostate cancer cells (LNCaP95 cells) overexpressing SF3B2 or GFP, when the tumor size reached 200 to 400 mm ³ , Pradienolide B was administered intraperitoneally. As a result, the mice having LNCaP95 cells stably overexpressing SF3B2 were more stable than those having LNCaP95 cells overexpressing GFP only when treated with Pradienolide B. Mice with overexpressed LNCaP95 cells had significantly reduced tumor volume (FIG. 7B, P <0.01). That is, it was found that the overexpression of SF3B2 increased the sensitivity of LNCaP95 cells to Pradienolide B.
In addition, the same results were obtained when using 22Rv1 cells (data not shown).
In other words, by utilizing such a cell-based screening system for a novel compound, a dramatic anti-cancer growth effect targeting AR-V7 splicing regulation is produced in the same manner as in Pradienolide B, and cytotoxicity is reduced. Compounds can be screened.

Claims (10)

A method for screening or evaluating a therapeutic agent for prostate cancer, comprising a step of adding a candidate compound to cells expressing the SF3B2 gene and a step of measuring the expression level of the SF3B2 gene in the cell to which the candidate compound has been added. The method according to claim 1, wherein a compound that reduces the expression level of the SF3B2 gene as compared to a control is selected or evaluated as a candidate for a therapeutic agent for prostate cancer. The SF3B2 gene according to claim 1 or 2, wherein the SF3B2 gene is represented by a DNA having a nucleotide sequence of SEQ ID NO: 1 or a DNA hybridizing under stringent conditions with a DNA having a complementary sequence to the nucleotide sequence of SEQ ID NO: 1. The described method. The method according to any one of claims 1 to 3, wherein the expression level of the SF3B2 gene is measured by measuring the expression level of a reporter gene linked to a promoter of the SF3B2 gene. A method for screening or evaluating a therapeutic agent for prostate cancer, comprising the steps of: adding a candidate compound to cells expressing the SF3B2 gene; and measuring the expression level of the AR-V7 gene in the cell to which the candidate compound has been added. The method according to claim 5, wherein a compound that reduces the expression level of the AR-V7 gene as compared to a control is selected or evaluated as a candidate for a therapeutic agent for prostate cancer. The SF3B2 gene according to claim 5 or 6, wherein the SF3B2 gene is represented by a DNA having a nucleotide sequence of SEQ ID NO: 1 or a DNA hybridizing under stringent conditions with a DNA having a complementary sequence to the nucleotide sequence of SEQ ID NO: 1. The described method. The method according to any one of claims 5 to 7, wherein the expression level of the AR-V7 gene is measured by measuring the expression level of a reporter gene linked to the promoter of the AR-V7 gene. A step of adding a candidate compound to an assay system containing an SF3B2 protein and an SF3b protein complex-forming molecule other than SF3B2, and a step of selecting a compound that inhibits SF3b protein complex formation as a candidate for a therapeutic agent for prostate cancer, A method for screening or evaluating a therapeutic drug. A kit for screening or evaluating a therapeutic agent for prostate cancer, comprising a reagent capable of measuring the expression level of the SF3B2 gene.

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