JP2009095343A - Method for detecting prostatic cancer and method for judging possibility of postoperative recurrence of prostatic cancer and therapeutic and/or prophylactic agent for prostatic cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly reliable method for detecting prostatic cancer and method for judging possibility of postoperative recurrence of prostatic cancer and to provide a new molecular target treatment and prophylactic method substantially different in mechanism from the conventionally known therapeutic agent for prostatic cancer. <P>SOLUTION: The method for detecting prostatic cancer and method for judging possibility of postoperative recurrence of prostatic cancer includes measurement of TFG gene expression in the prostate cell. The therapeutic and/or prophylactic agent for prostatic cancer includes an inhibitor for inhibiting expression of TGF gene as active ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting prostate cancer, a method for determining the possibility of postoperative recurrence of prostate cancer, and an agent for treating and / or preventing prostate cancer.

  Prostate cancer is the number one illness in male cancer prevalence in the United States. In Japan, it has been increasing due to changes in lifestyle habits such as westernization of eating habits. Prostate cancer has no specific symptoms in the early stages of development and is often associated with benign prostatic hyperplasia, which is similar to benign prostatic hyperplasia, and may have difficulty urinating, nocturia, urgency, etc. Therefore, even though there are no particular symptoms recently, it is almost always found by medical examination and measurement of PSA prostate specific antigen in blood by human dogs. Rarely, it is found in back pain from bone metastases in the form of advanced cancer. The stage before surgery is important as a factor that determines the prognosis of prostate cancer.

Stage A: If it is discovered by chance during surgery for benign prostatic hyperplasia or bladder cancer
Stage B: When cancer remains in the prostate
Stage C: When there is no metastasis to other organs (bones, lymph nodes, lungs, liver, etc.), but the cancer has gone beyond the capsule of the prostate (the surrounding membrane)
Stage D: When metastasized to other organs such as bones and lymph nodes

  For stage A-C, radical prostatectomy is basically performed, but there are a considerable number of cases of recurrence after surgery. In particular, local recurrence hormone therapy resistance and bone metastasis after surgery are important factors that worsen the prognosis of life. Predicting these at an early stage is clinically appropriate for patients' choice of treatment and follow-up. It has a big meaning. However, there are currently no molecular markers that predict prostate cancer prognosis at an early stage.

Conventionally, the prognosis or severity determination of prostate cancer, blood PSA (prostate specific antigen, p rostate- s pecific a ntigen) weight and prostate cancer tissues scoring (Gleason score) is used (Non-patent Document 1) However, the reliability is not necessarily high.

  As a prostate cancer therapeutic drug, for example, docetaxel belonging to taxanes is known (Non-patent Document 2).

  On the other hand, the association between the TFG gene, which is a marker for the prostate cancer detection method of the present invention described later, and anaplastic large cell lymphoma, thyroid papillary carcinoma or extraskeletal mucinous chondroma has been reported (Non-patent Document 3). ~ 7). However, no association between TFG gene and prostate cancer has been reported.

A.W.Partin et al., The Journal of the Americal Medical Association, Vol. 277 No. 18, May 14, 1997 Picus J et al., Semin Oncol. 1999 Oct; 26 (5 Suppl 17): 14-8 The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.Greco A, Mariani C, Miranda C, Lupas A, Pagliardini S, Pomati M, Pierotti MA., Mol Cell Biol 1995; 15: 6118-6127. Characterization and chromosomal mapping of the human TFG gene involved in thyroid carcinoma.Mencinger M, Panagopoulos I, Andreasson P, Lassen C, Mitelman F, Aman P., Genomics 1997; 41: 327-331. TRK-fused gene (TFG) is a new partner of ALK in anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.Hernandez L, Pinyol M, Hernandez S, Bea S, Pulford K, Rosenwald A, Lamant L, Falini B , Ott G, Masson DY, Delsol G, Campo E., Blood 1999; 94: 3265-3268 Diversity of genomic breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a new TFG-ALK (XL) chimeric gene with transforming activity.Hernandez L, Bea S, Bellosillo B, Pinyol M, Falini B, Carbone A, Ott G, Rosenwald A, Fernandez A, Pulford K, Mason D, Morris SW, Santos E, Campo E., Am J Pathol 2002; 160: 1487-1494. TFG is a novel fusion partner of NOR1 in extraskeletal myxoid chondrosarcoma. Hisaoka M, Ishida T, Imamura T, Hashimoto H., Genes Chromosomes Cancer 2004; 40: 325-328.

  An object of the present invention is to provide a highly reliable method for detecting prostate cancer and a method for determining the possibility of postoperative recurrence of prostate cancer. Another object of the present invention is to provide a novel molecular targeted treatment and prevention method for prostate cancer, which is completely different in mechanism from known prostate cancer therapeutic agents.

  The present inventors have undergone phosphorylation modification specifically for prostate cancer and bind to Pin1 by a phosphorylation proteome analysis method using peptidylprolyl cis-trans isomerase (Pin1), which is a phosphorylation site-binding enzyme, as a molecular probe. TFG was identified as a protein. TFG was found to have higher mRNA expression in prostate cancer than in non-cancerous areas, and its expression level was highly correlated with the recurrence rate after surgery. In addition, it was clarified that immunostaining with an antibody against TFG can distinguish a non-cancerous part from a prostate cancer part. Moreover, in the prostate cancer cell line PC3 cells, when siRNA was used to suppress the expression of TFG, growth inhibition, morphological changes, and induction of cellular aging were observed. As a result of newly obtaining these findings, the present invention has been achieved.

  That is, the present invention provides a method for detecting prostate cancer, which is a method performed on a specimen isolated from a living body, which comprises measuring the expression of the TFG gene in prostate cells. The present invention also provides a method for determining the possibility of postoperative recurrence of prostate cancer, comprising measuring a TFG gene expression in prostate cells, which is performed on a specimen isolated from a living body. . Furthermore, the present invention relates to a prostate cancer comprising a polynucleotide that specifically hybridizes with mRNA or cDNA of a TFG gene, or an antibody or antigen-binding fragment thereof that antigen-antibody reacts with a TFG gene product (simply referred to as “TFG”). A detection reagent is provided. Furthermore, the present invention provides a reagent for determining the possibility of postoperative recurrence of prostate cancer, comprising a polynucleotide that specifically hybridizes to mRNA or cDNA of TFG gene, or an antibody or antigen-binding fragment thereof that reacts with TFG for antigen-antibody reaction I will provide a. Furthermore, the present invention provides a therapeutic and / or prophylactic agent for prostate cancer containing an inhibitor that suppresses the expression of the TFG gene as an active ingredient. Furthermore, the present invention comprises a step of contacting an isolated prostate cancer cell with a candidate substance, a step of measuring the expression of the TFG gene in the cell, and reducing the expression of the TFG gene from the candidate substance. Provided is a method for screening a prostatic cancer therapeutic and / or prophylactic agent comprising the step of selecting a candidate substance.

  According to the present invention, a novel method for detecting prostate cancer using a gene specifically expressed in prostate cancer as a marker and a method for determining the possibility of recurrence of prostate cancer after surgery have been provided. As specifically described in the Examples below, TFG gene expression is significantly higher in prostate cancer cells compared to normal prostate cells. Therefore, prostate cancer can be detected with high reliability by the method of the present invention. Further, as described above, there was no method for determining the possibility of postoperative recurrence of prostate cancer, but the present invention provided for the first time a method for determining the possibility of postoperative recurrence of prostate cancer. Furthermore, as specifically described in the following examples, when the expression of the TFG gene is suppressed by siRNA, proliferation of prostate cancer cells is suppressed and aging is promoted. A new therapeutic and / or prophylactic agent for prostate cancer has been provided by molecular targeting, which has a mechanism completely different from that of drugs.

  Although details are described in the following Examples, it was found as follows that it is possible to detect prostate cancer and determine the possibility of recurrence of prostate cancer after the operation using TFG gene expression as an index.

  Soluble protein was extracted from prostate cancer cell lines and normal prostate tissue. Among these soluble proteins, a protein that specifically binds to the phosphorylation site-binding enzyme peptidylprolyl cis-trans isomerase (Pin1) was proteome-analyzed. That is, a recombinant fusion protein (GST-Pin1) of Pin1 and glutathione-S-transferase (GST) is immobilized on a glutathione-agarose gel column, and the soluble protein that binds to Pin1 is passed through this column. Protein was captured. Soluble protein bound to the column is excised from the column by thrombin digestion, subjected to one-dimensional SDS-PAGE, and after CBB staining, when prostate cancer cells are used as samples, they are separated as proteins that bind to Pin1, but normal prostate cells Found a protein that does not exist in. When this protein was digested in a gel and analyzed by ESI-Q-TOF-MS, it was identified as TFG.

  Therefore, when the amount of TFG mRNA in prostate cancer cell lines and normal prostate epithelial cells was measured by quantitative RT-PCR, the amount of TFG mRNA was significantly higher in prostate cancer cell lines than in normal prostate cells. It was. Moreover, when the amount of mRNA of the TFG gene was measured in the cancerous part and non-cancerous part of the prostate gland extracted from the same prostate cancer patient, the amount of TFG gene mRNA in the cancerous part was significantly high. Thus, it was revealed that prostate cancer can be detected using TFG gene expression as an index.

  Further, when an antibody against TFG was prepared and immunostaining was performed using about 20 prostate cancer tissues, no staining was observed in the non-cancerous part, but strong staining was observed in the cancerous part. Thus, the expression of the TFG gene can be examined by immunoassay using an antibody against TFG, and it has been clarified that prostate cancer can be detected by measuring TFG as well as measuring mRNA.

  Furthermore, in order to investigate whether TFG gene expression is an indicator of prostate cancer malignancy, among patients who underwent total prostatectomy, groups with high and low TFG gene expression (between the intermediate values) And the presence or absence of recurrence was examined. As a result, prostate cancer with high TFG expression was found to have a significantly higher (P <0.05) recurrence rate after surgery than prostate cancer with low expression. This revealed that the possibility of recurrence of prostate cancer after surgery can be determined using the expression of the TFG gene as an index.

  Furthermore, when an siRNA that specifically cleaves the mRNA of the TFG gene was administered to a prostate cancer cell line, the proliferation of the prostate cancer cells was suppressed and the aging of the prostate cancer cells was promoted. Thereby, it was shown that the inhibitor which suppresses the expression of TFG gene is effective as a treatment and / or prevention agent for prostate cancer.

  As described above, the method for detecting prostate cancer and the method for determining the possibility of postoperative recurrence of prostate cancer of the present invention include measuring the expression of the TFG gene in a specimen separated from a living body. The specimen is not particularly limited as long as it can contain tumor cells derived from the prostate, and examples thereof include blood, urine, cellular tissues such as prostate collected by surgery, biopsy, and the like.

  The TFG gene itself is known, its base sequence and amino acid sequence encoded by it are also known, and are registered as GenBank Accession No. BT007428. SEQ ID NOs: 1 and 2 show the base sequence of the TFG gene and the amino acid sequence encoded by it, which are registered as GenBank Accession No. BT007428.

  In the present invention, the term “measurement” is used in a sense encompassing all of detection, quantification, and semi-quantification. Therefore, “measuring the expression of the TFG gene” includes both the detection of the expression of the TFG gene and the measurement of the expression level, and further whether or not the expression level is a predetermined value or more. In the case of determination, in other words, the case where the expression is detected when the expression level is a predetermined value or more is also included.

  Measurement of TFG gene expression can be performed by measuring mRNA of the TFG gene or by measuring the gene product of the TFG gene. Since the TFG gene has a known base sequence and an amino acid sequence encoded by the TFG gene, its expression can be easily measured by various known methods.

  Measurement of TFG gene mRNA can be performed, for example, by quantitative RT-PCR (quantitative reverse transcription PCR). RT-PCR is a method of synthesizing cDNA using the mRNA to be measured as a template and amplifying it by PCR using this cDNA as a template. Quantitative RT-PCR is performed by, for example, performing PCR using a primer combined with a quencher fluorescent dye and a reporter fluorescent dye. For example, a method for measuring the amount of template DNA in a sample (real-time PCR) based on the number of cycles increasing rapidly. The quantitative RT-PCR method itself is well known in this field, and kits for this purpose are also commercially available, so that those skilled in the art can easily carry out if the nucleotide sequence of the mRNA to be measured is known. In addition, TFG gene mRNA can also be measured semi-quantitatively by performing normal RT-PCR or NASBA amplification of mRNA, subjecting the amplified product to gel electrophoresis, staining, and measuring band intensity. Can be done. Furthermore, TFG gene mRNA can also be measured by a method of detecting or semi-quantifying TFG gene mRNA or cDNA using a DNA chip.

  TFG gene expression can also be measured by measuring TFG. In this case, examples of the sample used for the immunoassay include proteins extracted from prostate cells by a conventional method. As described above, the TFG gene cDNA can be easily amplified by RT-PCR, so that TFG can be prepared by a well-known genetic engineering technique using the amplified cDNA. An anti-TFG antibody can be obtained by immunizing an animal using the obtained TFG as an immunogen and collecting the antibody from the obtained antiserum. TFG in a specimen can be measured by such an immunoassay using an anti-TFG antibody. The antibody recovered from the antiserum is a polyclonal antibody, and includes antibodies against all the exposed epitopes of TFG, and thus binds to TFG reliably. On the other hand, monoclonal antibodies can also be used to increase the reproducibility of the immunoassay. A method for producing a monoclonal antibody has already been established, and can be produced according to a conventional method by the method of Kohler and Milstein. Since TFG used as an immunogen can be prepared by genetic engineering techniques, a sufficient amount of purified immunogen can be obtained, so monoclonal antibodies that react with TFG for antigen-antibody can be easily prepared according to conventional methods. Can be prepared.

  Immunoassays themselves are well known in this field, and classified by reaction mode include sandwich method, competition method, agglutination method, Western blot method and the like. Further, when classified by label, there are radioimmunoassay, fluorescent immunoassay, enzyme immunoassay, biotin immunoassay and the like, and TFG immunoassay can be performed using any method. Although not particularly limited, the sandwich ELISA and the agglutination method can be preferably applied as an immunoassay method for TFG in the method of the present invention because the operation is simple and does not require a large-scale apparatus.

These immunoassays are well known and need not be described in the present specification, but briefly described, for example, in the sandwich method, an anti-TFG antibody (which may be a polyclonal antibody or a monoclonal antibody) is immobilized on a solid phase. And then react with the sample, wash, react with a labeled second anti-TFG antibody (either polyclonal or monoclonal), wash, and then measure the labeled second antibody bound to the solid phase To do. When both the immobilized antibody and the labeled antibody are monoclonal antibodies, those that can bind to TFG at the same time are selected and used. In place of the antibody, an antigen-binding fragment of the antibody can also be used. Here, the “antigen-binding fragment” means an antibody fragment having the ability to bind to an antigen, such as a Fab fragment or F (ab ′) ₂ fragment contained in an antibody molecule.

  As specifically described in the following examples, in the prostate cancer cells, the expression level of the TFG gene is higher than the expression level in the normal prostate cells. Based on this, prostate cancer can be detected. For example, the expression level of the TFG gene in normal prostate cells derived from a number of healthy individuals is measured in advance, and the expression level of the TFG gene in prostate cells in the specimen is determined from the average value of the expression levels in normal prostate cells. If it is statistically significant, it can be judged that the possibility of developing prostate cancer is high, and thereby prostate cancer can be detected. Alternatively, when a normal part and a tumorous part are observed in the prostate tissue of the patient who collected the specimen, the TFG amount in the cells collected from the normal part can be used as a reference. In the latter case, it is useful, for example, when the therapeutic effect of an anticancer drug or surgery is followed over time.

  Similarly, as specifically described in the following examples, when the expression level of the TFG gene in prostate cells is large, the possibility of recurrence of prostate cancer after surgery increases. The possibility of postoperative recurrence of prostate cancer can be determined based on the expression of the TFG gene. In this case, measure the amount of TFG expression in prostate cells for many patients who have undergone prostate cancer surgery, and determine the possibility of postoperative recurrence of prostate cancer based on whether it is higher or lower than the intermediate value (See examples below).

  The present invention also includes a polynucleotide that specifically hybridizes with mRNA or cDNA of a TFG gene, or an antibody or antigen-binding fragment thereof that antigen-antibody reacts with TFG, which is used in the method of the present invention. Also provided are reagents for detection or determination of the likelihood of postoperative recurrence of prostate cancer.

  The polynucleotide that specifically hybridizes with mRNA or cDNA of TFG gene (SEQ ID NO: 1) is a primer for amplifying the TFG gene mRNA or cDNA by a nucleic acid amplification method such as PCR or NASBA, or the mRNA or cDNA. It is used as a probe for specific detection. Here, “specifically hybridize” means that it hybridizes only with the target mRNA or cDNA under normal hybridization conditions and does not substantially hybridize with other nucleic acids. is there.

“Normal hybridization conditions” refer to the conditions used for normal PCR annealing and probe detection. For example, in the case of PCR using Taq polymerase, 50 mM KCl, 10 mM Tris- The reaction is performed using a general buffer solution such as HCl (pH 8.3 to 9.0) and 1.5 mM MgCl _{2 at} an appropriate annealing temperature of about 54 ° C. to 60 ° C. For example, in the case of Northern hybridization Is to perform the reaction at an appropriate hybridization temperature of about 42 ° C. to 65 ° C. using a general hybridization solution such as 5 × SSPE, 50% formamide, 5 × Denhardt's solution, 0.1 to 0.5% SDS. . However, the appropriate annealing temperature or hybridization temperature is not limited to the above examples, and is determined based on the Tm value of the polynucleotide for cancer detection used as a primer or probe and the rule of thumb of the experimenter. Can be determined. In addition, “substantially does not hybridize” means that it does not hybridize at all, or even if it is significantly less than the amount hybridized to the target partial region, and hybridizes only in a relatively negligible amount. It means that.

  Examples of the polynucleotide that specifically hybridizes under such conditions include a polynucleotide having a certain degree of homology with the base sequence of the partial region of the nucleic acid having the base sequence shown in SEQ ID NO: 1, for example, 90% As mentioned above, a polynucleotide having a homology of 95% or more, most preferably 100% is mentioned. Here, “homology” is a percentage expressed by dividing both base sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of bases is the number of bases obtained by counting one gap as one base. When the total number of bases counted in this way differs between the two sequences to be compared, the homology (%) is calculated by dividing the matching bases by the total number of bases of the longer sequence. . Even if the end of the polynucleotide includes a region that does not hybridize with the nucleic acid having the base sequence shown in SEQ ID NO: 1, in the case of a probe, the hybridized region occupies approximately half or more of the entire probe. Can be used for detection, and in the case of a primer, if the hybridizing region occupies approximately half or more of the entire primer and is on the 3 ′ end side, it is normally annealed to cause an extension reaction. So that it can be used for detection. As such, when a region that does not hybridize is included at the end of the polynucleotide for cancer detection, when calculating the homology with the target base sequence, the region that does not hybridize is not considered and hybridizes. It is assumed that the calculation is performed focusing on only the area. In the above description, “partial region” refers to a partial region in the base sequence shown in SEQ ID NO: 1, and is preferably a continuous region of 18 bases or more. In the present invention, the term “base sequence shown in SEQ ID NO: 1” includes the base sequence actually shown in SEQ ID NO: 1 as well as a complementary sequence thereto. Therefore, for example, when saying “polynucleotide having the base sequence shown in SEQ ID NO: 1”, a single-stranded polynucleotide having the base sequence actually shown in SEQ ID NO: 1, its complementary base sequence And double-stranded polynucleotides comprising these.

  The number of bases of the polynucleotide for cancer detection is preferably 18 bases or more from the viewpoint of ensuring specificity. When used as a probe, the size is preferably 18 bases or more, more preferably 20 bases or more, and preferably not more than the entire length of the coding region, and when used as a primer, it is preferably 18 bases or more, and preferably 50 bases or less. Preferable examples of the polynucleotide for cancer detection include a polynucleotide comprising 18 or more consecutive bases in the base sequence shown in SEQ ID NO: 1.

  The anti-TFG antibody or antigen-binding fragment thereof used for TFG immunoassay described above can also be used as a reagent for detecting prostate cancer or determining the possibility of postoperative recurrence of prostate cancer. The ability to detect a cancerous part in prostate tissue with an anti-TFG antibody is as specifically shown in the Examples below.

  As described above, when siRNA that specifically cleaves the mRNA of TFG gene was administered to the prostate cancer cell line, proliferation of prostate cancer cell was suppressed and aging of prostate cancer cell was promoted. An inhibitor that suppresses the expression of is effective as a therapeutic and / or prophylactic agent for prostate cancer. Examples of the inhibitor that suppresses the expression of the TFG gene include iRNA targeting the mRNA of the TFG gene, preferably siRNA. The iRNA is a double-stranded RNA containing a strand complementary to the target mRNA, and binds to and cuts the target mRNA. siRNA is a small iRNA having a size of about 21 to 23 bases. siRNA is preferable because it is small in size and easy to synthesize, and it is easy to set the cleavage site of mRNA. The technology for suppressing gene expression by siRNA is already well known, and as long as the mRNA sequence (cDNA sequence) is presented, a siRNA targeting it is designed and a recombinant vector in which the siRNA is incorporated into an expression vector is prepared. There are so many service providers. Therefore, since the cDNA sequence of the TFG gene is as described in SEQ ID NO: 1, siRNA for it can be easily set by those skilled in the art. Briefly, siRNAs are double-stranded RNAs that contain a complementary strand to the target mRNA and are usually 21-23 bases in size, usually with hangovers at both ends of the double-stranded RNA. . The size of the hangover is 1 to 2 bases each, and the hangover portion may be a deoxynucleotide. The complementarity with mRNA is preferably perfect complementarity, but often exhibits a sufficient cleavage action even if there is a mismatch of about 1 to 2 bases. Moreover, the hangover part does not need to be complementary. In many cases, siRNA is preferably set as 19 to 21 bases following aa in the base sequence of mRNA, and preferably has a gc content of about 50% (usually about 45 to 55%). Moreover, it is often set at a site separated by 50 bases or more from the 5 ′ end so as not to be set at a portion cleaved by a mature protein.

  The siRNA targeting the TFG gene mRNA has a target sequence in the region of 1 nt to 1200 nt in the base sequence described in SEQ ID NO: 1. Here, “having a target sequence in the region” means that there is a sequence in the region that is identical to one strand of siRNA double strands or homologous to the extent that mRNA can be cleaved. (However, thymine t and uracil u are considered the same base). Since the untranslated region of mRNA may not have a large sequence difference between genes, in order to cleave the mRNA of the TFG gene more specifically, the target sequence is within the coding region in SEQ ID NO: 1, ie, It preferably exists in the region of 1nt to 1200nt.

  The siRNA can be administered as it is, but the siRNA is produced in the cell by incorporating the siRNA-expressing DNA into an expression vector for mammalian cells and administering the resulting recombinant vector to express the TFG gene. May be suppressed. Various expression vectors for mammalian cells are commercially available, and the above DNA can be inserted into the multiple cloning site. In addition, as described above, a service of a manufacturer that produces an expression vector incorporating a DNA that expresses siRNA can also be used.

  Examples of inhibitors other than iRNA include antisense RNA and antibodies against TFG.

  The dose is appropriately selected according to the degree of prostate cancer, the patient's condition, body weight, etc. When the suppressor is siRNA, the dose is usually 0.01 mg / kg to adult (60 kg body weight) per day. In the case of a recombinant vector expressing siRNA, about 10 mg / kg, especially 0.1 mg / kg to 5 mg / kg, about 0.01 mg / kg to 10 mg / kg, especially 0.1 mg / kg to 5 mg / day per adult throughout the treatment Of course, the dose is not limited to these.

  In addition, as described above, in prostate cancer cells in which the TFG gene was suppressed, proliferation was suppressed and aging was promoted. That is, a substance that can promote cancer, that is, a substance useful as an agent for treating and / or preventing prostate cancer can be screened. Therefore, the present invention also provides a novel screening method for agents for treating and / or preventing prostate cancer.

  The screening method of the present invention comprises a step of contacting an isolated prostate cancer cell with a candidate substance, a step of measuring the expression of the TFG gene in the cell, and reducing the expression of the TFG gene from the candidate substance. Selecting a candidate substance to be made. As described above, the expression of the TFG gene can be measured by measuring the mRNA of the TFG gene, or by measuring the gene product of the TFG gene. If the expression of the TFG gene is lower than that of non-contact prostate cancer cells due to contact with the candidate substance, it can be considered that the candidate substance suppresses the growth of prostate cancer cells. It may be useful as a therapeutic and / or prophylactic agent. The prostate cancer cell to be used is not particularly limited, and may be any known tumor cell line derived from prostate cancer.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Materials and methods
(1) Materials and specimens Human prostate cancer cell line PC3 (source: ATCC)
Human prostate cancer cell line LNCaP (source: ATCC)
Human prostate cancer cell line DU145 (Source: ATCC)
Normal human prostate epithelial cells (PrEC) (commercially available from ATCC)
Dunning R cells: Dunning R3327 system (AT1, AT2, AT3, MAT-Lu and MAT-Ly-Lu) (Thelma R. Tennant, Hyung Kim, Mitchell Sokoloff, and Carrie W. Rinker-Schaeffer. The Dunning Model. Prostate, 43: 295-302, 2000)
Patient surgical specimen with sufficient informed consent

(2) Purification of GST-Pin1 fusion protein
A known recombinant vector pGEX-KG-Pin1 (J. Biol. Chem., Vol. 277, Issue 25, 23054-23064, June 21, 2002) containing a region encoding the GST-Pin1 fusion protein was transformed into E. coli. Transformed into BL21 strain, induced protein in the presence of IPTG (0.5 mM), recovered using glutathione-conjugated agarose beads (Sigma), eluted with reduced glutathione-containing buffer, dialyzed, desalted and buffer exchanged Was done. GST used as a control was purified in the same manner as described above using pGEX-KG (J. Biol. Chem., Supra), which is a known recombinant vector containing a region encoding GST.

(3) Separation of Pin1-binding protein (GST pull-down assay)
GST pull-down assay was performed as follows for the soluble proteins present in the above-mentioned human prostate cancer cell lines PC3, LNCaP and normal human prostate epithelial cells. GST pull-down buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100 (registered trademark), 1.5 mM MgCl ₂ , 1 mM EGTA, 100 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM dithiothreitol, 0.5 μg / ml leupeptin, 1.0 μg / ml pepstatin, 0.2 mM phenylmethylsulfonyl fluoride) and GST-Pin1 or GST-bound 70 μL glutathione-conjugated agarose beads for 2 hours at 4 ° C. did. Next, proteins bound to the beads were separated by thrombin digestion. In addition, Pin1-binding proteins were similarly isolated from soluble proteins in multiple types of rat prostate cancer cell lines contained in the Dunning R3327 system containing multiple cell lines with different malignancy.

(4) Proteomics analysis
The Pin1 binding protein obtained by GST pull-down is developed on one-dimensional SDS-PAGE, CBB staining is performed, and the band that binds to Pin1 is recovered only in cancer cells. After digestion in gel, ESI-Q-TOF MS Proteins were identified by accurate mass spectrometry (Q-Tof micro (trade name), Micromass, Manchester, UK).

(5) TFG knockdown using siRNA
An siRNA expression system (OligoEngine) using a pSuper-retro-puro (trade name) vector was used.
TFG-siRNA sequence (double stranded region)
siRNA1: 5'-gtcaggtgaaatatctccg-3 '(SEQ ID NO: 3)
siRNA2: 5'-gtctgcttctgattcttct-3 '(SEQ ID NO: 4)
siRNA3: 5'-ggtcagatgtaccaacagt-3 '(SEQ ID NO: 5)
Control siRNA sequence
5'-tcgtatgttgtgtggaatt-3 '(SEQ ID NO: 6)
Each oligo DNA was cloned into siRNA expression vector pSUPER-puro (oligo engine), a retroviral vector was prepared using packaging cells (293T), and the target cells were infected.

  The time course of the number of cells of the human prostate cancer cell line introduced with siRNA was measured as follows. The number of cells was counted using a hemocytometer (3 well / sample).

(6) Quantitative RT-PCR method According to the clinical specimen experiment protocol approved by the Yokohama City University Ethics Committee, mRNA is extracted from patient surgical specimens with sufficient informed consent, and quantitative RT-PCR is performed. I did it. mRNA was extracted using ISOGEN reagent (Nippon Gene). The reverse transcription reaction was performed at 45 ° C. for 1 hour using oligo dT18 and SuperScript3 reverse transcriptase (Invitrogen). RT-PCR was ABI7700 (Applied Biosystem) real-time PCR. Primer set; (forward side: 5'-ggaacacaaaagaccaaaatgg-3 '(SEQ ID NO: 7), reverse side 5'-agggctctactttagtacatc-3' (SEQ ID NO: 8), reaction conditions: 95 ° C x 30 seconds, 64 ° C x 1 minute 40 cycles were repeated.

(7) Senescence-associated β-galactosidase staining
This was performed according to Cellular Senescence Assay Kit (Chemicon).

(8) Preparation of TFG antibody
A 15-amino acid peptide (QQPPYTGAQTQAGQC, SEQ ID NO: 9) in which cysteine was added to the carboxyl terminus of the amino acid sequence of TFG (14 amino acids from 225-glutamine to 238-glutamine) was chemically synthesized by a peptide synthesizer. The obtained peptide fragment was mixed with an incomplete Freund's adjuvant in an equal amount, and an emulsion was prepared by sonication. The emulsion with increased viscosity was subcutaneously administered to the back of a rabbit (Japanese white breed). All blood was collected 39 days after the first immunization. The collected blood was allowed to stand at room temperature for 1 hour, stored at 4 ° C. overnight, and centrifuged at 3000 rpm for 10 minutes to obtain serum.

The antibody was purified from the obtained antiserum using a commercially available antibody purification kit (Amersham Bio., HiTrap Protein A HP). The buffers used were as follows:
Binding buffer 20 mM sodium phosphate pH 7.0
Elution buffer 0.1M sodium citrate pH3.0
Neutralization buffer 1.0M Tris-HCl, pH9.0

The specific purification operation was performed as follows.
[1] Column preparation: Connect a syringe to prevent bubbles from entering the column, and deliver 25 ml of ultrapure water at a flow rate of 5 drops / second.
[2] Column equilibration: 25 ml of binding buffer was sent at a flow rate of 5 drops / second.
[3] Sample addition: Prepared sample (antiserum diluted solution, __- fold diluted with buffer) at a flow rate of 5 drops / 2 seconds to wash non-adsorbed components, 25 ml of binding buffer at a flow rate of 5 drops / second Liquid feeding.
[4] Antibody elution: Send 25 ml of elution buffer at a flow rate of 5 drops / second, collect 3 ml of eluate (add 300 μL of neutralization buffer to the collection tube), and measure the concentration. The absorbance at 280 nm of each eluted fraction was measured, and the antibody fraction was dialyzed overnight against recovered PBS (−) and buffer exchanged.

(9) Immunohistochemical staining Using a commercially available immunohistochemical staining kit (VECTASTAIN ABC Kit), immunohistochemical staining of prostate tissue isolated from a patient was performed with the TFG-specific antibody obtained in (8) above. As prostate tissue, prostate cancer surgical specimens (26 cases in total) removed at Yokohama City University Hospital and related hospitals were used. Specifically, immunohistochemical staining was performed as follows.

[1] Deparaffinized and hydrophilized tissue sections with xylene and ethanol.
[2] Wash 2 times [5 minutes each]: Wash with 100 ml each of Tris-buffered saline (TBS).
[3] Inactivation of endogenous peroxidase [1 hour]: 2 ml of 30% -H ₂ O ₂ (1%) in 60 ml of TBS.
[4] Wash twice with 100 ml each of TBS [5 minutes each].
[5] Blocking [1 hour]: goat serum (final concentration 10%) in TBS (3 ml).
[6] Wash once with 100 ml of TBST (TBS containing 0.1% Tween20 (trade name)) [5 minutes].
[7] A primary antibody (TFG-specific antibody prepared in (8) above) diluted with a blocking solution (50-200 times) is added to the sample and reacted at room temperature for 2 hours or overnight at 4 ° C.
[8] Wash twice with 100 ml each of TBS [5 minutes each].
[9] A secondary antibody (a solution obtained by adding 2 μL of a biotinylated second antibody (goat anti-rabbit IgG antibody; attached to the kit) to 500 μL of TBS) was added to the specimen and reacted for 2 hours.
[10] Wash twice with 100 ml each of TBS [5 minutes each].
[11] Prepare AB solution (A: avidin solution (C solution) and B: biotinylated peroxidase (D solution) added in two drops to 10 ml of TBS). Prepare 30 minutes before reaction.
[12] Add AB solution to the sample and react for 2 hours.
[13] Wash twice with 100 ml each of TBS [5 minutes each].
[14] Sample of DAB solution (containing 50 μg (10 μg / ml) diaminobenzidine (DAB), a substrate for peroxidase, and 5 μL (0.03%) H ₂ O _{2 in} TBS (5 ml)) In addition, react for 5-10 minutes in the dark. Check the color occasionally.
[15] Wash once with 100 ml of TBS [5 minutes].
[16] Stain the cell nucleus blue with hematoxylin reagent.
[17] Ethanol substitution, xylene, and mounting agent added dropwise, sealed with cover glass.

2. result
(1) Prostate cancer-specific Pin1 binding protein is TFG A plurality of phosphorylated proteins capable of binding to Prostate cancer-specific Pin1 were identified by the above proteome analysis using Pin1 as a molecular probe. One of them, TFG, binds to Pin1 in two prostate cell lines, PC3 and LNCaP cells, but not in human normal prostate cells. A similar experiment using rat prostate cancer cells with different malignancy revealed that TFG significantly bound to Pin1 in prostate cancer with high malignancy. This result indicates that TFG binds to Pin1 specifically for prostate cancer.

(2) TFG is highly expressed in prostate cancer cells Next, the expression level of TFG in prostate cancer tissues was examined by the quantitative RT-PCR. Three prostate cell lines, PC3, DU145 and LNCaP cells, showed 2 to 3 times higher TFG expression compared to normal prostate epithelial cells (PrEC) (FIG. 1). In addition, in order to observe TFG expression in prostate cancer tissue, cancerous and non-cancerous parts were excised from prostate cancer tissue of the same patient, mRNA was extracted, and quantitative RT-PCR analysis was performed. As a result, TFG was found to be significantly higher in the cancerous part than in the prostate non-cancerous part (FIG. 2). However, there was no significant correlation with prostate cancer differentiation, stage, and Gleason Score.

(3) TFG was tested for TFG suppression using prostate cancer cell line PC3, which shows male hormone-independent growth, which can be a therapeutic target for prostate cancer. In PC3 cells, siRNA was used to steadily suppress TFG expression (FIG. 3). As a result, in TFG-suppressed cells, the size of the cells increased with age and a marked decrease in cell proliferation was observed. In addition, aging-related β-galactosidase (SABG) staining (Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995; 92: 9363-9367.), SABG positive cells were significantly increased in the TFG inhibitory cell group (FIG. 4). Moreover, cell proliferation was significantly suppressed (FIG. 5). This indicates that TFG plays an important role in preventing aging of prostate cancer cells and maintaining cell proliferation.

(4) TFG can be a biomarker for recurrence after prostate cancer surgery
To investigate whether TFG expression is an indicator of prostate cancer malignancy, 28 patients with total prostatectomy were treated with high and low TFG expression in prostate cells ( Classification was made with the intermediate value as the boundary, and the presence or absence of recurrence was examined. As a result, it became clear that the recurrence rate after surgery was significantly higher in prostate cancer with high TFG expression than in prostate cancer with low expression (P <0.05) (FIG. 6). In FIG. 6, the horizontal axis indicates the number of days after surgery, the vertical axis indicates the survival rate without recurrence, and the broken line indicates the results for the group with high TFG expression and the low solid line.

(5) TFG antibody staining in prostate cancer tissue Using the TFG-specific antibody prepared as described above as the primary antibody, immunohistochemical staining of prostate cancer surgical specimens (26 cases in total) was performed. As a result, no staining was observed in the non-cancerous part (hyperplastic part) in the prostate cancer tissue, and the expression of TFG was not confirmed, but in the cancerous part, some of the cells forming the gland duct were strongly stained. A strong expression of TFG was confirmed. FIG. 7 shows a representative example of the 26 tissue specimens.

It is a figure which shows the amount of mRNA of the TFG gene in a prostate cancer cell and a normal prostate epithelial cell measured by quantitative RT-PCR. It is a figure which shows the mRNA amount of the TFG gene in the cancer part and non-cancer part of the prostate cancer tissue extract | collected from the same patient measured by quantitative RT-PCR. It is a figure which shows the amount of mRNA of the TFG gene in a cell when the vector which expresses siRNA which cut | disconnects mRNA of TFG gene or the vector which expresses control siRNA is introduce | transduced into human prostate cancer cell line PC3. When cells that are stained with aging-related β-galactosidase (SABG) are stained when siRNA is expressed by introducing a vector expressing siRNA that cleaves TFG mRNA or control siRNA into human prostate cancer cell line PC3 It is a figure which shows a ratio (%). The time-dependent change of the number of cells when siRNA is expressed by introducing a vector expressing siRNA that cleaves mRNA of TFG gene or control siRNA into human prostate cancer cell line PC3 is shown. It is a figure which shows the relationship between the number of days after an operation | movement, and the survival rate without a recurrence about the group where the wavy line of the patient case which performed total prostatectomy is a group with a high expression of TFG, and a solid line is low. A representative example of immunohistochemical staining of prostate cancer tissue with a TFG-specific antibody is shown. A: Stained image in prostate non-cancerous part (hyperplastic) (negative) (200x), B: Stained image in prostate cancer part (200x), C: Magnified image of B (1400x), D; Magnified image (2400 ×)

Claims (15)

  A method for detecting prostate cancer, comprising: measuring a TFG gene expression in prostate cells, the method being performed on a specimen isolated from a living body.   A method for determining the possibility of postoperative recurrence of prostate cancer, comprising a method performed on a specimen isolated from a living body, comprising measuring the expression of a TFG gene in prostate cells.   The method according to claim 1 or 2, wherein the expression of TFG gene is measured by measuring mRNA of TFG gene or TFG gene product.   The method according to claim 3, wherein the expression of TFG gene is measured by measuring mRNA of TFG gene.   The method according to claim 4, wherein the mRNA of the TFG gene is measured by quantitative RT-PCR.   The method according to claim 3, wherein the expression of the TFG gene is measured by measuring the TFG gene product.   The method according to claim 6, wherein the measurement of the TFG gene product is performed by immunoassay using an antibody or antigen-binding fragment thereof that reacts with the TFG gene product.   A detection reagent for prostate cancer comprising a polynucleotide that specifically hybridizes with mRNA or cDNA of TFG gene, or an antibody or antigen-binding fragment thereof that reacts with a TFG gene product by antigen-antibody reaction.   A reagent for determining the possibility of postoperative recurrence of prostate cancer, comprising a polynucleotide that specifically hybridizes with mRNA or cDNA of a TFG gene, or an antibody or antigen-binding fragment thereof that reacts with an antigen-antibody with a TFG gene product.   The reagent according to claim 8 or 9, comprising a polynucleotide that specifically hybridizes with mRNA or cDNA of TFG gene.   The reagent according to claim 10, wherein the polynucleotide is a primer or a probe.   The reagent according to claim 8 or 9, comprising an antibody or an antigen-binding fragment thereof that reacts with a TFG gene product by an antigen-antibody reaction.   A therapeutic and / or prophylactic agent for prostate cancer comprising an inhibitor that suppresses the expression of TFG gene as an active ingredient.   The agent for treating and / or preventing prostate cancer according to claim 13, wherein the inhibitor is an siRNA that cleaves mRNA of the TFG gene or an siRNA-containing vector that expresses the siRNA in a cell.   A step of contacting an isolated prostate cancer cell with a candidate substance; a step of measuring expression of a TFG gene in the cell; and a step of selecting a candidate substance that decreases TFG gene expression from the candidate substance A screening method for a therapeutic and / or prophylactic agent for prostate cancer.