JP6425942B2 - Prostate cancer determination, treatment selection method, prophylactic or therapeutic agent - Google Patents

Description

  The present invention relates to prostate cancer determination, treatment selection methods, and prophylactic or therapeutic agents. More specifically, the determination of prostate cancer based on the expression of ABHD2 (Abhydrolase domain-containing protein 2; ABHD2 (hereinafter sometimes referred to simply as ABHD2)) gene, a method for determining prostate cancer, a therapeutic selection method, or prevention of prostate cancer by suppression of this expression Or a therapeutic agent.

In recent years, with the westernization of diet and the aging of the population, the number of prostate cancer patients is also rapidly increasing in Japan. Clinically, surgical treatment including prostatectomy, chemotherapy with anticancer agents, and radiation treatment are widely used in treatment techniques for prostate cancer.
Among the treatment methods, surgical treatment is the first choice, but if the cancer has already been diagnosed or if surgery is difficult due to recurrence after surgery, other treatment methods Is selected.

Generally, prostate cancer cells are known to be stimulated by androgen and proliferate. Therefore, hormonal therapy that inhibits androgen production and function is often used. This is an operation to remove the testicular and to reduce the level of androgen in the blood secreted from the testis using LHRH analogue, and to remove the adrenal gland-derived androgen secreted in trace amounts by internal medicine. is there.
Although this hormonal therapy is extremely effective, it has been a problem to relapse in several years as a castration-resistant prostate cancer (CRPC), and in the treatment of prostate cancer Control of dependent prostate cancer has become the most important issue.

  Androgen-independent prostate cancer is sensitive to extremely low concentrations of androgen, antiandrogens, and other steroid hormones by mutation or amplification of the androgen receptor (Androgen Receptor; AR (hereinafter sometimes simply referred to as AR)). It is suggested to show. However, the mechanism and the like are still unclear, and at present, anticancer drug therapy using docetaxel is regarded as the only treatment. However, with this treatment method, cases showing an effect are limited, and since the effect can not be predicted in advance, it is desired to provide a marker useful for predicting the effect.

In addition, in order to prevent the progression to androgen-independent prostate cancer, it is also desired to provide an effective therapeutic method other than hormonal therapy for androgen-dependent prostate cancer.
Since prostate cancer cells expressing AR enhance expression of androgen response genes, in recent years, antisense nucleic acids and the like are administered to androgen response genes to make the expression of these genes unstable. Are being developed to treat prostate cancer. However, since antisense nucleic acids and the like are easily degraded in the living body and are unstable, they have not been enough methods for treating prostate cancer.

  Therefore, the present inventors pay attention to PI polyamide having high stability even in vivo, and suppress the expression of fusion gene of TMPRSS2 which is an androgen response gene and EGR gene of ETS family which is a transcription regulator. We have developed (see Patent Document 1, etc.) and confirmed the usefulness for the prevention, treatment, etc. of prostate cancer.

As a result of further investigations for the purpose of enabling comprehensive prevention, treatment and the like of prostate cancer, the present inventors found the ABHD2 gene as one of the new AR target genes in the present invention. The
The ABHD2 gene is known to be involved in phospholipid metabolism and to influence the formation of alveolar structure, but its detailed function is unknown. In addition, as a result of exhaustive analysis of genes overexpressed in prostate cancer (PRC) cells in Patent Document 2, although the ABHD2 gene is mentioned as one of the genes, it is possible to specifically identify with prostate cancer. The relationship was unknown.

JP, 2013-234135, A Japanese Patent Application Publication No. 2006-500950

An object of the present invention is to solve the problems in the prior art and achieve the following objects.
That is, the present invention aims to provide a novel therapeutic effective for androgen-dependent prostate cancer. Another object of the present invention is to provide a marker useful for predicting the effect of treatment of androgen independent prostate cancer.

As a result of intensive studies to solve the above problems, the present inventors focused on the ABHD2 gene, which was found as one of new AR target genes.
In the present invention, when the ABHD2 gene is highly expressed in a specimen biopsied for the purpose of diagnosing prostate cancer, the present invention does not significantly respond to docetaxel treatment in the subsequent treatment course, and relapse of prostate cancer ( We found that it causes PSA recurrence). Therefore, by using the expression of this ABHD2 gene as a marker, it has been found that the effect of treatment of prostate cancer can be predicted in advance, and the present invention has been completed.
In addition, the present inventors have suggested that this ABHD2 gene is a gene that promotes the progression of prostate cancer and is resistant to docetaxel, which is currently the only therapeutic agent for advanced prostate cancer (castration-resistant prostate cancer). As a result, it has been found that double-stranded nucleic acid molecules such as siRNA capable of suppressing the expression of this ABHD2 gene can be used to suppress the progression of prostate cancer, thereby completing the present invention.

That is, the present invention relates to the agent for preventing or treating prostate cancer described in the following (1) to (9), a marker for determining the therapeutic effect of prostate cancer, and the like.
(1) An agent for the prophylaxis or treatment of prostate cancer, which comprises a double stranded nucleic acid molecule for suppressing the expression of ABHD2 gene as an active ingredient.
(2) The prophylactic or therapeutic agent for prostate cancer according to the above (1), wherein the double stranded nucleic acid molecule is a double stranded nucleic acid molecule comprising the following (a) and (b):
(A) a sense strand comprising a nucleotide sequence corresponding to a target sequence shown in any of SEQ ID NOs: 2 to 8 (b) an antisense strand comprising a nucleotide sequence complementary to the sense strand of (a) above (3) The agent for preventing or treating prostate cancer according to the above (2), wherein the 3 'end of each strand of the double-stranded nucleic acid molecule is a protruding end with an overhang of 2 bases to 6 bases.
(4) The agent for preventing or treating prostate cancer according to any one of the above (1) to (3), wherein the length of the double stranded nucleic acid molecule is at least one of 23 bases to 29 bases.
(5) The agent for preventing or treating prostate cancer according to any one of the above (1) to (4), wherein the double stranded nucleic acid molecule is a double stranded RNA.
(6) The agent for preventing or treating prostate cancer according to the above (5), wherein the double stranded nucleic acid molecule is siRNA.
(7) The agent for preventing or treating prostate cancer according to any one of the above (1) to (6) for suppressing the growth of prostate cancer cells.
(8) A method of determining the risk of prostate cancer in a subject animal, wherein ABHD2 protein or a fragment thereof in a biological sample derived from the subject animal or a nucleic acid encoding them is used as a determination marker for prostate cancer.
(9) A marker for determining prostate cancer, which comprises the ABHD2 protein or a fragment thereof, or a nucleic acid encoding them.

  Provision of the determination marker of the present invention also makes it possible to provide a novel diagnosis and treatment selection method for prostate cancer. Furthermore, provision of double stranded nucleic acid molecules such as siRNA targeting the ABHD2 gene of the present invention makes it possible to provide new therapeutic agents, treatment methods and the like useful in the treatment of prostate cancer. In addition, by combining the double-stranded nucleic acid molecule of the present invention with PI polyamide or the like that is effective for treatment of prostate cancer, comprehensive treatment of prostate cancer can be achieved.

(A) It is the figure which showed the result of having evaluated the relationship with the expression of ABHD2 gene and cancer specific survival rate in a prostate cancer tissue (Example 1). (B) It is the figure which showed the result of having evaluated the relationship between the expression of ABHD2 gene in prostate cancer tissue, and the PSA non-relapse rate after docetaxel introduction | transduction (Example 1). (C) It is the figure which showed the result of the immunostaining in the prostate cancer sample obtained by radical prostatectomy (Example 1). (D) It is the figure which showed the result of the immunostaining in the prostate cancer sample obtained by transrectal prostate biopsy operation (Example 1). It is the figure which showed the examination result of ABHD2 gene expression suppression effect by 2 nucleic acid molecules (Example 3). It is the figure which showed the examination result of the LNCaP cell growth inhibitory effect by 2 nucleic acid molecules (Example 4). It is the figure which showed the examination result of the growth inhibitory effect of LNCaP cell by 2 nucleic acid molecules in combination with docetaxel (Example 5). It is the figure which showed the examination result of the migration ability inhibitory effect of LNCaP cell by 2 nucleic acid molecules (Example 6). It is the figure which showed the photograph in examination of the tumor growth inhibitory effect by 2 nucleic acid molecules (Example 7). It is the figure which showed the examination result of the tumor growth inhibitory effect by 2 nucleic acid molecules (Example 7). It is the figure which showed the analysis result of the amount of ABHD2 protein in examination of the tumor growth inhibitory effect by 2 nucleic acid molecules (Example 7).

The “prophylactic or prophylactic agent for prostate cancer” of the present invention is an agent containing a double stranded nucleic acid molecule for suppressing the expression of the ABHD2 gene as an active ingredient, and this double stranded nucleic acid molecule causes the expression of the ABHD2 gene. An agent that can prevent or treat prostate cancer by suppressing
As long as it is an agent capable of preventing or treating prostate cancer, it may contain, in addition to the active ingredient, another ingredient, a pharmaceutically acceptable carrier, and the like.

The "ABHD2 gene" is known to be involved in phospholipid metabolism and to influence the formation of alveolar structure. In the present invention, the “target gene” of the double stranded nucleic acid molecule is the “ABHD2 gene” because its mRNA sequence is the target of the double stranded nucleic acid molecule and its expression is suppressed by the double stranded nucleic acid molecule. It may be indicated.
As a reference, the base sequence of the above-mentioned "ABHD2 gene" of human is shown in SEQ ID NO: 1.

The "double-stranded nucleic acid molecule" of the present invention refers to a double-stranded nucleic acid molecule formed by hybridizing a desired sense strand and an antisense strand.
The “double-stranded nucleic acid according to the present invention is a double-stranded nucleic acid consisting of a sense strand and an antisense strand containing any nucleotide sequence as long as it is a double-stranded nucleic acid molecule capable of suppressing the expression of the ABHD2 gene. It may be a molecule.
It is particularly preferred that such "double stranded nucleic acid molecule" of the present invention is a double stranded nucleic acid molecule comprising the following (a) and (b):
(A) a sense strand comprising a nucleotide sequence corresponding to a target sequence shown in any of SEQ ID NOs: 2-8 (b) an antisense strand comprising a nucleotide sequence complementary to the sense strand of (a) above The antisense strand may be an RNA strand, an RNA-DNA chimeric strand or the like. These sense and antisense strands can be hybridized to each other to form the "double-stranded nucleic acid molecule" of the present invention.

The “double-stranded nucleic acid molecule” of the present invention is not particularly limited and can be appropriately selected depending on the purpose. For example, double-stranded RNA (dsRNA), double-stranded RNA-DNA chimera Or the like.
"Double stranded RNA" refers to a double stranded nucleic acid molecule wherein both the sense and antisense strands consist of RNA sequences. In addition, "double-stranded RNA-DNA chimera" refers to a double-stranded nucleic acid molecule in which both of the sense strand and the antisense strand are composed of a chimeric sequence of RNA and DNA.

When the "double stranded nucleic acid molecule" of the present invention is a double stranded RNA, it is particularly preferred that it is a siRNA (small interfering RNA).
The “siRNA” is a small double-stranded RNA of 18 to 29 bases in length, and an antisense strand (guide strand) having a sequence complementary to the mRNA of a target gene cleaves the mRNA of the target gene , Has the function of suppressing the expression of the target gene.
The “siRNA” in the present invention is not particularly limited in its terminal structure as long as it can suppress the expression of this target gene, using the ABHD2 gene as a target gene, and can be appropriately selected according to the purpose. For example, the "siRNA" of the present invention may have a blunt end or an overhang (overhang).
In particular, the "siRNA" of the present invention preferably has a structure in which the 3 'end of the sense strand and the antisense strand has an overhang of 2 to 6 bases, and that the 3' end of each strand has an overhang of 2 bases More preferable.

In addition, when the “double stranded nucleic acid molecule” of the present invention is a double stranded RNA, it may be a shRNA (short hairpin RNA).
The “shRNA” is a single-stranded RNA comprising a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases, and is expressed in vivo to form a base pair to form a hairpin It becomes double stranded RNA. Thereafter, the shRNA is cleaved by Dicer (RNase III enzyme) to form siRNA, which can function to suppress the expression of the target gene.
The terminal structure of the "shRNA" in the present invention can also be appropriately selected according to the purpose, similarly to the "siRNA" of the present invention, may have a blunt end, and has an overhang (overhang) It may be one.

Furthermore, when the "double stranded nucleic acid molecule" of the present invention is a "double stranded RNA-DNA chimera", it is particularly preferred that it is a chimeric siRNA.
The term "chimeric siRNA" refers to a small-molecule double-stranded RNA-DNA chimera of 18-29 bases in length, in which a portion of the RNA sequence of the siRNA has been converted to DNA. In particular, it is a small molecule double-stranded RNA-DNA chimera of 21 to 23 bases in length in which 8 bases on the 3 'side of the sense strand of siRNA and 6 bases on the 5' side of the antisense strand are converted to DNA. Is preferred.
The terminal structure of the “chimeric siRNA” in the present invention can also be appropriately selected according to the purpose, similarly to the “siRNA” of the present invention, and may have blunt ends, and the overhanging ends (overhangs) You may have.

The "double-stranded nucleic acid molecule" of the present invention may be appropriately modified depending on the purpose. For example, 2'O-methylated modification, phosphorothioated (S-modified) modification, LNA (Locked Nucleic Acid) modification, etc. are applied to "double-stranded nucleic acid molecule" to impart resistance to a nucleolytic enzyme (nuclease) or The stability in the culture solution or the living body can be improved.
In addition, modifications such as nanoparticles, cholesterol, cell membrane passage peptides and the like can be applied to the 5 'end or 3' end of the sense strand of the double stranded nucleic acid molecule to enhance the efficiency of introduction into cells.
Such modification can be appropriately performed by any method known in the art.

Such "double-stranded nucleic acid molecule" of the present invention can be appropriately prepared by any method known in the art. For example, when the "double-stranded nucleic acid molecule" is "siRNA", a single-stranded RNA having a length of 18 to 29 bases corresponding to the sense strand and the antisense strand can be used with an existing automatic DNA / RNA synthesizer Can be chemically synthesized and prepared by annealing them.
In addition, “siRNA” in the present invention may be one produced by requesting a synthesis contract company for synthesis, or may be a commercially available double-stranded siRNA. Furthermore, the siRNA expression vector may be constructed, and the expression vector may be introduced into cells to produce "siRNA" produced using the reaction in cells.
When the "double-stranded nucleic acid molecule" of the present invention is a "chimeric siRNA", it is prepared, for example, by chemically synthesizing the sense strand and the antisense strand, which are chimeric nucleic acid molecules, respectively, and annealing them. be able to.

Among such “double-stranded nucleic acid molecules” of the present invention, “a sense strand comprising a nucleotide sequence corresponding to the target sequence shown in any of (a) SEQ ID NOs: 2 to 8”, for example, Mention may be made of the sense strand shown in 9, 11, 13, 15, 17, 19 or 21.
In addition, “(b) an antisense strand comprising a nucleotide sequence complementary to the sense strand of (a)” includes the antisense strand represented by SEQ ID NO: 10, 12, 14, 16, 18, 20 or 22 It can be mentioned.
Furthermore, as a "double-stranded nucleic acid molecule" of the present invention, a sense strand comprising a nucleotide sequence shown in SEQ ID NO: 23, 25 or 27, which is a double-stranded nucleic acid molecule capable of suppressing the expression of ABHD2 gene, The antisense strand shown in Sequence Listing 24, 26, 28 can also be mentioned as an antisense strand containing a complementary nucleotide sequence.

By including such a "double stranded nucleic acid molecule" of the present invention as an active ingredient, the "prostate cancer preventive or therapeutic agent" of the present invention can be obtained.
The “prophylactic cancer preventive or therapeutic agent” of the present invention may be an agent consisting only of this active ingredient, or may contain other components in addition to this active ingredient.
As the "other components", components that can maintain or improve the effect of the active ingredient of the present invention can be appropriately selected according to the purpose. For example, physiological saline for diluting the "double-stranded nucleic acid molecule" of the present invention to a desired concentration, a diluting agent such as a culture solution, or transfection for introducing (transfecting) into a target cell Reagent etc. are mentioned.
The content of these other components is also not particularly limited, and can be appropriately selected according to the purpose.

Moreover, as the "other components", for example, a pharmaceutically acceptable carrier can be included, and such a carrier is not particularly limited, and can be appropriately selected according to the dosage form etc. The amount can also be appropriately selected according to the purpose.
The pharmaceutical dosage form of the “prophylactic or prophylactic agent for prostate cancer” of the present invention is not particularly limited, and can be appropriately selected depending on the desired administration method. As such dosage forms, for example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquids (internal solutions, syrups, elixirs etc.), injections (solutions, suspensions) Solids for postscript dissolution, etc.), ointments, patches, gels, creams, external powders, sprays, inhaled powders and the like.
These agents include, according to the purpose, conventionally known excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, buffers, stabilizers, pH adjusters, etc. Additives such as a tonicity agent, a base, a wetting agent, a preservative, a cream, a gel, a paste and the like can be added, and each dosage form can also be produced by a conventionally known method.

There are no particular limitations on the target animal of administration of the "prophylactic or prophylactic agent for prostate cancer" of the present invention, and it can be appropriately selected according to the purpose. For example, humans, mice, rats, cattle, pigs, monkeys, dogs, cats and the like can be mentioned, and among these, it is particularly preferable to target humans.
Also, this administration method is not particularly limited, and it is systemic administration by local administration or oral administration etc. by directly administering the agent of the present invention to a tumor site etc. according to the dosage form, condition of prostate cancer patient etc. You can also.
The dose of this agent is not particularly limited, and can be appropriately selected according to the age, weight, desired effect, etc. of the patient to be administered. For example, when an adult is to be administered, the amount of the active ingredient (double-stranded nucleic acid molecule) per administration on a daily basis is preferably 10 to 15 mg.
Further, the number of times of administration of this agent is also not particularly limited, and can be appropriately selected according to the age, weight, desired effect and the like of the patient to be administered.
The “prophylactic or prophylactic agent for prostate cancer” of the present invention can appropriately select the administration time for the prophylaxis or treatment of prostate cancer.

Furthermore, the “method for determining the risk of prostate cancer in a subject animal” of the present invention refers to, for example, when a human being is used as a subject animal, prostate tissue derived from human, proteins extracted from the tissue, nucleic acid etc. It is a method of determining the risk of prostate cancer by qualitatively or quantitatively measuring ABHD2 protein or a fragment thereof or a nucleic acid encoding them as a chemical sample and contained in the sample.
The greater the amount of ABHD2 protein or a fragment thereof or the nucleic acid encoding them contained in a biological sample as described above, the higher the risk of suffering from prostate cancer or the risk of suffering from it. The case may be determined to be at high risk of developing treatment-resistant prostate cancer in the future.
In this determination, "ABHD2 protein or a fragment thereof, or a nucleic acid encoding them" can be used as a "determination marker" for prostate cancer.
When this "determination marker" is "ABHD2 protein or a fragment thereof", it can be measured qualitatively or quantitatively using an ABHD2 antibody or the like. In addition, when the “decision marker” is “a nucleic acid encoding these”, it is amplified by using a probe containing a nucleic acid complementary to this nucleic acid or a primer that can amplify this nucleic acid by RT-PCR or the like. It can be measured qualitatively or quantitatively.
From the results obtained by this "method for determining the risk of prostate cancer in a subject animal", it is also possible to select a method for treating prostate cancer.

In the present invention, among samples obtained from patients with inoperable advanced prostate cancer, it is confirmed that the sample with high recurrence rate of PSA after docetaxel introduction for hormone-resistant advanced prostate cancer, in particular, the staining intensity of ABHD2 is strong Using the sample as a biological sample and qualitatively and quantitatively examining the presence of the determination marker ABHD2 protein contained in the sample to predict the risk of future treatment-resistant prostate cancer Has been confirmed.
In addition, it is also possible to predict the risk of future treatment-resistant prostate cancer by qualitatively and quantitatively examining the mRNA of the ABHD2 gene obtained by the expression of the ABHD2 gene as a determination marker.
In the “method for determining the risk of prostate cancer in a subject animal” of the present invention, any animal can be used as the subject animal regardless of its type as long as it can be prostate cancer. In addition, any conventionally known device, method or the like can be used to qualitatively or quantitatively examine the determination marker.

Hereinafter, the present invention will be described in more detail by way of examples of the present invention and comparative examples, but the present invention is not limited to these.

Example 1
1-1. Immunostaining using clinical specimens and evaluation of association between ABHD2 gene expression and cancer-specific survival rate in prostate cancer patients In order to verify the expression of ABHD2 gene in prostate cancer tissue, 102 radical prostatectomy were performed Immunostaining was performed using the obtained prostate cancer sample and a prostate cancer sample obtained by 37 cases of transrectal prostate biopsy.
The prostate cancer specimens obtained by transrectal prostate biopsy are those obtained from patients with inoperable advanced prostate cancer, and in all these patients docetaxel has been introduced after initial hormonal therapy And later progressed to hormone resistance (CRPC) and has been given docetaxel chemotherapy.

Paraffin sections of 5 mm slices obtained from each sample were deparaffinized and heat-treated in a microwave oven to activate the antigen.
Perform endogenous peroxidase removal with 0.3% H ₂ O ₂ and react overnight with ABHD antibody (Abcam; 1: 200 dilution) used in Western blot, and react with secondary antibody (biotinylated antibody) for 1 hour And visualized using 3,30-diaminobenzidine 209 (DAB) solution (1 mM DAB, 50 mM Tris-HCl buffer pH 7.6210, and 0.006% H ₂ O ₂ ).
For quantitative evaluation of ABHD2 gene expression, expression intensity and the ratio of expression were scored. That is, the staining intensity was classified into three levels, and the ratio of expression in one field of view was classified into four levels and added.

From the results of the above-mentioned quantitative evaluation, the relationship between the expression of the ABHD2 gene and the cancer specific survival rate was evaluated in 102 prostatectomy cases.
In addition, in 37 cases obtained by transrectal prostate biopsy, the association between the expression of the ABHD2 gene and the PSA recurrence rate after docetaxel introduction was evaluated. This evaluation was performed by the Kaplan-Meier method and Log-Rank test, respectively. In addition, the association with the main patient background factors was evaluated.

1-2. Results FIG. 1 (A) shows the result of evaluating the relationship between the expression of ABHD2 gene in prostate cancer tissue and cancer specific survival rate in 102 prostatectomy cases. Among the 102 cases, the group with strong expression of the ABHD2 gene (36 cases) had a significantly lower cancer-specific survival rate after radical prostatectomy compared with the group with weak expression of the ABHD2 gene (66 cases).
FIG. 1 (B) shows the result of evaluating the relationship between the expression of ABHD2 gene in prostate cancer tissue and the PSA recurrence rate after docetaxel introduction in 37 cases obtained by transrectal prostate biopsy. . Among 37 cases, the group with strong expression of ABHD2 gene (27 cases) has significantly higher PSA recurrence rate after docetaxel introduction for hormone-resistant advanced prostate cancer compared to group with weak expression of ABHD2 gene (10 cases) The

Also, FIG. 1 (C) shows the results of immunostaining in a prostate cancer sample obtained by radical prostatectomy, and FIG. 1 (D) shows an immunity in a prostate cancer sample obtained by transrectal prostate biopsy. The results of staining were shown.
Among these immunostaining results, among the samples obtained from the patient with inoperable advanced prostate cancer on the left in FIG. 1 (D), the sample with a high PSA recurrence rate after docetaxel introduction to hormone-resistant advanced prostate cancer In particular, it was confirmed that the staining intensity of ABHD2 is strong.
Therefore, based on these results, the risk of developing future treatment-resistant prostate cancer by qualitatively and quantitatively examining ABHD2 protein in prostate cancer tissue collected for the purpose of diagnosis of prostate cancer by immunostaining, Western blot, etc. It is suggested that you can predict

In addition, as shown in Tables 1 and 2, in patients with total prostatectomy (102 patients), expression of the ABHD2 gene was significantly correlated with the Gleason Score, which was recognized to be a significant prognostic factor in multivariate analysis. It was done.
Furthermore, as shown in Tables 3 and 4, although inoperable advanced prostate cancer cases do not show any correlation with clinical stage, they are considered to be significant docetaxel resistance factors in multivariate analysis. It was done.
Therefore, these results suggest that the expression of the ABHD2 gene is involved in docetaxel treatment resistance in addition to the progression of prostate cancer.

Example 2
Preparation of Double-Stranded Nucleic Acid Molecule (siRNA) A double-stranded nucleic acid molecule (siRNA) for suppressing the expression of the ABHD2 gene was prepared as follows.
The siRNAs (siABHD2) # 1-7 against the ABHD2 gene have the nucleotide sequences (Table 6, sense strand, SEQ ID NOs 9, 11, 13) corresponding to the target sequences of the respective siRNAs (Table 5, SEQ ID NOs: 2-8). , 15, 17, 19, 21) and their complementary nucleotide sequences (Table 6, antisense strand, SEQ ID NO: 10, 12, 14, 16, 18, 20, 22) It was synthesized as a double-stranded RNA that would hang (RNAi).
In addition, as a negative control, 20 μM negative control (Invitrogen negative control high GC duplex, # 1151484) was used.

[Example 3]
Examination of the ABHD2 gene expression suppression effect by two nucleic acid molecules 2-1. Cell Culture AR positive human prostate cancer cell line LNCaP cells (hereinafter simply referred to as LNCaP cells) were used.
LNCaP cells are 5% in air using RPMI-1640 medium (Sigma) containing 10% fetal bovine serum (FBS, Sigma), 100 μg / ml streptomycin, 100 U / ml penicillin (Invitrogen) as a cell culture medium The culture was carried out at 37.degree. C. in an incubator containing carbon dioxide gas.

2-2. Transfection LNCaP cells cultured in the above were seeded at 3 × 10 ⁵ cells / well in a 6-well plate. Then, within 24 hours, 50 μM siABHD2 # 1 to # 7 (seven types) prepared in Example 2 or 20 μM siRNA as a negative control was prepared to 10 nM each and added to each well for transfection went.
Within 72 hours after transfection, total RNA was recovered from cells by ISOGEN (Nippon Gene), and cDNA was synthesized from 500 ng of the RNA by Primescript (registered trademark) RT reagent kit (TaKaRa Bio).

2-3. Quantitative Real-time PCR method Above 2. The cDNA synthesized in was diluted 10-fold and 2 μl of it was used for quantitative real-time PCR. The internal control was corrected using GAPDH and analysis of expression level was performed.
ABHD2 gene and internal control GAPDH gene of LNCaP cells transfected with each Si RNA using Step one (R) real-time PCR (Applied biosystem) and KAPA SYBR (R) Fast PCR kit (NIPPON Genetics) Expression levels were measured.
The expression level for the GAPDH gene was calculated from Cycle number using the ΔΔCt method, and correction was performed to examine the expression level of the ABHD2 gene.

2-4. Results As shown in FIG. 2, in the cells transfected with siABHD2 # 1 to # 7, the expression of ABHD2 gene was knocked down at the mRNA level compared to Reagent (blank) and negative control, and siABHD2 # It was confirmed that high effects were obtained when any of 1 to # 7 was transfected.

Example 4
Examination of the growth inhibitory effect of LNCaP cells by two nucleic acid molecules 3-1. MTS assay
2-1 of Example 3 And LNCaP cells cultured in the same manner as above were seeded at 3 × 10 ³ cells / well in a 96-well plate. Then, within 24 hours, each siRNA prepared in Example 2 and siRNA of the negative control were subjected to 2-2. The transfection was carried out by adding each well to the same concentration as above.
Then, after 1 day, 4 days, 5 days, or 6 days, Cell titer 96 (registered trademark) (Promega) was added and allowed to react for 2 hours. Thereafter, the number of living cells was measured at an absorbance of 490 nm with a microplate reader to examine the cell proliferation ability.

3-2. Results As shown in FIG. 3, in cells transfected with siABHD2 # 2, # 3, # 4, # 5, # 7, all cells were significantly more effective than Reagent (blank) or negative control. Growth was suppressed. In particular, in cells transfected with siABHD2 # 5 and # 7, it was observed that the proliferation after 4 days was strongly suppressed.

[Example 5]
Examination of LNCaP cell proliferation suppression effect by two nucleic acid molecules in combination with docetaxel 4-1. MTS assay
2-1 of Example 3 And LNCaP cells cultured in the same manner as above were seeded at 3 × 10 ³ cells / well in a 96-well plate. Then, within 24 hours, each siRNA # 1 or # 7 prepared in Example 2 and the siRNA of the negative control were subjected to 2-2. The transfection was carried out by adding each well to the same concentration as above.
About 12 hours after transfection, docetaxel (Docetaxel (Sigma-Aldrich)) is added to each well to be 1 nM or 10 nM, and after 1 day, 2 days or 4 days, Cell titer 96 (Promega) (Promega) ) Was added and allowed to react for 1 hour. Thereafter, the number of living cells was measured at an absorbance of 490 nm with a microplate reader to measure the cell proliferation ability.

4-2. Results FIG. 4 shows the results when 1 nM of docetaxel was added. As shown in FIG. 4, in cells transfected with siABHD2 # 1 and # 7, it was observed that the combined use of docetaxel significantly suppressed the cell growth compared to the case without combined use with docetaxel. The

[Example 6]
Inhibitory effect of LNCaP cell migration ability by dual nucleic acid molecule 5-1. Evaluation of Cell Migration Ability Among the siRNAs prepared in Example 2, cells into which siABHD2 # 1 or # 7 were introduced, or cells into which negative control siRNA (Invitrogen negative control high GC duplex, # 1151484) was introduced were cultured. Then, the migration ability of each cell was examined by Cell migration assay using Cell Cultuer Insert and 8.0 μm pore size PET filter (manufactured by Becton Dickinson).
That is, fibronectin (manufactured by Sigma) diluted to 10 μg / ml with PBS was allowed to act on culture dishes for 30 minutes to prepare a lower layer filter, and 700 μl of phenol red-containing medium RPMI 1640 medium was added to the lower layer chamber.
LNCaP cells cultured for 3 days in phenol red-containing medium supplemented with 5 μM of each siRNA were divided into 5 × 10 4 cells, and each cell was suspended in 300 μl of phenol red-containing medium and added to the upper chamber. The The culture was incubated at 37 ° C., 5% CO 2 for 24 hours, and the filter was removed.
The cells on the lower layer filter were fixed with methanol for 30 minutes, washed with PBS, and incubated for 30 seconds with Gimsa's stain solution (Muto Pure Chemicals). Thereafter, the cells were observed under a microscope at 200 magnification, and the cell migration ability was evaluated by counting the number of cells.

5-2. Results FIG. 5 shows the evaluation results of cell migration ability. As a result, it was found that introduction of the siRNA of the present invention, such as siABHD2 # 1 or # 7, significantly suppressed cell migration as compared to the case where a negative control siRNA was introduced.

[Example 7]
Examination of tumor growth inhibitory effect by two nucleic acid molecules 6-1. Subcutaneous implantation of cancer cells into nude mice Male 8-week-old BALB / c nude mice (CLEA Japan, Inc.) (20 mice) were used.
Per nude mouse, 2-1. Were mixed with 100 μl of PBS so that 1 × 10 ⁷ LNCaP cells cultured in the same manner as above were mixed with 100 μl of Matrigel (BD bioscience), and injected subcutaneously into each mouse using a 25 G injection needle.

6-2. Administration of Double-Stranded Nucleic Acid Molecule Above 6-1. 5 to 8 weeks after subcutaneous injection, and administration of double stranded nucleic acid molecules was performed when the tumor volume in each mouse reached a size of more than 100 mm ³ .
The double stranded nucleic acid molecule used siABHD2 # 7 among the produced in Example 2, and BannoNegaCon (RNAi, Inc.) (5'-GUACCGCACGUCAUUCGUAUC-3 ') was used as a negative control.
Each 5 μg of siRNA or negative control was mixed with 15 μl of RNAi MAX (Invitrogen) and phenol red-free OPTI-MEM to obtain each siRNA solution. 100 μl of this siRNA solution was administered by local injection into the tumor of each mouse. This administration was repeated twice / week for 4 weeks, and the tumor size of the mice was measured once a week.
The tumor diameter of the mouse measured the major axis (r1) and the minor axis (r2, r3) at two points, and the tumor size was measured using the formula r1 × r2 × r3 / 2. Five weeks after the start of administration, the patient was anaesthetized with Avatin and photographed to confirm the size of the tumor.
Furthermore, 8 weeks after transplantation of LNCaP cells, tumors were excised subcutaneously. The tumor was weighed, and partially RNA was removed by ISOGEN, and partially lysed by NP40 lysis buffer to extract proteins. For protein extraction, divide the tumor into 5 to 10 mm ³ size, add 500 μl NP40 lysis buffer, grind with a homogenizer, centrifuge the solution at 15,000 rpm, 4 ° C, 30 minutes, and collect the supernatant I did it by doing.

6-3. Western blot method 4-2. Then, proteins extracted from mice subcutaneously were electrophoresed on an 8% SDS-PAGE gel and blotted to Immobilon (registered trademark) -P Transfer Membrane (Millipore Corp., Billerica, MA).
The reaction was performed overnight using anti-ABHD2 (Abcam, 1: 500) as a primary antibody, and the peroxidase-conjugated anti-rabbit IgG antibody was reacted for 1 hour. Moreover, it was made to react overnight using anti- (beta) -actin (Sigma, 1: 1000), and the anti-mouse IgG antibody which couple | bonded with peroxidase was made to react for 1 hour.
The antigen-antibody complex was reacted with an ImmunoCruz (registered trademark) Western blotting detector system (Santa Cruz Biotechnology, Inc) and photographed on an X-ray film.

6-4. Result The result of photography was shown in FIG.
Also, the change in tumor size after siRNA or negative control administration is shown in FIG. As shown in FIG. 7, it was confirmed that administration of siABHD2 of the present invention can significantly suppress tumor growth.
Furthermore, the analysis results of the amount of ABHD2 protein by Western blotting are shown in FIG. As shown in FIG. 8, it could be confirmed that the intratumoral administration of siRNA suppressed the expression level of ABHD2 gene.

  Provision of the determination marker of the present invention makes it possible to provide a novel diagnosis and treatment selection method for prostate cancer. In addition, provision of a double-stranded nucleic acid molecule such as siRNA targeting the ABHD2 gene of the present invention makes it possible to provide new therapeutic agents, therapeutic methods and the like useful in the treatment of prostate cancer. In addition, by combining the double-stranded nucleic acid molecule of the present invention with PI polyamide or the like that is effective for treatment of prostate cancer, comprehensive treatment of prostate cancer can be achieved.

Claims (6)

An agent for the prophylaxis or treatment of prostate cancer, which comprises as an active ingredient a double-stranded nucleic acid molecule comprising the following (a) and (b) for suppressing the expression of the ABHD2 gene:
(A) a sense strand comprising a nucleotide sequence corresponding to the target sequence shown in any of SEQ ID NOs: 2 to 8 shown in SEQ ID NOs: 9, 11, 13, 15, 17, 19 or 21 (b) above (a) An antisense strand comprising a nucleotide sequence complementary to the sense strand of The agent for preventing or treating prostate cancer according to claim 1, wherein the 3 'end of each strand of the double-stranded nucleic acid molecule is a protruding end with an overhang of 2 to 6 bases. The agent for preventing or treating prostate cancer according to claim 1 or 2, wherein the length of the double stranded nucleic acid molecule is at least one of 23 bases to 29 bases. The agent for preventing or treating prostate cancer according to any one of claims 1 to 3, wherein the double stranded nucleic acid molecule is a double stranded RNA. The agent for preventing or treating prostate cancer according to claim 4, wherein the double stranded nucleic acid molecule is siRNA. The agent for preventing or treating prostate cancer according to any one of claims 1 to 5, which is for suppressing the growth of prostate cancer cells.