WO2010123124A1 - Recurrence and prognosis factor for solid cancer, and use thereof for clinical purposes - Google Patents

Recurrence and prognosis factor for solid cancer, and use thereof for clinical purposes Download PDF

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WO2010123124A1
WO2010123124A1 PCT/JP2010/057298 JP2010057298W WO2010123124A1 WO 2010123124 A1 WO2010123124 A1 WO 2010123124A1 JP 2010057298 W JP2010057298 W JP 2010057298W WO 2010123124 A1 WO2010123124 A1 WO 2010123124A1
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pls3
test
cancer
expression level
cells
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PCT/JP2010/057298
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French (fr)
Japanese (ja)
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武彦 横堀
功士 三森
森　正樹
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国立大学法人九州大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is intended for patients with solid cancer, particularly colorectal cancer or breast cancer, factors relating to epithelial-mesenchymal transition (EMT) of cancer cells (hereinafter referred to as “EMT factor”), and recurrence after treatment or Regarding factors effective for predicting prognosis (hereinafter referred to as “relapse prognostic factors”), these factors (hereinafter collectively referred to as “factors of the present invention”) are detected. It relates to a reagent.
  • the present invention also relates to a method for predicting epithelial-mesenchymal transition of cancer cells and postoperative recurrence or prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer, using the factor of the present invention as an index.
  • the present invention relates to a component capable of suppressing cancer cell epithelial-mesenchymal transition (EMT) for solid cancer, particularly colorectal cancer or breast cancer, that is, an effective component for suppressing cancer progression such as invasion and metastasis, or
  • EMT cancer cell epithelial-mesenchymal transition
  • the present invention relates to an ingredient effective for preventing recurrence after treatment in these cancer patients and improving prognosis, and a pharmaceutical composition containing the ingredient as an active ingredient.
  • the present invention relates to the above components, that is, components effective for suppressing cancer cell epithelial-mesenchymal transition (EMT) and cancer progression for solid cancer, particularly colon cancer or breast cancer, and these
  • EMT cancer cell epithelial-mesenchymal transition
  • the present invention relates to a screening method for searching for effective components for preventing recurrence after treatment and improving prognosis in cancer patients.
  • lymph nodes lymph node metastasis
  • lymph nodes lymph nodes
  • Those with such metastases clearly have a poor prognosis.
  • rectal cancer with invasion beyond the muscle layer without lymph node metastasis (Dukes B) has a 79-year survival rate of 79%, but rectal cancer with lymph node metastasis The 5-year survival rate of Dukes C drops to 52%.
  • Dukes B rectal cancer with lymph node metastasis
  • Non-patent Document 1 The cause of the recurrence or poor prognosis in spite of negative lymph node metastasis is the presence of potential cancer cells that cannot be detected at the current level of medical technology, and the potential cancer cells It has been suggested that it has treatment resistance and has the ability to complete metastasis (Non-patent Document 1).
  • minute cancer cells micrometastasis
  • lymphatics using immunohistological staining for gastrointestinal cancers such as gastric cancer, rectal cancer, and esophageal cancer. It has been reported that there is a significant relationship between the presence of nodal micrometastasis and its prognosis.
  • Non-patent Document 2 Reported the presence of free cancer cells in pathologically diagnosed metastasis-negative lymph nodes (Non-patent Document 2), and the presence of free cancer cells in peripheral blood and bone marrow Clinical significance has also been clarified.
  • Non-patent Document 3 Reported the presence of free cancer cells in the bone marrow, they are not directly related to recurrence or prognosis, and both are reported to be independent of each other (Non-patent Document 3).
  • Non-patent Document 3 there is still no conclusion as to whether the presence of free cancer cells in the bone marrow is directly related to recurrence or poor prognosis.
  • Known prognostic factors other than lymph node metastasis include cancer tissue type, cell atypia, gene mutation (oncogene), tumor suppressor gene, hormone receptor, and the like.
  • prognostic predictor over or comparable to lymph node metastasis, and there is an urgent need to find a highly accurate prognostic predictor.
  • the degree of cancer progression EMT, invasion, metastasis, etc.
  • recurrence and prognosis can be predicted, the patient can be more accurate without causing pain. It is possible to select a treatment policy and postoperative adjuvant therapy.
  • the tumor markers that have been used in clinical practice include carcinoembrionic antigen (CEA), CA19-9 (colon cancer) and CA15-3 (breast cancer) in blood. Cancer is diagnosed by detecting a protein with an antibody, follow-up after surgery, or determination of a therapeutic effect. Further, it has been reported that mRNA expression of CEA and CA19-9 in peripheral blood and portal vein can be a prognostic factor of colorectal cancer (Non-patent Document 4).
  • Plastin 3 Plastin 3 (PLS3) is one of three isoforms of actin-binding proteins that play an important role in maintaining the cytoskeleton, and is known to serve as a scaffold when bacteria migrate into cells. .
  • the gene is expressed locally throughout the solid tissue, unlike the Plastin 1 (PLS1) gene expressed in the kidney and small intestine and the plastin 2 (PLS2) gene expressed in hematopoietic cells.
  • PLS1 Plastin 1
  • PLS2 plastin 2
  • hematopoietic cells a relationship with skin T-cell lymphoma (Non-Patent Document 5) and a relationship with bladder cancer, prostate cancer, and head and neck cancer (Non-Patent Documents 6 and 7) have been reported. So far, there are no reports of the presence or significance of PLS3 expression in clinical specimens such as colorectal cancer and breast cancer, and peripheral blood.
  • the present invention identifies in vivo molecules related to epithelial-mesenchymal transition (EMT) of cancer cells and in vivo molecules related to recurrence and prognosis in patients with such solid cancers, particularly colorectal cancer and breast cancer. It is intended to provide EMT factors relating to solid cancer, particularly colorectal cancer or breast cancer, and factors for predicting recurrence or prognosis in patients with these solid cancers (recurrence prognosis factor).
  • EMT epithelial-mesenchymal transition
  • the present invention uses these EMT factors and recurrence prognostic factors (generic name: factor of the present invention) as an index, and EMT of cancer cells in solid cancer, particularly colorectal cancer, and the degree of cancer progression (wet) thereby,
  • the purpose is to provide a useful test drug for predicting recurrence or prognosis of cancer patients such as colorectal cancer and breast cancer.
  • the present invention uses the above-described factor of the present invention as an index, and a method for predicting EMT and cancer progression (wet) due to these solid cancers, particularly colon cancer cells, and solid cancer, In particular, it aims to provide a method for predicting recurrence or prognosis for patients with colorectal cancer and breast cancer.
  • the present invention relates to an active ingredient for suppressing the EMT of cancer cells and suppressing cancer progression (invasion) for solid cancer, particularly colon cancer, or a patient with solid cancer, particularly colon cancer or breast cancer.
  • An object of the present invention is to provide an active ingredient for preventing recurrence after treatment and improving prognosis, and to provide a pharmaceutical composition containing the ingredient.
  • the present invention relates to a solid cancer, particularly colorectal cancer, an ingredient effective for suppressing cancer cell EMT and inhibiting cancer progression (invasion), and solid cancer, particularly colorectal cancer or breast cancer. It is an object of the present invention to provide a screening method for searching for an effective component for preventing recurrence after treatment and improving the prognosis in patients of the present invention.
  • PLS3 protein Plastin 3
  • exogenous PLS3 gene By introducing and expressing exogenous PLS3 gene, cells of solid cancer such as colorectal cancer are transferred from epithelial phenotype to mesenchymal phenotype (induction of epithelial-mesenchymal transition (EMT)), and mesenchymal marker gene
  • EMT epithelial-mesenchymal transition
  • mesenchymal marker gene The expression level of (2) Transduction and expression of exogenous PLS3 gene enhances the invasion and migration of solid cancer cells such as colorectal cancer, (3)
  • EMT of epithelial cancer cells was induced by TGF ⁇ stimulation, PLS3 mRNA expression increased.
  • PLS3 siRNA is administered to the EMT-induced cells, the expression level of the mesenchymal marker gene decreases and the expression level of the epithelial marker gene increases (induction of mesenchymal epithelial transition).
  • the PLS3 gene was thought to be a gene that induces epithelial-mesenchymal transition (EMT) related to cancer cell invasion and migration in solid tumors such as colorectal cancer (EMT-inducible gene).
  • EMT epithelial-mesenchymal transition
  • the present inventors targeted PLS3 mRMA expression in primary tumor tissue, non-tumor tissue, portal vein blood / tumor reflux venous blood, and peripheral blood for more than 100 colon cancer patients, The following findings were obtained when investigating the clinicopathological factors (lymph node metastasis, lymph fluid invasion, venous invasion, liver metastasis, Dukes classification) and the recurrence status and prognosis after 5 years of treatment.
  • PLS3 mRNA expression is observed not only in the primary focus tissue and portal blood / tumor reflux venous blood but also in peripheral blood, (5) The presence of free cancer cells in the peripheral blood of colorectal cancer patients, and the expression of PLS3 mRNA is observed in the cells, (6) High expression of PLS3 mRNA in primary tissue, portal vein / tumor reflux venous blood and peripheral blood (including free cancer cells), lymph node metastasis, lymph fluid infiltration, venous invasion, liver metastasis, and recurrence and There is a positive correlation with poor prognosis.
  • the present invention has been completed based on these findings, and has the following specific aspects.
  • Test drugs and test drug kits for measuring recurrence prognostic factors for solid cancer patients (I-1) Stringent conditions for the base sequence of the PLS3 gene (SEQ ID NO: 1) or its complementary base sequence A test agent for measuring PLS3, a prognostic factor for recurrence in solid cancer patients, comprising a polynucleotide having at least 15 bases that hybridizes with.
  • I-2 A diagnostic drug for measuring PLS3, a prognostic factor for recurrence in solid cancer patients, having an antibody that recognizes PLS3 protein (anti-PLS3 antibody).
  • (I-4) A test drug kit for measuring the prognosis factor PLS3 in patients with solid cancer, comprising at least the test drug described in any of (I-1) to (I-3).
  • test drug and test drug kit can also be used to detect epithelial-mesenchymal transition cells as described below.
  • I'-1 Detects epithelial-mesenchymal transition cells containing a polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of the PLS3 gene (SEQ ID NO: 1) or a complementary base sequence thereof. To test drugs.
  • I′-2 A test agent for detecting epithelial-mesenchymal transition cells having an antibody that recognizes PLS3 protein (anti-PLS3 antibody).
  • (I'-3) A test agent kit for detecting epithelial-mesenchymal transition cells, containing at least the test agent described in (I'-1) or (I'-2).
  • test sample is a solid cancer tissue, bone marrow, or blood.
  • test sample is bone marrow or blood free cancer cells.
  • test sample (1) measuring PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient, (2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production) Have (3) When the test expression level or test production level is higher than the control expression level or control production level, it is determined that the subject is likely to relapse or has a poor prognosis, (II-1) Thru
  • III Method for detecting epithelial-mesenchymal transition cells
  • Epithelial-mesenchymal step comprising measuring PLS3 gene expression level or PLS3 protein production level in a sample collected from a solid cancer patient (test sample)
  • a method of detecting cancer (tumor) cells that have migrated comprising measuring PLS3 gene expression level or PLS3 protein production level in a sample collected from a solid cancer patient (test sample)
  • test sample is a solid cancer tissue, bone marrow, or blood.
  • test sample is a free cancer cell in bone marrow or blood.
  • (III-5) (1) a step of measuring the PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient, (2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production) Have (3) When the test expression level or the test production level is higher than the control expression level or the control production level, it is determined that the cancer cells or free cancer cells of the solid cancer patient have transitioned to epithelial mesenchyme ( The method according to any one of (III-1) to (III-4).
  • compositions for suppressing recurrence or improving prognosis of solid cancer or pharmaceutical composition for suppressing cancer progression (IV-1)
  • Effective siRNA or PLS3 neutralizing antibody that suppresses expression of PLS3 gene A pharmaceutical composition comprising as an ingredient.
  • (IV-2) The pharmaceutical composition according to (IV-1), which is used for suppressing recurrence or improving the prognosis of solid cancer, or for suppressing cancer progression of solid cancer.
  • siRNA that suppresses the expression of PLS3 gene, which is used to suppress recurrence or improve the prognosis of solid cancer, or to suppress cancer progression of solid cancer, or neutralizing antibody against PLS3 .
  • siRNA that suppresses expression of the PLS3 gene described in (IV-4) where the solid cancer is colon cancer or breast cancer (IV-4), or a neutralizing antibody against PLS3.
  • (V) effective ingredient preventing recurrence or prognosis improving solid cancer patients, and methods of screening for active ingredients for cancer suppression of progression solid cancer having (V-1) the following step, the expression of PLS gene
  • Methods for screening for inhibitors (1) contacting the test substance with a cell capable of expressing the PLS3 gene; (2) a step of measuring the expression level (test expression level) of the PLS3 gene in the cell contacted with the test substance, and (3) the expression level of the PLS3 gene in the control cell where the test expression level is not in contact with the test substance.
  • V-2 A screening method for a substance that suppresses the function of PLS3, comprising the following steps: (1 ′) contacting a test substance with a cell capable of producing PLS3 protein or a cell fraction prepared from the cell, (2 ′) a step of measuring the PLS3 protein production amount (test production amount) of the cell contacted with the test substance or its cell fraction, and (3 ′) a control cell in which the above test production quantity does not contact the test substance. Alternatively, a step of selecting a test substance when the PLS3 protein production amount (control production amount) of the cell fraction is smaller.
  • V-3) The screening method according to (V-1) or (V-2), wherein the cell capable of expressing the PLS3 gene or the cell capable of producing the PLS3 protein is a non-hematopoietic cell.
  • (V-4) The screening method according to any one of (V-1) to (V-3), which is a method for searching for a component effective in preventing recurrence or improving prognosis in patients with solid cancer .
  • (V-6) The screening method according to any one of (V-1) to (V-3), which is a method for searching for an effective component for suppressing cancer progression (wetting and metastasis) of solid cancer.
  • the present invention provides plastin 3 (PLS3) as a new independent factor (relapse prognosis factor) related to postoperative recurrence or prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer.
  • the test agent of the present invention or a method for predicting recurrence using the same is a method using the above-mentioned recurrence prognostic factor PLS3 as an index, and according to the method, blood in patients with solid cancer, particularly colorectal cancer or breast cancer.
  • an appropriate treatment strategy can be established.
  • the prognosis can be improved by performing appropriate follow-up and adjuvant therapy after radical surgery for patients who have been found to have a recurrence possibility or poor prognosis by the method of the present invention.
  • by omitting useless treatment for patients with a low possibility of recurrence or patients with a good prognosis it is possible to reduce the physical or mental pain of the patient and to reduce the medical cost.
  • by measuring the likelihood of recurrence and prognosis for postoperative patients it is possible to take appropriate measures to prevent recurrence and improve prognosis, such as designing appropriate postoperative therapy for each patient. it can.
  • epithelial-mesenchymal transition cells the presence of cancer (tumor) cells that have migrated to epithelial-mesenchymal cells (hereinafter referred to as “epithelial-mesenchymal transition cells”) can be detected by using the above-mentioned relapse prognostic factor PLS3 as an index.
  • PLS3 epithelial-mesenchymal transition cells
  • the pharmaceutical composition (cancer progression inhibitor, recurrence preventive agent or prognostic agent) provided by the present invention is used to prevent cancer progression in patients with solid cancer, particularly colorectal cancer or breast cancer, and postoperatively. Can be used effectively to prevent recurrence and improve prognosis.
  • an effective component for suppressing cancer progression invasion, metastasis
  • prevention of recurrence and improvement of prognosis in patients with solid cancer particularly colorectal cancer or breast cancer. Therefore, it is possible to obtain effective components. That is, the screening method can be effectively used for development of a new drug effective in suppressing the progression of solid cancer and preventing recurrence after treatment or improving the prognosis.
  • LoVo-PLS3 cells are PLS3-expressing LoVo cells (LoVo-PLS3 cells) (right side) with control LoVo cells (LoVo-mock cells) (left side) ( ⁇ 200, scale bar: 100 ⁇ m).
  • LoVo-PLS3 cells are images of LoVo cells immunostained with anti-PLS3 antibody (left side) and anti-vimentin antibody (right side) for LoVo-PLS3 cells (bottom) and LoVo-mock cells (top), respectively ( ⁇ 400, scale bar: 50 ⁇ m).
  • Results of measuring the proliferation of LoVo cells for each of LoVo-PLS3 cells and LoVo-mock cells using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MMT) assay Indicates.
  • the horizontal axis represents the cell culture time (hour), and the vertical axis represents the cell growth rate (relative ratio when the cell growth is 1 when the culture time is 0 hour).
  • Data represent mean ⁇ s.d. (* p ⁇ 0.05).
  • the sensitivity (resistance) to the anticancer drug (5-FU) was determined as 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide. (MTT)
  • the result measured using assay is shown.
  • the horizontal axis represents the 5-FU concentration ( ⁇ g / ml), and the vertical axis represents the cell growth rate (relative ratio when the cell growth is 1 when the 5-FU concentration is 0 ⁇ g / ml).
  • Data represent mean ⁇ s.d. (* p ⁇ 0.05).
  • FIG. (b) is a diagram comparing the expression levels of EMT-related gene mRNAs of vimentin, FN1, N-cadherin and FOXC2 in PLS3-expressing LoVo cells (LoVo-PLS3 cells) or control LoVo cells (LoVo-mock cells). is there.
  • FIG. 1 shows vimentin, E-cadherin, TWIST and SNAIL mRNA in CaR-1 cells treated with PLS3 siRNA (CaR-1 PLS3 siRNA) and CaR-1 cells treated with control siRNA (CaR-1 control siRNA) It is the figure which contrasted the expression level. All data are expressed as mean ⁇ s.d. All data are normalized to GAPDH (* p ⁇ 0.05, compared to control). The results of RT-PCR (a) and Western blot analysis (b) of LoVo cells and CaR-1 cells are shown.
  • (a) shows LoVo-PLS3 cells (left), LoVo-mock cells (left), CaR-1 control siRNA cells (right), and CaR-1 PLS3 siRNA cells (right) The expression level of PLS3 mRNA is shown. Data show mean values ⁇ s.d. All data were normalized with GAPDH (* -p ⁇ 0.05).
  • the expression level of PLS3 mRNA is shown as a value normalized by the expression level of GAPDH mRNA.
  • A shows the results of immunohistochemical staining of cancer (T) tissue and non-cancer (N) tissue of colorectal cancer patients using anti-PLS3 antibody. Arrows indicate cancer cells overexpressing PLS3 ( ⁇ 200, scale bar: 200 ⁇ m). N: Non-cancerous tissue side, T: Vaginal cancer tissue side.
  • the left figure shows the result of cytokeratin staining
  • the middle figure shows the result of PLS3 staining
  • the right figure shows the result of overlaying both.
  • PLS3 mRNA expression level in cancer (T) tissue and non-cancer (N) tissue of breast cancer patients (n 34) measured by real-time quantitative RT-PCR. All values are standardized by the expression level of GAPDH mRNA in each tissue (PLS3 mRNA / GAPDH mRNA).
  • PLS3 protein and “PLS3 gene” mean a protein that binds to actin that controls cell morphology and skeletal structure that affect cell migration ability: Plastin 3 and its gene, respectively. In the present specification, both may be collectively referred to simply as “PLS3”.
  • the “PLS3 gene” basically means the PLS3 gene derived from human shown in SEQ ID NO: 1.
  • a protein having a biological function equivalent to a human-derived PLS3 protein such as an ortholog gene conserved between species such as human, mouse, and rat (for example, a homolog (such as a homolog or a splice variant). ), Variants and derivatives) are also included.
  • genes of other species such as mouse and rat corresponding to the human PLS3 gene encoding the PLS3 protein can be exemplified.
  • genes can be identified by HomoloGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, the specific human gene name (PLS3 gene) and the accession number (GenBank Accession No. NM_005032.4) of LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) Search for relevant human genetic data. From the list showing the correlation of gene homologs between other species and human genes displayed by accessing the homologene in the link menu of the data, select genes of other species such as mice and rats that correspond to the human PLS3 gene. It can be selected as a gene (homolog).
  • mouse PLS3 gene (GenBank Accession No. NM_145629.1) can be exemplified as a mouse homologue of the human PLS3 gene (GenBank Accession No. NM_005032.4) (SEQ ID NO: 1).
  • gene refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
  • gene expression means that mRNA is transcribed and synthesized based on genetic information.
  • DNA refers to double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand) including cDNA and synthetic DNA, and a sequence complementary to the sense strand. These include both single-stranded DNA (antisense strand) and fragments thereof.
  • RNA includes not only single-stranded RNA but also single-stranded RNA having a sequence complementary thereto, and further double-stranded RNA composed thereof, unless otherwise specified. Used for purposes.
  • the RNA includes synthetic RNA such as total RNA, mRNA, rRNA, siRNA and the like.
  • nucleotide refers to nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.
  • PLS3 protein basically means a human-derived PLS3 protein (GenBank Accession No. NP_005023.2) represented by SEQ ID NO: 2.
  • the present invention is not limited to this, but it is limited to the "protein” that is a translation product from orthologous genes conserved among species such as humans, mice, and rats, and the biological function equivalent to the human PLS3 protein. And their homologues (homologs and splice variants), mutants, derivatives, amino acid modifications and the like.
  • homologs include proteins of other species such as mouse and rat corresponding to the human PLS3 protein (SEQ ID NO: 2).
  • variants are homologous genes (http://www.ncbi.nlm.nih.gov/HomoloGene). It can be identified a priori from the base sequence of the gene identified by /). Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein having no mutation. As a mouse homologue to the above human PLS3, mouse PLS3 (GenBank Accession No. NP_663604.1) can be mentioned.
  • antibody includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or an antibody having an antigen binding property such as a Fab fragment or a fragment generated by a Fab expression library. Some are included.
  • colon cancer is a carcinoma that occurs in the large intestine (cecum, colon, rectum), and usually includes cecal cancer, colon cancer, and rectal cancer. Cancers that develop are also included.
  • breast cancer is a carcinoma that occurs in breast tissue. Such breast cancer includes wet duct cancer and non-wet breast cancer, preferably wet breast cancer.
  • free cancer cell ITC: Isolated Tumor Cell
  • ITC Isolated Tumor Cell
  • recurrence means that cancer cells that have recurred or potentially existed after a radical treatment such as curative resection or radiotherapy grow to a size that can be found again by examination.
  • the recurrence includes any of local recurrence, regional recurrence, and distant metastasis (organ metastasis).
  • prognosis means the prospect of how a patient will recover after cancer radical treatment such as curative resection or radiotherapy, in particular, how long he can survive (life prognosis).
  • the prognosis includes functional prognosis and life prognosis.
  • the life prognosis includes both a case where the cancer survives without recurrence (non-disease survival rate) and a case where the cancer survives while recurrence (relapse survival rate).
  • the “prognosis prediction” means predicting the life prognosis.
  • “poor prognosis” means that the probability of death due to cancer recurrence is relatively high within 5 years after cancer root treatment.
  • “good prognosis” means that the probability of death due to cancer recurrence within 5 years after cancer root treatment is relatively low.
  • the “test agent” refers to the degree of cancer progression (infiltration, metastasis) of solid cancer, particularly colorectal cancer or breast cancer, or the possibility of recurrence in patients with solid cancer, particularly colorectal cancer or breast cancer, or It is used directly or indirectly to predict prognosis.
  • the "test drug” is an effective component (cancer progression) that suppresses the degree of cancer progression (invasion, metastasis) of solid cancer, particularly colon cancer or breast cancer, suppresses recurrence, and improves the prognosis. It is also used directly or indirectly as a tool for screening a candidate substance for a suppressor, a preventive agent for recurrence, or a prognostic agent.
  • PLS3 protein PLS3 protein
  • PLS3 protein PLS3 protein
  • PLS3 protein PLS3 protein
  • Possible (poly) (oligo) nucleotides or antibodies are included. Based on the above properties, these (poly) (oligo) nucleotides and antibodies are used as probes for detecting the PLS3 mRNA and PLS3 protein expressed in vivo in vivo and in vivo, and (poly) (oligo) Nucleotides can be used effectively as primers for amplifying the PLS3 gene expressed in vivo.
  • biological tissue and “biological sample” to be diagnosed include tissues in which expression of PLS3 mRNA increases in association with solid cancer, particularly colon cancer or breast cancer, and expression products (PLS3 Proteins are produced or secreted (including blood).
  • tissue include solid cancer cancer tissues (primary lesions), bone marrow, and blood (portal vein blood and peripheral peripheral blood). Peripheral blood is preferable.
  • control person is used as a term that opposes cancer patients (cancer carriers) suffering from solid cancer, particularly colorectal cancer or breast cancer. Specifically, it means a non-cancer-bearing person who does not suffer from solid cancer.
  • Such controls include healthy individuals as well as those suffering from benign diseases other than cancer (such as hernias and gallstones).
  • PLS3 gene polynucleotide
  • PLS3 protein
  • Test agent present invention for measuring the recurrence prognostic factors solid cancer, solid cancers, in particular to test agents that are used to predict recurrence or prognosis after cancer therapy for patients with colon cancer or breast cancer.
  • the test agent is used for detecting and quantifying the expression (mRNA) of PLS3 gene or its expression product PLS3 protein in bone marrow and blood, preferably peripheral blood, using relapse prognosis factor PLS3 as an index.
  • the test drug searches for active ingredients (candidate substances for recurrence prevention or prognosis improvement) for patients with solid cancer to suppress postoperative recurrence and improve prognosis. Also used for.
  • the reagent is an active ingredient (candidate substance for cancer progression inhibitor) for examining the degree of cancer progression (invasion, metastasis) of solid cancer, and for suppressing cancer progression of solid cancer in the screening method described later. ) Is also used for searching.
  • the prognosis of recurrence is based on the presence or absence of increased expression of PLS3 gene or the expression level (mRNA expression level) in a biological sample (bone marrow or blood) collected from a subject, preferably peripheral blood. Done by evaluating.
  • the test agent of the present invention specifically serves as a primer for specifically recognizing and amplifying the RNA produced by the expression of the gene or a polynucleotide derived therefrom, or specifically the polynucleotide derived therefrom. Used as a probe for detection.
  • the test agent of the present invention only needs to selectively (specifically) recognize PLS3 mRNA or a polynucleotide (cDNA) derived from the mRNA, and is a polynucleotide comprising the base sequence (full-length sequence) of the PLS3 gene. It may be a polynucleotide consisting of its complementary sequence. Moreover, the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.
  • “selectively (specifically) recognize” means that, for example, in Northern blotting, PLS3 mRNA or a polynucleotide (cDNA) derived therefrom can be specifically detected, and RT-PCR method.
  • PLS3 mRNA or a polynucleotide (cDNA) derived therefrom is specifically produced, but the present invention is not limited thereto, and those skilled in the art can detect the product or product derived from PLS3 mRNA. Anything that can be determined to be.
  • the test agent of the present invention is characterized by comprising a polynucleotide having at least 15 consecutive bases and / or a polynucleotide complementary thereto in the base sequence of the PLS3 gene.
  • the complementary polynucleotide (complementary strand, reverse strand) is the full-length sequence of a polynucleotide consisting of the base sequence of the PLS3 gene, or a partial sequence thereof having a base sequence of at least 15 bases in length in the base sequence (
  • positive strands refer to polynucleotides that are in a base-complementary relationship based on base pair relationships such as A: T and G: C.
  • such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions.
  • stringent conditions here combine complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.” can be mentioned.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
  • the more stringent hybridization conditions are about “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”
  • the more severe hybridization conditions are “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.”.
  • a complementary strand a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand.
  • An example is a chain composed of a base sequence having the same.
  • the test agent of the present invention can be designed using, for example, the vector NTI (manufactured by Infomax) based on the base sequence of the human-derived PLS3 gene represented by SEQ ID NO: 1. Specifically, a primer or probe candidate sequence obtained by applying the base sequence of the PLS3 gene (SEQ ID NO: 1) to the vector NTI software, or a sequence partially including the sequence is used as a primer or probe. be able to.
  • test agent of the present invention may be any polynucleotide having a length of at least 15 consecutive bases as described above, and specifically, the length can be appropriately selected and set according to the application. .
  • test agent of the present invention should be used as a primer or probe according to a conventional method in known methods for specifically detecting the expression of a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, etc. Can do. By using this, it is possible to evaluate the presence or absence of expression increase or expression level (expression level) of PLS3 gene in a sample collected from a subject.
  • the sample to be measured can be obtained by collecting a part of cancer tissue or bone marrow of a subject's solid cancer with a biopsy or the like, or blood (portal blood or peripheral blood) ) And the like, and total RNA prepared therefrom according to a conventional method may be used, or various polynucleotides (cDNA) prepared based on the RNA may be used.
  • biological sample biological sample
  • cDNA polynucleotides
  • test agent of the present invention When used as a primer for the measurement of the recurrence prognosis factor PLS3, it hybridizes under stringent conditions to the base sequence of the PLS3 gene (SEQ ID NO: 1) or a base sequence complementary thereto, at least 15
  • the polynucleotide having a base include those having a base length of usually 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp.
  • detection probe those having a base length of usually 15 bp to the entire sequence, preferably 15 bp to 1 kb, more preferably 100 bp to 1 kb can be exemplified.
  • An example of a primer used in the present invention is a polynucleotide having the following sequence.
  • PLS3 F primer ccttccgtaactggatgaactc (SEQ ID NO: 3)
  • PLS3 R primer ggatgcttccctaattcaacag (SEQ ID NO: 4).
  • the test agent (probe or primer) of the present invention includes a label suitable for detecting PLS3 mRNA or its derivative (cDNA), such as a fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance, biotin, etc. Is included.
  • a label suitable for detecting PLS3 mRNA or its derivative such as a fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance, biotin, etc. Is included.
  • fluorescent dye used in the present invention those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used.
  • HEX 4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto.
  • TMR tetramethylrhodamine
  • a method for labeling a nucleotide with a fluorescent dye an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)].
  • Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system, manufactured by Amersham Pharmacia).
  • the probe oligo or polynucleotide
  • the test agent of the present invention is a probe in which the above probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to.
  • the immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good.
  • the immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • a commercially available spotter such as Amersham
  • a DNA microarray for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from biological tissue Presence / absence or expression level (expression level) of the PLS3 gene in living tissue by detecting a complex of the (inspection drug of the present invention) and labeled DNA or RNA using the label of the labeled DNA or RNA as an indicator Can be evaluated.
  • the above-mentioned DNA chip or the like only needs to contain one or more types of probes (test agents) that can bind to PLS3 mRNA or its derivative (cDNA).
  • test agents probes
  • cDNA PLS3 mRNA or its derivative
  • the test agent of the present invention is useful for evaluating the degree of cancer progression (infiltration and metastasis) of solid cancer and predicting the possibility of recurrence and prognosis after treatment for patients with solid cancer.
  • the evaluation of the degree of cancer progression and the prediction of the prognosis of recurrence using the test drug are based on the difference in the expression level of the PLS3 gene in the biological sample of the solid cancer patient and the biological sample of the control person (non-bearing patient). This can be done by determining.
  • the expression of the PLS3 gene is elevated in the solid tumor primary lesion, bone marrow, and blood (portal vein blood, peripheral blood).
  • the subject may have a high degree of cancer progression (invasion, metastasis), risk of recurrence after surgery, and poor prognosis Increases nature.
  • the PLS3 expression level in the primary lesion is greater than 2 compared to the internal control level of GAPDH, the PLS3 expression level in peripheral blood and portal vein blood
  • the expression level is larger than the “average value ⁇ 2 sd”
  • the above possibility increases as a PLS3 high expression person.
  • the upper limit of the 95% confidence interval of the control person is the cut off value, and if the expression level of PLS3 is higher than that value, the above possibility becomes high as a PLS3 high expression person.
  • Antibody provides an antibody capable of specifically recognizing PLS3 protein as a test agent.
  • Specific examples of the antibody include an antibody capable of specifically recognizing a human-derived PLS3 protein having the amino acid sequence set forth in SEQ ID NO: 2.
  • the present invention relates to the fact that the expression of the PLS3 gene is related to EMS induction, PLS3 in solid tumors, particularly the primary lesions of colon cancer or breast cancer, bone marrow, blood (portal blood or peripheral blood).
  • PLS3 in solid tumors
  • the primary lesions of colon cancer or breast cancer particularly the primary lesions of colon cancer or breast cancer, bone marrow, blood (portal blood or peripheral blood).
  • To detect the presence or extent of increased PLS3 protein production based on the knowledge that high gene expression is closely related to cancer progression (invasion, metastasis), postoperative recurrence, and poor prognosis Is based on the idea that the degree of cancer progression (invasion, metastasis) of solid cancer can be evaluated, and the risk and prognosis of postoperative recurrence can be predicted.
  • the antibody detects the presence or extent of the above-mentioned increase in PLS3 protein production in patients with solid cancer, thereby assessing the degree of cancer progression in the patient or predicting the presence or prognosis of postoperative recurrence Used as a testing agent for.
  • the form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using PLS3 protein as an immunizing antigen or a monoclonal antibody thereof.
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)).
  • the antibody of the present invention is a polyclonal antibody
  • an oligopeptide having a partial amino acid sequence of the PLS3 protein is synthesized using a PLS3 protein expressed and purified in Escherichia coli according to a conventional method, or according to a conventional method.
  • a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method.
  • spleen cells and bone marrow obtained by immunizing a non-human animal such as a mouse with PLS3 protein expressed and purified in Escherichia coli or the like according to a conventional method or an oligopeptide having a partial amino acid sequence of the protein are obtained. It can be obtained from hybridoma cells prepared by cell fusion with tumor cells (Current protocol, in molecular molecular biology, edit. Ausubel et al. (1987) Publishing, John Wiley, and Sons, Section 11.1-11.11).
  • the PLS3 protein used as an immunizing antigen for antibody production is based on DNA sequence information (for example, SEQ ID NO: 1) provided by the present invention, DNA cloning, construction of each plasmid, transfection into a host, transformation It can be obtained by manipulation of body culture and protein recovery from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
  • a recombinant DNA (expression vector) capable of expressing a gene encoding PLS3 protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured.
  • the protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture.
  • the partial peptide of PLS3 protein can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information (SEQ ID NO: 2) provided by the present invention.
  • the PLS3 protein of the present invention includes not only the protein related to the amino acid sequence shown in SEQ ID NO: 2, but also homologues thereof.
  • the homologue consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and is immunologically equivalent to the protein shown in SEQ ID NO: 2. Can be mentioned.
  • examples of the protein having equivalent immunological activity include a protein having an ability to induce a specific immune reaction in an appropriate animal or its cells and specifically bind to an antibody against PLS3 protein.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of PLS3 protein.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as PLS3 protein.
  • An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the PLS3 protein can be exemplified.
  • Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
  • the antibody of PLS3 protein can be obtained commercially, for example, from “Santa Cruz Biotechnology Inc., CA, USA”.
  • the antibody of the present invention Since the antibody of the present invention has the property of specifically binding to PLS3 protein, it was secreted into PLS3 protein or blood expressed and produced in the tissues of colon cancer patients or breast cancer patients by using the antibody. PLS3 protein can be specifically detected. That is, the antibody is useful as a probe for detecting the presence and level of PLS3 protein in a biological tissue or biological sample (including blood) of a colon cancer or breast cancer patient.
  • a portion of solid cancer tissue or bone marrow is collected with a biopsy, etc., or blood (peripheral blood, portal vein blood, tumor reflux venous blood), etc. is collected and prepared according to a conventional method.
  • the PLS3 protein can be detected and quantified using the extracted extract or protein by using the antibody of the present invention as a probe in a known detection method such as Western blotting or ELISA.
  • the difference in the amount of PLS3 protein in the biological sample of the solid cancer patient and the amount of PLS3 protein in the biological sample of the control person may be determined.
  • PLS3 protein is produced in solid tumor patients' primary lesions, bone marrow or blood (peripheral blood, tumor reflux venous blood, portal vein blood) in comparison with those of controls. It is rising.
  • the subject has a high degree of cancer progression (invasion, metastasis), a risk of recurrence after surgery, and a poor prognosis.
  • Test drug kit provides a test drug kit for use in the evaluation of the degree of cancer progression of solid cancer, or the possibility of postoperative recurrence and prognosis for patients with solid cancer, including the above test drugs. Also related.
  • the test drug kit contains at least one oligo or polynucleotide (which may be labeled or immobilized on a solid phase) or antibody used as the probe or primer.
  • the reagent kit of the present invention includes, in addition to the probe or primer described above as the test agent of the present invention, a hybridization reagent, a probe label, a label detection agent, a buffer solution, etc., if necessary. Other reagents and instruments necessary for the implementation may be included as appropriate.
  • the present invention provides a method for predicting recurrence prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer, using the above-described test agent of the present invention.
  • the recurrence prognosis prediction method of the present invention detects the expression level of a PLS3 gene and a protein derived from this gene (PLS3 protein) contained in a biological sample collected from a subject, and the expression level or the protein thereof. By measuring the amount, the prognosis for recurrence is predicted for the subject.
  • PLS3 protein protein derived from this gene
  • the recurrence prognosis prediction method of the present invention includes the following steps (1) and (2): (1) measuring a PLS3 gene expression level or PLS3 protein production level in a biological sample (test sample) collected from a patient with solid cancer; (2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured as described above is the PLS3 gene expression level (control expression level) in a biological sample (control sample) collected from a control person or The process of comparing with PLS3 protein production (control production).
  • test expression level or test expression level is high compared to the control expression level or control production level obtained by such a method, the subject is likely to relapse after surgery or the prognosis is determined to be poor. Can do.
  • the biological sample used here corresponds to the sample (test sample) prepared from the primary tumor, bone marrow, or blood (tumor reflux vein, peripheral blood) for the subject, and to the test sample for the control.
  • Biological tissue, bone marrow, blood can be mentioned. Peripheral blood is preferable.
  • free cancer cells isolated and recovered from bone marrow or blood can also be used as test samples.
  • an RNA-containing sample prepared from the tissue, bone marrow or blood, a sample containing a polynucleotide further prepared therefrom, or a sample containing a protein prepared from the tissue can be mentioned.
  • a sample containing such RNA, polynucleotide or protein can be prepared according to a conventional method by collecting a part of cancer tissue or bone marrow of a subject with a biopsy or the like, or collecting blood therefrom.
  • the method of the present invention is specifically performed as follows according to the type of biological sample used as a measurement target.
  • relapse prognosis method specifically includes the following steps (a), (b) and (c) Can be implemented by: (a) a step of binding RNA prepared from a biological sample collected from a subject (test sample) or a complementary polynucleotide transcribed therefrom and the test agent (poly (oligo) nucleotide) of the present invention, (b) a step of measuring RNA derived from a test sample bound to the test agent or a complementary polynucleotide transcribed from the RNA using the test agent as an index, (c) Results obtained from the steps (a) and (b) for the biological sample (control sample) derived from the control person (non-cancer-bearing person) in the same manner as the measurement result (test result) in (b) above. Step to contrast with (control result).
  • This method is carried out by detecting and measuring the expression level of the PLS3 gene in the RNA.
  • known methods such as Northern blot method, RT-PCR method, DNA chip analysis method, in-situ hybridization analysis method, etc. using the above-described test agent of the present invention comprising poly (oligo) nucleotides as a primer or probe. It can implement by performing the method of.
  • the presence or absence of the expression of the PLS3 gene in RNA and its expression level can be detected and measured by using the above-described test agent of the present invention as a probe.
  • the test agent of the invention composite strand radioisotopes (such as 32 P, 33 P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method
  • the formed diagnostic agent (DNA) and RNA duplex is radiated to a signal derived from the label of the diagnostic agent (labeled substance such as RI or fluorescent substance).
  • a method of detecting and measuring with a detector BAS-1800II, manufactured by Fuji Film) or a fluorescence detector can be exemplified.
  • the test drug probe DNA
  • the test drug is labeled according to the protocol and hybridized with RNA derived from the biological sample of the subject, and then labeled with the test drug.
  • the presence or absence and expression level of the PLS3 gene in RNA can be detected and measured by using the above-described test agent of the present invention as a primer.
  • a pair of primers prepared from the test agent of the present invention is prepared so that cDNA can be prepared from RNA derived from a biological sample of a subject according to a conventional method, and the target PLS3 gene region can be amplified using this as a template.
  • An example of a method for detecting the amplified double-stranded DNA obtained by hybridizing the above cDNA (-strand) to the positive strand and the reverse strand binding to the + strand and performing PCR according to a conventional method. can do.
  • the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance.
  • a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled test agent is used as a probe and hybridized with this to detect it.
  • the produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems).
  • DNA chip analysis When using DNA chip analysis, prepare a DNA chip to which the above-mentioned test agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and then apply RNA from RNA derived from the biological tissue of the subject by a conventional method. A method of detecting a double strand of DNA and cRNA formed by hybridization with the prepared cRNA by binding with a labeled probe prepared from the test agent of the present invention can be mentioned. In addition, a DNA chip that can detect and measure the gene expression level of the PLS3 gene can also be used as the DNA chip.
  • the method for predicting recurrence of the present invention is carried out by detecting PLS3 protein in the biological sample and measuring the amount thereof. Is done. Specifically, it can be carried out by a method comprising the following steps (a), (b) and (c): (a) a step of binding PLS3 protein in a biological sample collected from a subject (test sample) with the test agent of the present invention relating to an antibody (an antibody recognizing PLS3 protein), (b) measuring the PLS3 protein in the test sample bound to the test agent using the test agent as an index, (c)
  • the measurement result (test result) in (b) above is the same as the result (control result) obtained from the steps (a) and (b) for the biological sample (control sample) collected from the control person. Contrast process.
  • Western blotting uses a test antibody of the present invention as a primary antibody, followed by a labeled antibody (primary antibody labeled with a radioisotope such as 125 I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), etc. as a secondary antibody.
  • a radiometer BAS-1800II: manufactured by Fuji Film Co., Ltd.
  • a fluorescence detector etc., detect the signal derived from the labeling substance such as a radioisotope of the obtained labeled compound or a fluorescent substance. And can be implemented by measuring.
  • the function or activity of the PLS3 protein to be measured in the above has a positive correlation with the amount of the protein. Therefore, instead of measuring the amount of the protein, the method of the present invention can also be carried out by measuring the function or activity of the protein. That is, the method of the present invention also includes a method for predicting recurrence, comprising measuring and evaluating according to the method disclosed in the present specification using the function or activity of PLS3 as an index.
  • PLS3 is known to have a function of binding actin fibers, and its measurement method is also known (Giganti, A., et al. Actin-filament cross-linking protein T-plastin increases Arp2 / 3-mediated actin-based movement. J Cell Sci 118, 1255-1265 (2005); Oprea, GE, et al. Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy. Science 320, 524-527 (2008)).
  • the possibility of recurrence and prognosis after surgery is determined by biological samples collected from the subject (primary tissue, bone marrow, blood, etc.)
  • the expression level of the PLS3 gene in the sample) or the amount, function, or activity of the PLS3 protein that is the expression product of this gene (hereinafter, these may be collectively referred to as “protein level”) It can be performed by comparing the gene expression level or the protein level in a biological sample (tissue, bone marrow, blood or the like corresponding to the test sample) and determining the difference between the two.
  • the biological sample is preferably peripheral blood because of its ease of collection.
  • Comparison of PLS3 gene expression level (level) or PLS3 protein level (level) between the biological sample of the subject (test sample) and the biological sample of the control (control sample) is based on the measurement of the test sample and the control sample. This can be done in parallel. If not performed in parallel, the expression level of the PLS3 gene obtained by measurement under uniform measurement conditions using a plurality (at least 2, preferably 3 or more, more preferably 5 or more) of control samples, or The average value or statistical intermediate value of the amount (level) of PLS3 protein, which is the gene expression product, can be used for comparison as the gene expression level or protein amount of the control.
  • Whether a subject has a high possibility of recurrence after surgery or has a poor prognosis depends on whether the expression level of the PLS3 gene in the test sample or the level of the PLS3 protein, which is the expression product, corresponds to the test sample. It can be performed using as an index that the amount is significantly higher than the level of the control sample. In this case, the subject can be determined to have a high possibility of recurrence after surgery or a poor prognosis.
  • Cancer progression inhibitor, recurrence preventive or prognostic improver for solid cancer drug of the present invention
  • the PLS3 gene siRNA shown below suppresses the expression of PLS3 mRNA in solid cancer cells expressing the PLS3 gene.
  • sc-43215 A CCACGGAUAGAUAUUAACAtt (SEQ ID NO: 5) UGUUAAUAUCUAUCCGUGGtt (SEQ ID NO: 6) sc-43215 B GAACUCUUGGUGUCAAUtt (SEQ ID NO: 7) AUUGACACCAAGAGAGUUCtt (SEQ ID NO: 8) sc-43215 C GAAGAGUGGCAAUCUAACAtt (SEQ ID NO: 9) UGUUAGAUUGCCACUCUUCtt (SEQ ID NO: 10).
  • the siRNA can be used as an active ingredient of a drug that suppresses cancer progression of a solid cancer (cancer progression inhibitor) by blocking EMS induction by PLS3 mRNA expression.
  • the siRNA suppresses PLS3 mRNA expression to prevent postoperative recurrence (relapse prevention agent) or improve prognosis (prognosis) for patients with solid cancer, especially colorectal cancer and breast cancer. It can be used as an active ingredient of an improver.
  • these drugs are collectively referred to as “the drug of the present invention”.
  • the present invention provides the above-mentioned agent of the present invention comprising siRNAsi that suppresses the expression of PLS3 gene as an active ingredient.
  • the siRNA RNA targeted by the present invention is double-stranded, and is formed by hybridizing a sense strand that is complementary to the target sequence of the PLS3 gene and an antisense strand that is complementary to the sense strand. Yes.
  • Such siRNA is not particularly limited as long as it suppresses the expression of the PLS3 gene, and preferred examples include siRNA (SEQ ID NOs: 5 to 10) described above.
  • siRNA that suppresses the expression of the PLS3 gene but also those having an action of suppressing the expression of the PLS3 gene can be used as the active ingredient of the drug of the present invention.
  • Antisense polynucleotide can be mentioned as what has this effect
  • the antisense polynucleotide has a base sequence complementary to or substantially complementary to the base sequence of the PLS3 gene, or a part thereof, and hybridizes with the RNA of the PLS3 gene to thereby It has an effect of inhibiting synthesis or function, or regulating / controlling the expression of PLS3 gene through interaction with the RNA.
  • the antisense polynucleotide may be any antisense polynucleotide as long as it has the above action, and includes antisense RNA, antisense DNA, and the like.
  • the antisense polynucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases.
  • phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are converted to chemically modified phosphate residues such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted.
  • These antisense polynucleotides can be produced using a known DNA synthesizer.
  • siRNA that suppresses the expression of the PLS3 gene but also those having an action of suppressing the function of PLS3 can be used as the active ingredient of the drug of the present invention.
  • examples of such an action include antibodies against PLS3, particularly neutralizing antibodies.
  • the neutralizing antibody means an antibody having the property of inhibiting the function or activity originally possessed by the antigen by binding to the antigen.
  • the neutralizing antibody against PLS3 refers to an antibody having a property of inhibiting the function or activity of PLS3 by binding to PLS3.
  • the agent of the present invention is an active ingredient (siRNA, antisense polynucleotide, antibody, etc.) that suppresses the expression of PLS3 gene or suppresses its function, and any carrier or additive, for example, pharmaceutically acceptable Carriers and additives can be included.
  • active ingredient siRNA, antisense polynucleotide, antibody, etc.
  • any carrier or additive for example, pharmaceutically acceptable Carriers and additives can be included.
  • Examples of pharmaceutically acceptable carriers and additives include excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; Fragrances such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersants such as surfactants Diluents such as water and physiological saline; base wax and the like, but are not limited thereto.
  • excipients such as sucrose and starch
  • binders such as cellulose and methylcellulose
  • disintegrants such as starch and carboxymethylcellulose
  • lubricants such as magnesium stearate and aerosil
  • Fragrances such as citric acid and menthol
  • preservatives such as sodium benzoate and sodium bisulfite
  • stabilizers such
  • Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained.
  • parenteral administration preparations include aqueous and non-aqueous sterile suspensions, including suspensions, solubilizers, thickeners, stabilizers, preservatives, and the like. It may be included.
  • the dose of the drug of the present invention varies depending on the body weight and age of the subject to be administered, the severity of the disease, etc., and cannot be set unconditionally.
  • the amount of the active ingredient per day for an adult is several mg to Several tens mg / kg body weight can be mentioned (for example, the range of 0.1 to 90 mg / kg body weight is exemplified), and this can be administered once to several times a day.
  • the above active ingredient is encoded by DNA
  • gene therapy can be performed as it is or by incorporating it into a gene therapy vector.
  • the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
  • substances that can suppress the expression of PLS3 gene (mRNA expression) and PLS3 protein function as active ingredients that suppress the progression of solid cancer, prevent recurrence after surgery, and prognosis. It is considered useful as an active ingredient that improves.
  • the screening method of the present invention described below includes: (3-1) suppression of PLS3 gene expression, (3-2) decrease in PLS3 protein production, or (3-3) PLS3 protein among test substances.
  • the drug of the present invention By searching for substances that exhibit these effects using the decrease in function as an indicator, it is possible to suppress postoperative recurrence or improve prognosis for cancer progression inhibitors of solid cancer or patients with solid cancer Therefore, it is intended to obtain an active ingredient of a drug (relapse inhibitor, prognosis improver) (hereinafter collectively referred to as “the drug of the present invention”).
  • the screening method of the present invention can be carried out on samples containing these candidate substances (collectively referred to as “test substances”).
  • the sample containing the candidate substance includes a cell extract, an expression product of a gene library, a microorganism culture supernatant, a cell component, and the like.
  • this method can be carried out by performing the following steps (1) to (3). (1) contacting the test substance with a cell capable of expressing the PLS3 gene; (2) a step of measuring the expression level (test expression level) of the PLS3 gene in the cell contacted with the test substance, and (3) the expression level of the PLS3 gene in the control cell not contacting the test substance. A step of selecting the test substance as the candidate substance when it is smaller than (control expression level).
  • the cells used for such screening may be cells that can express the PLS3 gene regardless of whether they are endogenous or exogenous.
  • the origin of the PLS3 gene is not particularly limited, and may be a human-derived PLS3 gene (SEQ ID NO: 1), or may be derived from mammals such as mice other than humans or other biological species. PLS3 gene derived from human is preferable.
  • cells capable of expressing PLS3 gene other than hematopoietic cells that is, solid tissue cells (including those derived from humans and other species), and the tissue cells isolated and prepared therefrom Of primary cultured cells.
  • a transformed cell prepared by introducing an expression vector having cDNA of PLS3 gene and capable of expressing PLS3 mRNA can be used.
  • the category of cells used for screening includes tissues that are aggregates of cells.
  • the condition for bringing the test substance into contact with the cell capable of expressing the PLS3 gene is not particularly limited, but the culture condition (temperature) in which the cell does not die and the PLS3 gene can be expressed. , PH, medium composition, etc.).
  • Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of expressing the PLS3 gene under the above conditions and searching for a substance that suppresses the expression of the PLS3 gene and reduces the expression level of the mRNA. .
  • the expression level of PLS3 mRNA when cultivating cells capable of expressing PLS3 gene in the presence of a test substance is PLS3 obtained when culturing cells capable of expressing PLS3 gene corresponding to the above in the absence of test substance
  • the test substance that has been brought into contact with the cell can be selected as a candidate substance using as an index the expression level that is smaller than the expression level of mRNA (control expression level).
  • the measurement (detection and quantification) of the expression level of PLS3 mRNA is performed by measuring the expression level of PLS3 mRNA in a cell capable of expressing PLS3 gene with an oligonucleotide having a sequence complementary to the base sequence of the PLS3 mRNA (the reagent of the present invention described above) It can be carried out by carrying out a known method such as a Northern blot method using RT and the like, RT-PCR method, real-time quantitative RT-PCR method, or a measuring method using a DNA array.
  • the detection and quantification of the expression level of the PLS3 gene is performed using a cell line in which a fusion gene in which a marker gene such as a luciferase gene is connected to a gene region (expression control region) that controls the expression of the PLS3 gene is used. It can also be carried out by measuring the activity of a gene-derived protein.
  • the screening method for a PLS3 gene expression control substance of the present invention includes a method for searching for a target substance using the expression level of the marker gene as an index. In this sense, the method described in the section (V) described above is included.
  • the concept of “PLS3 gene” includes a fusion gene of an expression control region of the PLS3 gene and a marker gene.
  • the marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction.
  • reporter genes such as alkaline phosphatase gene, chloramphenicol acetyltransferase gene, ⁇ -glucuronidase gene, ⁇ -galactosidase gene, and aequorin gene can be exemplified in addition to the above luciferase gene. Creation of a fusion gene and measurement of activity derived from a marker gene can be performed by known methods.
  • the substance selected by the screening method of the present invention can be positioned as a gene expression inhibitor for the PLS3 gene.
  • These substances are drugs that inhibit EM induction by suppressing the expression of the PLS3 gene and suppress cancer progression such as invasion and metastasis, drugs that prevent postoperative recurrence, and drugs that improve prognosis.
  • this method can be carried out by performing the following steps (1 ′) to (3 ′).
  • the cells used for the screening may be any cells that can express PLS3 gene and produce PLS3 protein regardless of whether they are endogenous or exogenous.
  • the origin of the PLS3 gene is not particularly limited, and may be derived from humans, or derived from mammals such as mice other than humans or other biological species.
  • PLS3 protein derived from human is preferable. Specific examples of such cells include cells capable of expressing a PLS3 gene other than hematopoietic cells, that is, solid tissue cells, and primary cultured cells of the cells isolated and prepared.
  • transformed cells prepared in a state capable of producing PLS3 protein by introducing an expression vector having PLS3 gene cDNA according to a conventional method can also be used.
  • the category of cells used for screening includes tissues that are aggregates of cells.
  • the condition for bringing the test substance into contact with the cell capable of producing PLS3 protein is not particularly limited, but the cell does not die and the PLS3 gene is expressed.
  • Selection of candidate substances can be performed, for example, by searching for substances that reduce the production amount of PLS3 protein by contacting the test substance with cells capable of producing PLS3 protein or a cell fraction thereof under the above conditions. Specifically, when a PLS3 protein-producing cell or a cell fraction thereof is cultured in the presence of a test substance, the PLS3 protein production amount corresponding to the above in the absence of the test substance or a cell fraction thereof The test substance brought into contact with a cell or a cell fraction can be selected as a candidate substance by using as an index the production amount (control production amount) of PLS3 protein obtained when the fraction is cultured.
  • Measurement (detection and quantification) of PLS3 protein production is performed by measuring the amount of PLS3 protein obtained from cells capable of producing PLS3 protein or its cell fraction, and an antibody against the PLS3 protein (anti-PLS3 antibody) (the detection reagent of the present invention described above) Can be carried out by performing a known method such as Western blotting, immunoprecipitation or ELISA. Specifically, in Western blotting, the detection reagent of the present invention is used as a primary antibody, followed by labeling with a radioisotope such as 125 I as a secondary antibody, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), or the like.
  • a radioisotope such as 125 I as a secondary antibody
  • a fluorescent substance such as horseradish peroxidase (HRP), or the like.
  • HRP horseradish peroxidase
  • the labeling can be performed using an antibody that binds to the primary antibody, and signals derived from these labeling substances are measured with a radiation measuring instrument (BAS-1800II, manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like. Further, after using the detection reagent of the present invention as a primary antibody, ECL Plus Western Blotting Detction System (Amersham Pharmacia Biotech) was used for detection according to the protocol, and multi-bioimager STORM860 (Amersham Pharmacia Biotech) Can also be measured.
  • a radiation measuring instrument BAS-1800II, manufactured by Fuji Film Co., Ltd.
  • a fluorescence detector or the like.
  • ECL Plus Western Blotting Detction System was used for detection according to the protocol, and multi-bioimager STORM860 (Amersham Pharmacia Biotech) Can also be measured.
  • a substance selected by the screening method of the present invention can be positioned as a PLS3 protein production inhibitor.
  • These substances are drugs that inhibit EM induction by inhibiting the production of PLS3 protein and suppress cancer progression such as invasion and metastasis, drugs that prevent recurrence after surgery, and drug candidates that improve prognosis It becomes.
  • the method can be carried out by performing the following steps (1 ′′) to (3 ′′).
  • (1 ") a step of contacting a test substance with a cell that reacts with PLS3 or a cell fraction prepared from the cell
  • (2 ) a step of contacting PLS3 with a cell that reacts with PLS3,
  • (3 ′′) a step of detecting the function or activity of PLS3 in a cell or cell fraction in contact with the test substance and PLS3; and
  • the cells that react with PLS3 used for such screening may be cells that exhibit some biological activity when brought into contact with PLS3.
  • Specific examples of such cells include colon cancer or breast cancer-derived cell lines and isolated primary cultured cells.
  • the category of cells used for screening includes tissues that are aggregates of cells.
  • Selection of candidate substances can be performed by contacting a test substance with cells that react with PLS3 or a cell fraction thereof under the above conditions to search for substances that reduce the action of PLS3.
  • the function of PLS3 that occurs when cells that respond to PLS3 in the presence of a test substance are cultured is that of PLS3 obtained when cells that respond to PLS3 corresponding to the above are cultured in the absence of the test substance.
  • the test substance can be selected as a candidate substance using an index lower than the function (control function) as an index.
  • Substances selected by the above screening methods (i) to (iii) have the effect of suppressing EMS induction by suppressing the increase (enhancement) of PLS3 gene expression in solid cancers, especially colon cancer or breast cancer tissue cells. Therefore, it can be used as an active ingredient of a drug that suppresses cancer progression (invasion or metastasis) of colon cancer or breast cancer. Furthermore, the substance selected by the screening methods (1) to (3) can be used as an active ingredient of a drug for preventing postoperative recurrence of a solid cancer patient and a drug for improving the prognosis.
  • the candidate substance selected by the above screening method can be further screened using a non-human animal having a solid cancer.
  • Candidate substances thus selected may be further subjected to safety tests using non-human animals, and further to clinical trials for patients with solid cancer. By carrying out these tests, the drug of the present invention is more practical. It is possible to select and acquire the active ingredients.
  • the material selected in this way is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic engineering, And can be used for the preparation of the medicament of the present invention.
  • test samples used in the experimental examples described later were prepared according to the following methods.
  • the test samples collected at Kyushu University Hospital, Teikyo University Hospital and related hospitals during 1991-2002 were used after obtaining written informed consent.
  • non-cancerous tissue (N) corresponding to tumor tissue (T) of the primary colorectal cancer was collected from 110 cases during surgery, and tumor perfusion venous blood (VDT) was collected during surgery, peripheral blood (PB) were obtained from 177 cases each before surgery.
  • PB peripheral blood
  • Peripheral blood (PB) used as a control group (control PB) was collected from 25 healthy volunteers, and portal vein blood (control VDT) used as a control for tumor perfusion venous blood (VDT) was collected from 24 people. Collected from patients with benign diseases.
  • Tumor tissue (T) and non-tumor tissue (N) from the colorectal cancer primary lesion are embedded in Tissue Tek OCT medium (Sakura, Tokyo, Japan) at -80 °C until RNA extraction, and then RNA is extracted. CDNA was prepared. RNA extraction from VDT and PB was performed using PAXgene blood RNA kit (Qiagen K.K. GmbH, Germany).
  • colon cancer cell line LoVo and rectal cancer cell line CaR-1 were obtained from Cell Resource Center of Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). The cells were cultured in F12 medium and MEM medium containing 10% FBS, 100 units / mL penicillin and 100 units / mL streptomycin, respectively (5% CO 2 , 37 ° C). In the following experimental examples, these cell lines are collectively referred to as “colon cancer cell lines”.
  • PLS3 mRNA amplification was performed by first denaturing at 95 ° C for 10 minutes, followed by denaturation at 95 ° C for 10 seconds for 40 cycles, annealing for 10 seconds at 62 ° C, and extension reaction for 67 seconds. C. for 10 seconds.
  • the amplification of mRNA of genes other than PLS3 is the same as described above except that the temperature used for annealing is changed to 60 ° C instead of 62 ° C, and the temperature used for the extension reaction is changed to 67 ° C instead of 67 ° C. Performed under conditions.
  • the amount of mRNA was standardized using GAPDH mRNA as an internal control.
  • LoVo-PLS3 cells into which PLS3 had been introduced were selected using neomycin (600 mg / mL) (Invitrogen Crop., Carlsbad, Calif., USA). Further, as a control cell, a mock vector was used instead of the above vector, and the colon cancer cell line LoVo was similarly transfected to prepare “LoVo-mock cells”.
  • Nonspecific proteins were blocked with 3% FBS (in PBS) for 30 minutes, washed, and PLS3-expressing cells and vimentin-expressing cells were removed using the avidin-biotin-peroxidase method (LSAB kit; DAKO, Kyoto, Japan) It was detected (see Masuda, TA, et al., Cancer Res 62, 3819-3825 (2002)). All sections were nuclear stained with hematoxylin.
  • LoVo-PLS3 cells and LoVo-mock cells were placed on a glass cover slip at a rate of 1.0 ⁇ 10 5 cells / mL and incubated for 48 hours. Thereafter, it was washed with PBS, and fixed by treatment with 3.7% formaldehyde for 10 minutes and -20 ° C. with 90% methanol for 5 minutes. After washing with PBS, cells were treated with anti-F-actin antibody (Molecular Probes, Eugene, OR, USA) and anti-E-cadherin antibody (BD Biosciences Pharmingen, San Diego, CA, USA) for 1 hour at room temperature. Reacted. All test cells were nuclear stained with DAPI. The cells were photographed after excitation with a confocal LSM510 microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) ( ⁇ 63magnification).
  • the fluorescence intensity of the invasive cells that migrated to the lower surface through the cell membrane was measured with a fluorescence plate reader (excitation wavelength 485 nm / emission wavelength 530 nm) using Multilabel Plate Counter VICTOR3 (PerkinElmer, Inc., USA).
  • PLS3 RNA interference are shown below PLS3-specific siRNA (Santa Cruz Biotechnology Inc., CA, USA) and PLS3 inhibition experiments (SEQ ID NO: 5-10) was performed.
  • sc-43215 A CCACGGAUAGAUAUUAACAtt (SEQ ID NO: 5)
  • UGUUAAUAUCUAUCCGUGGtt SEQ ID NO: 6
  • sc-43215 B GAACUCUCUUGGUGUCAAUtt (SEQ ID NO: 7) AUUGACACCAAGAGAGUUCtt (SEQ ID NO: 8)
  • sc-43215 C GAAGAGUGGCAAUCUAACAtt (SEQ ID NO: 9) UGUUAGAUUGCCACUCUUCtt (SEQ ID NO: 10).
  • siRNA Silencer TM Negative Control # 1 siRNA (Ambion, USA) was used as siRNA for negative control.
  • the transfection reagent Lipofectamine RNAi MAX (Invitrogen Crop., Carlsbad, CA, USA) and PLS3-specific siRNA (or Silencer TM Negative Control # 1 siRNA for negative control) were mixed with After adding and incubating in 6 mL wells of 2 mL capacity of the titer plate, human colon cancer cell line CaR-1 (1.5 ⁇ 10 5 cells / well) is added and humidified (37 ° C, 5% CO 2 ). Incubated.
  • Lipofectamine RNAi MAX Invitrogen Crop., Carlsbad, CA, USA
  • PLS3-specific siRNA or Silencer TM Negative Control # 1 siRNA for negative control
  • Labeled mononuclear cells were analyzed and sorted using the BD FACS Vantage SE Cell Sorter System, and the data were analyzed with FACSDiva software (Becton Dickinson). Gating was performed based on a negative-control staining profile.
  • CD45 (-) peripheral blood fluorescence immunocytochemistry colon cancer patients circulating tumor cells sorted by BD FACSVantage SE Cell Sorter System, CD45 (-) were obtained circulating cells.
  • the obtained CD45 ( ⁇ ) circulating cells were seeded on a glass cover glass and incubated at 37 ° C. for 12 hours. After washing with PBS to remove non-adherent circulating cells other than cancer cells, incubation with anti-cytokeratin antibody (clone MNF-116, DAKO) and anti-PLS3 antibody (Santa Cruz Biotechnology) for 1 hour at room temperature, then- Immobilization with 90% methanol at 20 ° C.
  • anti-cytokeratin antibody clone MNF-116, DAKO
  • anti-PLS3 antibody Santa Cruz Biotechnology
  • anti-mouse and anti-goat fluorescently labeled rabbit antibodies were used for 1 hour incubation at room temperature. All sections were stained with DAPI. The immunostained cells were observed with a confocal LSM510 microscope (Carl Zeiss Microimaging) (magnification ⁇ 63).
  • FIG. 1 a shows images of LoVo-PLS3 cells (right side) and LoVo-mock cells (left side).
  • LoVo-PLS3 cells have undergone morphological changes, proteins related to PLS3 and epithelial-mesenchymal transition (EMT) are found in LoVo-PLS3 cells and control LoVo-mock cells. And E-cadherin) and F-actin expression levels were measured by immunocytostaining and fluorescent immunostaining.
  • the expression levels of PLS3 and mesenchymal marker vimentin were determined by immunocytostaining according to the method described in experimental method (3), and the expression levels of epithelial markers E-cadherin and F-actin were determined by experimental method. According to the method described in (4), it was examined by fluorescent immunocell staining. The results of immune cell staining and fluorescent immune cell staining are shown in FIGS. 1b and 1c, respectively. As a result, the expression levels of PLS3 and Vimentin were enhanced in LoVo-PLS3 cells compared to LoVo-mock cells, but the expression levels of E-cadherin were suppressed ((FIG.
  • PLS3-expressing cells are switched from the epithelial cell phenotype to the mesenchymal cell phenotype, and epithelial-mesenchymal transition (EMT) is induced. It was confirmed that -actin was accumulated.
  • LoVo-PLS3 cells and LoVo-mock cells are also BD BioCoatTM Tumor Invasion System (Becton Dickinson, San San Jose, CA, USA) and Cultrex 24 Well Cell Cell Migration, GREthergen , MD, USA) was used to measure the invasion ability and migration ability of each cell according to their protocol.
  • the outline of the Invitro assay method used for measuring the invasion ability and migration ability of each cell is described in the experimental methods (5) and (6), respectively.
  • the results relating to invasive ability are shown on the left in FIG. 2, and the results relating to migration ability are shown on the right in FIG.
  • PLS3SmRNA expressing cells are both about 1.5 times more potent in invasion and migration than cells that do not express PLS3 mRNA (LoVo-mock cells).
  • LoVo cell line LoVo-PLS3 cell, LoVo-mock cell
  • an anticancer agent (5-FU).
  • LoVo-mock cells and LoVo-PLS3 cells were seeded in 96-well plates, and 24 hours later, 5-FU was treated with the concentrations shown in FIG. 4 (0, 1, 5, and 10 ⁇ g / ml).
  • the MTT assay described in (4) was performed, and the sensitivity (resistance) to 5-FU was evaluated from the cell proliferation rate. The results are shown in FIG. As shown in FIG.
  • LoVo-PLS3 cells cells that proliferate PLS3 mRNA grow more in spite of higher 5-FU concentrations than cells that do not express PLS3 mRNA (LoVo-mock cells). This suggests that EMT-induced LoVo-PLS3 cells are resistant to 5-FU.
  • EMT is induced in colon cancer cells by the expression of PLS3 mRNA, which is known to control cell morphology and migration ability while regulating actin polymerization and depolymerization, invasion ability Further, it was confirmed that the migration ability was enhanced, the cell proliferation ability was lowered, and the sensitivity to the anticancer agent was weakened (tolerated).
  • the expression levels of the mesenchymal markers vimentin, FN1, N-cadherin, and FOXC2 are expressed in cells that express PLS3 mRNA (LoVo-PLS3 cells) but do not express PLS3 mRNA (LoVo- mock cells) were confirmed to be significantly higher (p ⁇ 0.05).
  • the mRNA expression level and protein production level of PLS3 in LoVo-PLS3 cells were significantly higher than those in LoVo-mock cells.
  • the mRNA expression level and protein production level of PLS3 in CaR-1 PLS3 siRNA cells were significantly lower than those in CaR-1 control siRNA cells.
  • the PLS3 mRNA expression level was substantially reduced by 70% in cells treated with PLS3 siRNA (PLS3 siRNA cells), and it was confirmed that PLS3 siRNA suppresses PLS3 mRNA expression production.
  • EMT-related genes E-cadherin, TWIST, SNAIL, vimentin
  • PLS3 siRNA cells PLS3 siRNA cells
  • CaR-1 cells treated with negative control siRNA control siRNA cells
  • epithelial-mesenchymal transition switches to mesenchymal epithelial transition (MET) by suppressing the expression of PLS3PL mRNA with siRNA.
  • EMT epithelial-mesenchymal transition
  • MET mesenchymal epithelial transition
  • the PLS3 gene is a gene that induces EMT related to cancer cell invasion and migration (EMT-inducible gene).
  • lymph node metastasis 0.06
  • venous invasion 0.013
  • Fig. 7b shows the survival rate for 5 years after the surgery for the high PLS3 expression group (26 cases) and the low PLS3 expression group (84 cases) (Kaplan-Meier survival).
  • the following table shows the results of multivariate analysis of the effects of prognosis on the factors of PLS3 expression in tumor tissue (T), tumor size, lymph node metastasis, and liver metastasis using Cox proportional hazard regression.
  • the relative risk (RR) was ranked next to the risk due to lymph node metastasis.
  • the PLS3 mRNA expression level (mean ⁇ sd) in the primary lesion tissue of each case was Dukes A case: 1.22 ⁇ 0.93, Dukes B case: 1.29 ⁇ 0.9, Dukes C case: 1.36 ⁇ 1.07, Dukes D cases: 2.38 ⁇ 1.58, confirming that PLS3 mRNA expression in the primary tissue was significantly higher in Dukes D (with distant metastasis) cases.
  • This result indicates that cases with high PLS3 mRNA expression in the primary lesion tissue formed distant metastasis by EMT induction.
  • FIG. 9a shows the results of immunohistochemical staining of cancer (T) tissue and non-cancer (N) tissue of colorectal cancer patients using an anti-PLS3 antibody according to the method described in experimental method (10).
  • the “N” side is a non-cancer cell region
  • the “T” side is a cancer cell region.
  • Arrows indicate cancer cells that overexpressed PLS3.
  • FIG. 9b is an enlarged view of a part of FIG.
  • the PLS3 gene was expressed more strongly in cancer cells (T) than in normal cells (N) even in the same tissue.
  • the PLS3 gene was particularly strongly expressed in a group of small cancer cells called “budding tumor cells” that was distant from the main tumor cells and invaded the non-cancer cell side.
  • the cutoff value of VDT / portal blood was determined to be 0.0569 (average +2 sd), and the cutoff value of PB was determined to be 0.0546 (average +2 sd).
  • Fig. 10a shows the relationship between PLS3 expression status in tumor perfusion venous blood (VDT) and survival rate (non-disease survival rate (DFS), overall survival rate (OS)).
  • VDT tumor perfusion venous blood
  • DFS non-disease survival rate
  • OS overall survival rate
  • FIG. 10b The relationship between (disease survival rate (DFS), overall survival rate (OS)) is shown in FIG. 10b.
  • VDT tumor return venous blood
  • PB peripheral blood
  • DFS disease-free survival
  • OS overall survival
  • cancer stem cells CRC overexpressing PLS3 mRNA show high malignancy in both primary tumors and circulating cells.
  • Tables 8 and 9 show the relationship between the high PLS3 expression group and clinicopathological factors for reflux venous blood (VDT) (portal blood) and peripheral blood (PB).
  • VDT reflux venous blood
  • PB peripheral blood
  • FIG. 11 shows normal tissues (from the left in the figure, adrenal gland, bone marrow, cerebrum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, liver, placenta, peripheral blood, prostate, salivary gland, skeletal muscle, small intestine. , PLS3 mRNA expression distribution in spinal cord, spleen, stomach, testis, thyroid, trachea, uterus).
  • the PLS3 gene is a gene expressed in solid tissues and not expressed in hematopoietic cells (Delanote, V., et al., Acta Pharmacol Sin 26, 769-779 (2005), Delanote, V., et al. Traffic 6, 335-345 (2005).).
  • PLS3 mRNA expression is not originally observed in bone marrow, reflux venous blood (portal vein blood) and peripheral blood. Therefore, from the above results, by measuring the expression level of PLS3 mRNA in reflux venous blood (portal vein blood) and peripheral blood, it is possible to detect cancer of colorectal cancer without measuring the expression level of PLS3 mRNA in cancer cells (primary focus cells). The degree of progression (invasion and metastasis) and postoperative prognosis can be predicted.
  • PLS3 / GAPDH ⁇ PB cut-off value high PLS3 expression
  • PLS3 / GAPDH ⁇ PB low PLS3 expression group
  • CD45 ( ⁇ ) cells not expressing CD45 are cells other than blood cells.
  • the collected CD45 ( ⁇ ) cells were subjected to cytokeratin staining and fluorescent double-immunization of PLS3 using an anticytokeratin antibody and an anti-PLS3 antibody according to the method described in Experimental Procedure (10).
  • the results are shown in FIG. In FIG. 13, the left figure shows the result of cytokeratin staining, the middle figure shows the result of PLS3 staining, and the right figure shows the result of overlapping the two. This figure shows that cancer cells are present in the peripheral fluid, and the cancer cells express PLS3 mRNA.
  • Cancer cells that flow through the blood usually die immediately after undergoing apoptosis.
  • free cancer cells expressing PLS3 mRNA which is resistant to apoptosis, are present in the peripheral blood of colorectal cancer patients.
  • FIG. 14a The result is shown in FIG. 14a. As shown, there was no significant difference in the expression level of PLS3 mRNA between the cancer tissue (T) and the normal tissue (N).
  • PLS3 mRNA / GAPDH mRNA 13 cases in which the expression of PLS3 mRNA in cancer tissue (T) is 4.1 or more relative to the expression of GAPDH mRNA are referred to as “high PLS3 expression group” (PLS3 (T) / GAPDH (T) ⁇ 4.1).
  • 89 cases less than 4.1 were designated as “low PLS3 expression group” (PLS3 (T) / GAPDH (T) ⁇ 4.1).
  • FIG. 14b shows the survival rate for 10 years after surgery for the high PLS3 expression group (17 cases) and the low PLS3 expression group (77 cases) (Kaplan-Meier all survival). As shown in FIG. 14b, patients belonging to the high PLS3 expression group had a significantly worse prognosis (10-year survival rate) than patients belonging to the low PLS3 expression group (p ⁇ 0.05).
  • Cox proportional hazard regression was used to determine the effects of high and low PLS3 mRNA expression, lymph node metastasis, and progesterone receptor (negative / positive) components on prognosis (survival after surgery) in cancer tissues (T).
  • the table below shows the results of univariate analysis and multivariate analysis.
  • the relative risk (RR) was ranked next to the risk due to lymph node metastasis.
  • FIG. 15 shows the relationship between the PLS3 expression status in peripheral blood (PB) and the survival rate (overall survival rate (OS), non-disease survival rate (DFS)).
  • FIG. 16 shows the relationship between the PLS3 expression status in the bone marrow and the overall survival rate (OS) and the disease free survival rate (DFS).
  • Cox proportional hazard regression was used to determine the effects of high and low PLS3 mRNA expression, lymph node metastasis, and progesterone receptor (negative / positive) components on the prognosis (postoperative overall survival) in peripheral blood.
  • the table below shows the results of univariate analysis and multivariate analysis.
  • PLS3 gene is a gene that induces epithelial-mesenchymal transition (EMT)
  • EMT epithelial-mesenchymal transition
  • PLS3 mRNA expression in the primary tumor, bone marrow, and blood is related to recurrence and poor prognosis in patients with solid cancer, especially colorectal cancer and breast cancer, and the risk of poor prognosis It can be a highly accurate marker for identifying high patients.
  • EMT control by PLS3 is clinically useful in controlling cancer progression (invasion, metastasis) of solid cancer, particularly colorectal cancer.
  • PLS3 expression in peripheral blood can be a prognosis and recurrence predictor even in Dukes B stage colorectal cancer.
  • the expression of PLS3 in the peripheral blood is clinically useful because it exceeds the currently used progression classification.
  • SEQ ID Nos: 3 to 4 and 11 to 34 show the base sequences of primers specific to each gene described in Table 1.
  • SEQ ID Nos: 5 to 10 show the base sequences of siRNAs of the PLS3 gene.

Abstract

Disclosed is a recurrence/prognosis factor for solid cancer such as colorectal cancer and breast cancer, which is identified as a biomolecule involved in the epithelial-mesenchymal transition (EMT) of cancer cells of the solid cancer and the recurrence and prognosis of the solid cancer. Specifically disclosed are: a method for predicting the EMT of cancer cells of the solid cancer, the degree of progression (invasion and metastasis) of the solid cancer, and the recurrence or prognosis of the solid cancer using, as a measure, the increase in the expression of mRNA for PLS3 (which is employed as the factor) in peripheral blood; and a test agent which can be used in the method effectively.

Description

固形がんの再発および予後因子、およびその臨床利用Solid cancer recurrence and prognostic factors and their clinical use
　本発明は、固形がん、特に大腸がんまたは乳がんの患者を対象として、癌細胞の上皮間葉移行（EMT）に関わる因子（以下、「EMT因子」と称する）、ならびに治療後の再発または予後を予測するために有効な因子（以下、これを「再発予後因子」と称する）に関し、これらの因子（以下、これらの因子を総称して「本発明因子」と称する）を検出するための試薬に関する。また本発明は、当該本発明因子を指標として、固形がん、特に大腸がんまたは乳がんの患者について、癌細胞の上皮間葉移行、ならびに術後の再発または予後を予測する方法に関する。 The present invention is intended for patients with solid cancer, particularly colorectal cancer or breast cancer, factors relating to epithelial-mesenchymal transition (EMT) of cancer cells (hereinafter referred to as “EMT factor”), and recurrence after treatment or Regarding factors effective for predicting prognosis (hereinafter referred to as “relapse prognostic factors”), these factors (hereinafter collectively referred to as “factors of the present invention”) are detected. It relates to a reagent. The present invention also relates to a method for predicting epithelial-mesenchymal transition of cancer cells and postoperative recurrence or prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer, using the factor of the present invention as an index.
　さらに本発明は、固形がん、特に大腸がんまたは乳がんについて、癌細胞の上皮間葉移行（EMT）を抑制できる成分、すなわち浸潤や転移などの癌進展を抑制するために有効な成分、またはこれらのがん患者において治療後の再発を予防し、また予後を向上するために有効な成分、および当該成分を有効成分とする医薬組成物に関する。 Furthermore, the present invention relates to a component capable of suppressing cancer cell epithelial-mesenchymal transition (EMT) for solid cancer, particularly colorectal cancer or breast cancer, that is, an effective component for suppressing cancer progression such as invasion and metastasis, or The present invention relates to an ingredient effective for preventing recurrence after treatment in these cancer patients and improving prognosis, and a pharmaceutical composition containing the ingredient as an active ingredient.
　さらにまた本発明は、上記成分、すなわち固形がん、特に大腸がんまたは乳がんについて、癌細胞の上皮間葉移行（EMT）を抑制し、癌進展を抑制するうえで有効な成分、ならびにこれらのがん患者において治療後の再発を予防し、また予後を向上するために有効な成分を探索するためのスクリーニング方法に関する。 Furthermore, the present invention relates to the above components, that is, components effective for suppressing cancer cell epithelial-mesenchymal transition (EMT) and cancer progression for solid cancer, particularly colon cancer or breast cancer, and these The present invention relates to a screening method for searching for effective components for preventing recurrence after treatment and improving prognosis in cancer patients.
　固形がんの診断において、リンパ節中の癌細胞の存在（リンパ節転移）は、治療方法の決定や術後の化学療法の決定に有益な情報であるとともに、特に大腸がんや乳がんにおいては特に重要な予後予測因子となっている。その転移が認められたものは明らかに予後が悪い。例えば、日本の直腸がん患者の場合、リンパ節転移のない筋層を超えた浸潤のある直腸がん（Dukes B）の5年生存率は79％であるが、リンパ節転移のある直腸がん（Dukes C）の5年生存率は52％まで低下する。また乳がん患者についても同様の傾向がある。しかし、リンパ節転移のない癌症例（Dukes B）において、再発・予後不良群と無再発・予後良好群との違いはどこにあるのか、未だ明らかになっていない。 In the diagnosis of solid cancer, the presence of cancer cells in lymph nodes (lymph node metastasis) is useful information for deciding treatment methods and postoperative chemotherapy, especially in colorectal cancer and breast cancer. It is an especially important prognostic factor. Those with such metastases clearly have a poor prognosis. For example, in Japanese rectal cancer patients, rectal cancer with invasion beyond the muscle layer without lymph node metastasis (Dukes B) has a 79-year survival rate of 79%, but rectal cancer with lymph node metastasis The 5-year survival rate of Dukes C drops to 52%. There is a similar trend for breast cancer patients. However, in cancer cases without lymph node metastasis (Dukes B), it is not yet clear where the difference between the relapse / poor prognosis group and the non-relapse / good prognosis group is.
　リンパ節転移陰性にも関わらず、再発したり予後不良の症例が存在する原因として、現在の医療技術レベルでは検出不可能な潜在的癌細胞が存在していること、およびその潜在的癌細胞が治療抵抗性を有し、転移を完遂する能力を有していることが示唆されている（非特許文献１）。近年、様々な悪性腫瘍について微小な癌細胞（微小転移）の存在が検討されており、胃がん、直腸がんおよび食道がんなどの消化器がんに関して、免疫組織学的染色法を用いたリンパ節微小転移の存在とそれと予後との間に有意な関係があることが報告されている。また1995年に、本件発明者の三森らによって、病理診断転移陰性リンパ節において遊離癌細胞の存在が報告されて以来（非特許文献２）、末梢血液や骨髄中における遊離癌細胞の存在とその臨床的意義も明らかにされている。しかしながら、結論として確かに骨髄中に遊離癌細胞は存在するものの、それが再発や予後に直接関係することはなく、両者は互いに無関係であることが報告されている（非特許文献３）。しかしながら、単に骨髄中に遊離癌細胞が存在することが再発や予後不良に直接関係するか否かについては、未だに結論がでていない。 The cause of the recurrence or poor prognosis in spite of negative lymph node metastasis is the presence of potential cancer cells that cannot be detected at the current level of medical technology, and the potential cancer cells It has been suggested that it has treatment resistance and has the ability to complete metastasis (Non-patent Document 1). In recent years, the existence of minute cancer cells (micrometastasis) has been studied for various malignant tumors, and lymphatics using immunohistological staining for gastrointestinal cancers such as gastric cancer, rectal cancer, and esophageal cancer. It has been reported that there is a significant relationship between the presence of nodal micrometastasis and its prognosis. In 1995, the present inventor Mimori et al. Reported the presence of free cancer cells in pathologically diagnosed metastasis-negative lymph nodes (Non-patent Document 2), and the presence of free cancer cells in peripheral blood and bone marrow Clinical significance has also been clarified. However, as a conclusion, although free cancer cells certainly exist in the bone marrow, they are not directly related to recurrence or prognosis, and both are reported to be independent of each other (Non-patent Document 3). However, there is still no conclusion as to whether the presence of free cancer cells in the bone marrow is directly related to recurrence or poor prognosis.
　リンパ節転移以外の予後予測因子として、癌の組織型、細胞異型度、遺伝子の変異例（癌遺伝子）、癌抑制遺伝子、ホルモン受容体などが知られている。しかし、リンパ節転移にまさる、またはそれに匹敵する予後予測因子は知られておらず、精度の高い予後予測因子を見出すことが急務になっている。特に、固形がんの場合、末梢血を材料として、より精度高く、癌進展の度合い（EMT、浸潤、転移など）ならびに再発や予後が予測できれば、患者に苦痛を与えることなく、より適確な治療方針や術後の補助療法を選択することが可能になる。 Known prognostic factors other than lymph node metastasis include cancer tissue type, cell atypia, gene mutation (oncogene), tumor suppressor gene, hormone receptor, and the like. However, there is no known prognostic predictor over or comparable to lymph node metastasis, and there is an urgent need to find a highly accurate prognostic predictor. In particular, in the case of solid cancer, if peripheral blood is used as a material and the degree of cancer progression (EMT, invasion, metastasis, etc.) and recurrence and prognosis can be predicted, the patient can be more accurate without causing pain. It is possible to select a treatment policy and postoperative adjuvant therapy.
　ちなみに、従来、臨床で使用されている腫瘍マーカーとして、血中の癌胎児性蛋白抗原（CEA：carcinoembrionic antigen）、CA19-9（大腸がん）およびCA15-3（乳がん）などがあり、これらのタンパク質を抗体で検出することで、がんを診断したり、術後の経過観察または治療効果の判定が行われている。また、末梢血や門脈でのCEAおよびCA19-9のmRNA発現が、大腸がんの予後予測因子になりえることが報告されている（非特許文献４）
　ところで、Plastin 3（PLS3）は、細胞骨格維持に重要な役割を担うアクチン結合タンパクの３種のisoformの一つであり、細菌が細胞内に移行する際に足場になることが知られている。また当該遺伝子は、腎臓や小腸で発現するPlastin 1（PLS1）遺伝子や造血細胞で発現するplastin 2（PLS2）遺伝子とは異なり、固体組織全体で局在的に発現する。当該遺伝子について、従来、皮膚Ｔ細胞リンパ腫との関係（非特許文献５）、膀胱がん、前立腺がん、および頭頚部がんとの関係（非特許文献６および７）が報告されているが、これまで大腸がんや乳がんなどの臨床検体、および末梢血でのPLS3発現の有無ならびにその意義を調べた報告はない。 By the way, the tumor markers that have been used in clinical practice include carcinoembrionic antigen (CEA), CA19-9 (colon cancer) and CA15-3 (breast cancer) in blood. Cancer is diagnosed by detecting a protein with an antibody, follow-up after surgery, or determination of a therapeutic effect. Further, it has been reported that mRNA expression of CEA and CA19-9 in peripheral blood and portal vein can be a prognostic factor of colorectal cancer (Non-patent Document 4).
By the way, Plastin 3 (PLS3) is one of three isoforms of actin-binding proteins that play an important role in maintaining the cytoskeleton, and is known to serve as a scaffold when bacteria migrate into cells. . In addition, the gene is expressed locally throughout the solid tissue, unlike the Plastin 1 (PLS1) gene expressed in the kidney and small intestine and the plastin 2 (PLS2) gene expressed in hematopoietic cells. Regarding the gene, a relationship with skin T-cell lymphoma (Non-Patent Document 5) and a relationship with bladder cancer, prostate cancer, and head and neck cancer (Non-Patent Documents 6 and 7) have been reported. So far, there are no reports of the presence or significance of PLS3 expression in clinical specimens such as colorectal cancer and breast cancer, and peripheral blood.
　前述するように、固形がん、特に大腸がんや乳がんでは、根治術施行にも拘わらず、またリンパ節転移陰性症例にも拘わらず、再発したり予後が不良な症例が多く、再発ならびに予後不良に関わる因子、具体的には潜在的癌細胞に治療抵抗性を与え、また転移を完遂する能力を与える分子が存在することが示唆されている。 As mentioned above, in solid cancers, especially colorectal cancer and breast cancer, there are many cases of recurrence or poor prognosis regardless of whether radical surgery is performed or patients with negative lymph node metastasis. It has been suggested that there are molecules that contribute to the failure, specifically the ability to confer resistance to potential cancer cells and the ability to complete metastasis.
　本発明は、かかる固形がん、特に大腸がんや乳がんの患者について、癌細胞の上皮間葉移行（EMT）に関わる生体内分子、ならびに再発ならびに予後に関わる生体内分子を同定し、これらを、固形がん、特に大腸がんまたは乳がんに関するEMT因子、ならびにこれらの固形がんの患者における再発または予後を予測するための因子（再発予後因子）として提供することを目的とする。また、本発明は、これらのEMT因子および再発予後因子（総称：本発明因子）を指標として、固形がん、特に大腸がんにおける癌細胞のEMT化およびそれによる癌進展度（湿潤）や、大腸がんや乳がん等のがん患者について再発または予後を予測するために有用な検査薬を提供することを目的とする。また、本発明は、上記本発明因子を指標として、これらの固形がん、特に大腸がんの細胞についてEMT化およびそれによる癌進展度（湿潤）を予測するための方法、ならびに固形がん、特に大腸がんや乳がんの患者について再発または予後を予測するための方法を提供することを目的とする。 The present invention identifies in vivo molecules related to epithelial-mesenchymal transition (EMT) of cancer cells and in vivo molecules related to recurrence and prognosis in patients with such solid cancers, particularly colorectal cancer and breast cancer. It is intended to provide EMT factors relating to solid cancer, particularly colorectal cancer or breast cancer, and factors for predicting recurrence or prognosis in patients with these solid cancers (recurrence prognosis factor). In addition, the present invention uses these EMT factors and recurrence prognostic factors (generic name: factor of the present invention) as an index, and EMT of cancer cells in solid cancer, particularly colorectal cancer, and the degree of cancer progression (wet) thereby, The purpose is to provide a useful test drug for predicting recurrence or prognosis of cancer patients such as colorectal cancer and breast cancer. In addition, the present invention uses the above-described factor of the present invention as an index, and a method for predicting EMT and cancer progression (wet) due to these solid cancers, particularly colon cancer cells, and solid cancer, In particular, it aims to provide a method for predicting recurrence or prognosis for patients with colorectal cancer and breast cancer.
　さらに本発明は、固形がん、特に大腸がんについて、癌細胞のEMT化を抑制し、癌進展（浸潤）を抑制するための有効成分、または固形がん、特に大腸がんや乳がんの患者において治療後の再発を予防し、また予後を向上するための有効成分を提供すること、また当該成分を含有する医薬組成物を提供することを目的とする。 Furthermore, the present invention relates to an active ingredient for suppressing the EMT of cancer cells and suppressing cancer progression (invasion) for solid cancer, particularly colon cancer, or a patient with solid cancer, particularly colon cancer or breast cancer. An object of the present invention is to provide an active ingredient for preventing recurrence after treatment and improving prognosis, and to provide a pharmaceutical composition containing the ingredient.
　さらにまた本発明は、固形がん、特に大腸がんについて、癌細胞のEMT化を抑制し、癌進展（浸潤）を抑制するために有効な成分、ならびに固形がん、特に大腸がんまたは乳がんの患者において治療後の再発を予防し、また予後を向上するために有効な成分を探索するためのスクリーニング方法を提供することを目的とする。 Furthermore, the present invention relates to a solid cancer, particularly colorectal cancer, an ingredient effective for suppressing cancer cell EMT and inhibiting cancer progression (invasion), and solid cancer, particularly colorectal cancer or breast cancer. It is an object of the present invention to provide a screening method for searching for an effective component for preventing recurrence after treatment and improving the prognosis in patients of the present invention.
（Ａ）本発明者らは、上記目的を達成すべく、細胞の遊走能に影響を与える細胞の形態および骨格構造を制御するアクチンに結合するタンパク質Plastin 3（PLS3）に着目して、大腸癌細胞株および乳癌細胞株に、PLS3遺伝子をトランスフェクションしたところ、下記の知見を得た。
　（1）外因性PLS3遺伝子の導入発現により、大腸がんなどの固形がんの細胞が上皮表現型から間葉表現型に移行され（上皮間葉移行（EMT）の誘導）、間葉マーカー遺伝子の発現レベルが上昇すること、
（2）外因性PLS3遺伝子の導入発現により、大腸がんなどの固形がんの細胞の浸潤能および遊走能が亢進すること、
（3）TGFβ刺激によって上皮性の癌細胞のEMTを誘導すると、PLS3mRNA発現は上昇した。またそのEMT誘導細胞にPLS3 siRNAを投与すると間葉マーカー遺伝子の発現レベルが低下し、上皮マーカー遺伝子の発現レベルが上昇する（間葉上皮移行の誘導）。 (A) In order to achieve the above object, the present inventors have focused on a protein Plastin 3 (PLS3) that binds to actin that controls cell morphology and skeletal structure that affects cell migration ability. When the PLS3 gene was transfected into cell lines and breast cancer cell lines, the following findings were obtained.
(1) By introducing and expressing exogenous PLS3 gene, cells of solid cancer such as colorectal cancer are transferred from epithelial phenotype to mesenchymal phenotype (induction of epithelial-mesenchymal transition (EMT)), and mesenchymal marker gene The expression level of
(2) Transduction and expression of exogenous PLS3 gene enhances the invasion and migration of solid cancer cells such as colorectal cancer,
(3) When EMT of epithelial cancer cells was induced by TGFβ stimulation, PLS3 mRNA expression increased. In addition, when PLS3 siRNA is administered to the EMT-induced cells, the expression level of the mesenchymal marker gene decreases and the expression level of the epithelial marker gene increases (induction of mesenchymal epithelial transition).
　これらの知見から、PLS3遺伝子は、大腸がんなどの固形がんにおいて、癌細胞の浸潤や遊走に関係する上皮間葉移行（EMT）を誘導する遺伝子（EMT誘導遺伝子）であると考えられた。 Based on these findings, the PLS3 gene was thought to be a gene that induces epithelial-mesenchymal transition (EMT) related to cancer cell invasion and migration in solid tumors such as colorectal cancer (EMT-inducible gene). .
　（Ｂ）さらに本発明者らは、100名以上の大腸がん患者を対象として、原発巣組織、非腫瘍組織、門脈血/腫瘍還流静脈血、および末梢血中のPLS3 mRMAの発現と、臨床病理的要素（リンパ節転移、リンパ液浸潤、静脈浸潤、肝転移、Dukes分類）並びに治療後5年間の再発状況および予後との関係を調べたところ、下記の知見を得た。
　（4）原発巣組織および門脈血／腫瘍還流静脈血のみならず、末梢血液中にもPLS3 mRNAの発現が認められること、
（5）大腸がん患者の末梢血液中には、遊離癌細胞が存在し、当該細胞にはPLS3 mRNAの発現が認められること、
（6）原発巣組織、門脈血／腫瘍還流静脈血および末梢血液（遊離癌細胞も含まれる）におけるPLS3 mRNAの高い発現と、リンパ節転移、リンパ液浸潤、静脈浸潤、肝転移、ならびに再発および予後不良との間には正の相関関係があること。 (B) In addition, the present inventors targeted PLS3 mRMA expression in primary tumor tissue, non-tumor tissue, portal vein blood / tumor reflux venous blood, and peripheral blood for more than 100 colon cancer patients, The following findings were obtained when investigating the clinicopathological factors (lymph node metastasis, lymph fluid invasion, venous invasion, liver metastasis, Dukes classification) and the recurrence status and prognosis after 5 years of treatment.
(4) PLS3 mRNA expression is observed not only in the primary focus tissue and portal blood / tumor reflux venous blood but also in peripheral blood,
(5) The presence of free cancer cells in the peripheral blood of colorectal cancer patients, and the expression of PLS3 mRNA is observed in the cells,
(6) High expression of PLS3 mRNA in primary tissue, portal vein / tumor reflux venous blood and peripheral blood (including free cancer cells), lymph node metastasis, lymph fluid infiltration, venous invasion, liver metastasis, and recurrence and There is a positive correlation with poor prognosis.
　（Ｃ）さらに本発明者らは、乳がん患者を対象として、原発巣組織、非腫瘍組織、末梢血および骨髄におけるPLS3 mRMAの発現と、臨床病理的要素（リンパ節転移、ホルモン受容体（陰性/陽性）など）並びに治療後約10年間の再発状況および予後との関係を調べたところ、下記の知見を得た。
　（5）原発巣のみならず、末梢血液および骨髄中にもPLS3 mRNAの発現が認められること、
（6）原発巣、末梢血および骨髄中におけるPLS3 mRNAの高い発現と、リンパ節転移、ホルモン受容体陰性、ならびに再発および予後不良との間には正の相関関係があること。 (C) Furthermore, the present inventors have studied the expression of PLS3 mRMA in primary tumor tissue, non-tumor tissue, peripheral blood and bone marrow and clinicopathological factors (lymph node metastasis, hormone receptor (negative / negative) in breast cancer patients. Positive)) and the relationship with the recurrence status and prognosis about 10 years after treatment, the following findings were obtained.
(5) PLS3 mRNA expression is observed not only in the primary lesion but also in the peripheral blood and bone marrow,
(6) There is a positive correlation between high expression of PLS3 mRNA in the primary lesion, peripheral blood and bone marrow and lymph node metastasis, hormone receptor negative, recurrence and poor prognosis.
　すなわち、これらの知見からわかるように、固形がんについて、原発巣組織のみならず、門脈血、末梢血、および骨髄中におけるPLS3 mRNAの高い発現レベルは、癌進展（浸潤、転移）、再発および予後不良と正の相関関係を示すことから、これらの血液や骨髄中のPLS3 mRNAの多量発現が、当該癌進展（浸潤、転移）、ならびに再発および予後の独立した予測因子になると考えられる。 That is, as can be seen from these findings, for solid tumors, high expression levels of PLS3 mRNA in portal vein blood, peripheral blood, and bone marrow, as well as in the primary tissue, indicate cancer progression (invasion, metastasis), recurrence In addition, since it shows a positive correlation with poor prognosis, it is considered that the high expression of PLS3 mRNA in these blood and bone marrow becomes an independent predictor of the cancer progression (invasion, metastasis), recurrence and prognosis.
　また、上記（Ａ）のことから、固形癌組織においてPLS3 mRNAの発現を抑制してやれば、EMT誘導を阻止することができ、その結果、癌進展（浸潤や転移）が抑制できると考えられる。すなわち、PLS3 mRNAの発現やそのEMS誘導機能を抑制する物質によれば、固形がんの癌進展（浸潤や転移）を抑制することができ、また術後の再発や予後不良を低減することができると考えられる。 Also, from the above (A), it is considered that if the expression of PLS3 mRNA is suppressed in a solid cancer tissue, EMT induction can be prevented, and as a result, cancer progression (invasion and metastasis) can be suppressed. That is, a substance that suppresses the expression of PLS3 mRNA and its EMS-inducing function can suppress cancer progression (invasion and metastasis) of solid cancer, and reduce postoperative recurrence and poor prognosis. It is considered possible.
　本発明はこれらの知見に基づいて完成したものであり、下記の具体的態様を有するものである。 The present invention has been completed based on these findings, and has the following specific aspects.
　（Ｉ）固形がん患者について、再発予後因子を測定するための検査薬および検査薬キット
（I-1）PLS3遺伝子の塩基配列（配列番号１）またはそれに相補的な塩基配列にストリンジェントな条件でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチドを含む、固形がん患者の再発予後因子PLS3を測定するための検査薬。 (I) Test drugs and test drug kits for measuring recurrence prognostic factors for solid cancer patients (I-1) Stringent conditions for the base sequence of the PLS3 gene (SEQ ID NO: 1) or its complementary base sequence A test agent for measuring PLS3, a prognostic factor for recurrence in solid cancer patients, comprising a polynucleotide having at least 15 bases that hybridizes with.
　（I-2）PLS3タンパクを認識する抗体（抗PLS3抗体）を有する、固形がん患者の再発予後因子PLS3を測定するための検査薬。 (I-2) A diagnostic drug for measuring PLS3, a prognostic factor for recurrence in solid cancer patients, having an antibody that recognizes PLS3 protein (anti-PLS3 antibody).
　（I-3）固形がんが、大腸がんまたは乳がんである（I-1）または（I-2）に記載する検査薬。 (I-3) The diagnostic agent described in (I-1) or (I-2), wherein the solid cancer is colorectal cancer or breast cancer.
　（I-4）（I-1）乃至（I-3）のいずれかに記載する検査薬を少なくとも含有する、固形がん患者の再発予後因子PLS3を測定するための検査薬キット。 (I-4) A test drug kit for measuring the prognosis factor PLS3 in patients with solid cancer, comprising at least the test drug described in any of (I-1) to (I-3).
　また当該検査薬および検査薬キットは、下記に記載するように、上皮間葉移行細胞を検出するためにも使用することができる。
　（I’-1）PLS3遺伝子の塩基配列（配列番号１）またはそれに相補的な塩基配列にストリンジェントな条件でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチドを含む、上皮間葉移行細胞を検出するための検査薬。 In addition, the test drug and test drug kit can also be used to detect epithelial-mesenchymal transition cells as described below.
(I'-1) Detects epithelial-mesenchymal transition cells containing a polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of the PLS3 gene (SEQ ID NO: 1) or a complementary base sequence thereof. To test drugs.
　（I’-2）PLS3タンパクを認識する抗体（抗PLS3抗体）を有する、上皮間葉移行細胞を検出するための検査薬。 (I′-2) A test agent for detecting epithelial-mesenchymal transition cells having an antibody that recognizes PLS3 protein (anti-PLS3 antibody).
　（I’-3）少なくとも（I’-1）または（I’-2）に記載する検査薬を含有する、上皮間葉移行細胞を検出するための検査薬キット。 (I'-3) A test agent kit for detecting epithelial-mesenchymal transition cells, containing at least the test agent described in (I'-1) or (I'-2).
　　　（II）固形がん患者に対する再発予後予測方法
（II-1）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程を有する、当該患者について固形がんの再発または予後を予測する方法。 (II) Relapse prognosis prediction method for solid cancer patients (II-1) About the patient having a step of measuring PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient A method to predict the recurrence or prognosis of solid cancer.
　（II-2）上記被験試料が、固形がん組織、骨髄、または血液である（II-1）に記載する方法。 (II-2) The method according to (II-1), wherein the test sample is a solid cancer tissue, bone marrow, or blood.
　（II-3）上記被験試料が、骨髄または血液中の遊離癌細胞である（II-1）に記載する方法。 (II-3) The method according to (II-1), wherein the test sample is bone marrow or blood free cancer cells.
　（II-4）上記血液が、末梢血である（II-2）または（II-3）に記載する方法。 (II-4) The method according to (II-2) or (II-3), wherein the blood is peripheral blood.
　（II-5）（1）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程、
（2）上記で測定したPLS3遺伝子発現量（被験発現量）またはPLS3タンパク産生量（被験産生量）を、対照者から採取された上記被験試料に対応する試料（対照試料）のPLS3遺伝子発現量（対照発現量）またはPLS3タンパク産生量（対照産生量）と対比する工程、
を有し、
（3）対照発現量または対照産生量に比して被験発現量または被験産生量が高い場合に、被験者について再発の可能性が高いか、または予後不良であると決定する、（II-1）乃至（II-4）のいずれかに記載する方法。 (II-5) (1) measuring PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient,
(2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production)
Have
(3) When the test expression level or test production level is higher than the control expression level or control production level, it is determined that the subject is likely to relapse or has a poor prognosis, (II-1) Thru | or the method in any one of (II-4).
　（II-6）被験者から採取された試料（被験試料）におけるPLS3遺伝子発現量（被験発現量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3遺伝子発現量（対照発現量）を、（I-1）に記載する検査薬を用いて測定する、（II-1）乃至（II-5）のいずれかに記載する方法。 (II-6) PLS3 gene expression level (test expression level) in a sample (test sample) collected from a subject and PLS3 gene expression level (control expression) in a sample (control sample) corresponding to a test sample collected from a control person The method according to any one of (II-1) to (II-5), wherein the amount) is measured using the test agent described in (I-1).
　（II-7）被験者から採取された試料（被験試料）におけるPLS3タンパク産生量（被験産生量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3タンパク産生量（対照産生量）を、（I-2）に記載する検査薬を用いて測定する、（II-1）乃至（II-5）のいずれかに記載する方法。 (II-7) PLS3 protein production (test production) in the sample collected from the subject (test sample) and PLS3 protein production (control production) in the sample (control sample) corresponding to the test sample collected from the control The method according to any one of (II-1) to (II-5), wherein the amount) is measured using the test agent described in (I-2).
　（III）上皮間葉移行細胞を検出する方法
（III-1）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程を有する、上皮間葉移行した癌（腫瘍）細胞を検出する方法。 (III) Method for detecting epithelial-mesenchymal transition cells (III-1) Epithelial-mesenchymal step, comprising measuring PLS3 gene expression level or PLS3 protein production level in a sample collected from a solid cancer patient (test sample) A method of detecting cancer (tumor) cells that have migrated.
　（III-2）上記被験試料が、固形がん組織、骨髄、または血液である（III-1）に記載する方法。
方法。 (III-2) The method according to (III-1), wherein the test sample is a solid cancer tissue, bone marrow, or blood.
Method.
　（III-3）上記被験試料が、骨髄または血液中の遊離癌細胞である（III-1）に記載する方法。
方法。 (III-3) The method according to (III-1), wherein the test sample is a free cancer cell in bone marrow or blood.
Method.
　（III-4）上記血液が、末梢血である（III-2）または（III-3）に記載する方法。 (III-4) The method according to (III-2) or (III-3), wherein the blood is peripheral blood.
　（III-5）（1）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程、
（2）上記で測定したPLS3遺伝子発現量（被験発現量）またはPLS3タンパク産生量（被験産生量）を、対照者から採取された上記被験試料に対応する試料（対照試料）のPLS3遺伝子発現量（対照発現量）またはPLS3タンパク産生量（対照産生量）と対比する工程、
を有し、
（3）対照発現量または対照産生量に比して被験発現量または被験産生量が高い場合に、固形がん患者の癌細胞または遊離癌細胞が上皮間葉移行していると決定する、（III-1）乃至（III-4）のいずれかに記載する方法。 (III-5) (1) a step of measuring the PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient,
(2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production)
Have
(3) When the test expression level or the test production level is higher than the control expression level or the control production level, it is determined that the cancer cells or free cancer cells of the solid cancer patient have transitioned to epithelial mesenchyme ( The method according to any one of (III-1) to (III-4).
　（III-6）被験者から採取された試料（被験試料）におけるPLS3遺伝子発現量（被験発現量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3遺伝子発現量（対照発現量）を、（I’-1）に記載する検査薬を用いて測定する、（III-1）乃至（III-5）のいずれかに記載する方法。 (III-6) PLS3 gene expression level (test expression level) in the sample collected from the subject (test sample) and PLS3 gene expression level (control expression) in the sample (control sample) corresponding to the test sample collected from the control The method according to any one of (III-1) to (III-5), wherein the amount is measured using the test agent described in (I′-1).
　（III-7）被験者から採取された試料（被験試料）におけるPLS3タンパク産生量（被験産生量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3タンパク産生量（対照産生量）を、（I’-2）に記載する検査薬を用いて測定する、（III-1）乃至（III-5）のいずれかに記載する方法。 (III-7) PLS3 protein production (test production) in the sample collected from the subject (test sample) and PLS3 protein production (control production) in the sample (control sample) corresponding to the test sample collected from the control The method according to any one of (III-1) to (III-5), wherein the amount is measured using the test agent described in (I'-2).
　（IV）固形がんの再発抑制または予後向上のための医薬組成物、または癌進展抑制のための医薬組成物
（IV-1）PLS3遺伝子の発現を抑制するsiRNAまたはPLS3に対する中和抗体を有効成分として含む、医薬組成物。 (IV) Pharmaceutical composition for suppressing recurrence or improving prognosis of solid cancer, or pharmaceutical composition for suppressing cancer progression (IV-1) Effective siRNA or PLS3 neutralizing antibody that suppresses expression of PLS3 gene A pharmaceutical composition comprising as an ingredient.
　（IV-2）固形がんの再発抑制または予後向上のために用いられるか、または固形がんの癌進展を抑制するために用いられる（IV-1）記載の医薬組成物。 (IV-2) The pharmaceutical composition according to (IV-1), which is used for suppressing recurrence or improving the prognosis of solid cancer, or for suppressing cancer progression of solid cancer.
　（IV-3）固形がんが大腸がんまたは乳がんである（IV-1）または（IV-2）に記載する医薬組成物。 (IV-3) The pharmaceutical composition according to (IV-1) or (IV-2), wherein the solid cancer is colon cancer or breast cancer.
　（IV-4）固形がんの再発抑制または予後向上のために用いられるか、または固形がんの癌進展を抑制するために用いられるPLS3遺伝子の発現を抑制するsiRNA、またはPLS3に対する中和抗体。 (IV-4) siRNA that suppresses the expression of PLS3 gene, which is used to suppress recurrence or improve the prognosis of solid cancer, or to suppress cancer progression of solid cancer, or neutralizing antibody against PLS3 .
　（IV-5）固形がんが大腸がんまたは乳がんである（IV-4）に記載するPLS3遺伝子の発現を抑制するsiRNA、またはPLS3に対する中和抗体。 (IV-5) siRNA that suppresses expression of the PLS3 gene described in (IV-4) where the solid cancer is colon cancer or breast cancer (IV-4), or a neutralizing antibody against PLS3.
　（V）固形がん患者の再発予防または予後向上に有効な成分、および固形がんの癌進展抑制に有効な成分をスクリーニングする方法
（V-1）下記の工程を有する、PLS遺伝子の発現を抑制する物質のスクリーニング方法：
（1）被験物質と、PLS3遺伝子を発現可能な細胞とを接触させる工程、
（2）被験物質を接触させた細胞のPLS3遺伝子の発現量（被験発現量）を測定する工程、及び
（3）上記の被験発現量が、被験物質を接触させない対照細胞のPLS3遺伝子の発現量（対照発現量）よりも小さい場合の被験物質を選択する工程。 (V) effective ingredient preventing recurrence or prognosis improving solid cancer patients, and methods of screening for active ingredients for cancer suppression of progression solid cancer having (V-1) the following step, the expression of PLS gene Methods for screening for inhibitors:
(1) contacting the test substance with a cell capable of expressing the PLS3 gene;
(2) a step of measuring the expression level (test expression level) of the PLS3 gene in the cell contacted with the test substance, and (3) the expression level of the PLS3 gene in the control cell where the test expression level is not in contact with the test substance. A step of selecting a test substance when it is smaller than (control expression level).
　（V-2）下記の工程を有する、PLS3の機能を抑制する物質のスクリーニング方法：
（1’）被験物質とPLS3タンパクを産生可能な細胞またはこの細胞から調製した細胞画分とを接触させる工程、
（2’）被験物質を接触させた細胞またはその細胞画分のPLS3タンパク産生量（被験産生量）を測定する工程、及び
（3’）上記の被験産生量が、被験物質を接触させない対照細胞もしくはその細胞画分のPLS3タンパク産生量（対照産生量）よりも小さい場合の被験物質を選択する工程。 (V-2) A screening method for a substance that suppresses the function of PLS3, comprising the following steps:
(1 ′) contacting a test substance with a cell capable of producing PLS3 protein or a cell fraction prepared from the cell,
(2 ′) a step of measuring the PLS3 protein production amount (test production amount) of the cell contacted with the test substance or its cell fraction, and (3 ′) a control cell in which the above test production quantity does not contact the test substance. Alternatively, a step of selecting a test substance when the PLS3 protein production amount (control production amount) of the cell fraction is smaller.
　（V-3）PLS3遺伝子を発現可能な細胞またはPLS3タンパクを産生可能な細胞が、非造血細胞である（V-1）または（V-2）に記載のスクリーニング方法。 (V-3) The screening method according to (V-1) or (V-2), wherein the cell capable of expressing the PLS3 gene or the cell capable of producing the PLS3 protein is a non-hematopoietic cell.
　（V-4）固形がんの患者に対して再発予防または予後向上に有効な成分を探索するための方法である、（V-1）乃至（V-3）のいずれかに記載するスクリーニング方法。 (V-4) The screening method according to any one of (V-1) to (V-3), which is a method for searching for a component effective in preventing recurrence or improving prognosis in patients with solid cancer .
　（V-5）固形がんが大腸がんまたは乳がんである（V-4）に記載するスクリーニング方法。 (V-5) The screening method described in (V-4), wherein the solid cancer is colon cancer or breast cancer.
　（V-6）固形がんの癌進展（湿潤、転移）抑制に有効な成分を探索するための方法である、（V-1）乃至（V-3）のいずれかに記載するスクリーニング方法。 (V-6) The screening method according to any one of (V-1) to (V-3), which is a method for searching for an effective component for suppressing cancer progression (wetting and metastasis) of solid cancer.
　（V-7）固形がんが大腸がんまたは乳がんである（V-6）に記載するスクリーニング方法。 (V-7) The screening method described in (V-6), wherein the solid cancer is colon cancer or breast cancer.
　本発明は、固形がん、特に大腸がんまたは乳がんの患者について、術後の再発または予後に関わる新しい独立因子（再発予後因子）として、plastin 3（PLS3）を提供する。本発明の検査薬またはそれを用いた再発予後予測方法は、上記再発予後因子PLS3を指標とする方法であり、当該方法によれば、固形がん、特に大腸がんまたは乳がんの患者について、血液、特に末梢血を被験試料として、癌進展度（浸潤や転移）を測定したり、また治療後の再発可能性、ならびに予後を予測することが可能になる。本発明の方法によって癌進展度を評価することで、適切な治療戦略をたてることが可能になる。また、本発明の方法によって再発の可能性若しくは予後不良であることが判明した患者に対しては、根治施術後に適切な経過観察と補助療法を行うことで、予後向上を図ることができる。また再発の可能性が低い患者や予後良好な患者に対しては無駄な治療を省くことで、患者の肉体的または精神的苦痛を軽減できるとともに、医療費の削減を図ることが可能になる。すなわち、術後患者について再発可能性および予後を測定することにより、各患者に応じて適切な術後療法を設計するなど、再発を防ぎ、また予後を向上するための的確な対策を講じることができる。 The present invention provides plastin 3 (PLS3) as a new independent factor (relapse prognosis factor) related to postoperative recurrence or prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer. The test agent of the present invention or a method for predicting recurrence using the same is a method using the above-mentioned recurrence prognostic factor PLS3 as an index, and according to the method, blood in patients with solid cancer, particularly colorectal cancer or breast cancer. In particular, it is possible to measure the degree of cancer progression (invasion and metastasis) using peripheral blood as a test sample, and to predict the likelihood of recurrence and prognosis after treatment. By evaluating the degree of cancer progression by the method of the present invention, an appropriate treatment strategy can be established. In addition, the prognosis can be improved by performing appropriate follow-up and adjuvant therapy after radical surgery for patients who have been found to have a recurrence possibility or poor prognosis by the method of the present invention. Further, by omitting useless treatment for patients with a low possibility of recurrence or patients with a good prognosis, it is possible to reduce the physical or mental pain of the patient and to reduce the medical cost. In other words, by measuring the likelihood of recurrence and prognosis for postoperative patients, it is possible to take appropriate measures to prevent recurrence and improve prognosis, such as designing appropriate postoperative therapy for each patient. it can.
　また上記再発予後因子PLS3を指標とすることにより、上皮間葉移行した癌（腫瘍）細胞（以下、「上皮間葉移行細胞」という）の存在を検出することができる。こうすることで、前述するように、例えば末梢血を被験試料として、癌進展度（浸潤や転移）や治療後の再発可能性ならびに予後を予測することが可能になることに加え、腫瘍マーカーとして癌の診断、ならびに治療効果（抗がん剤への感受性を含む）の判定も可能になり得る。 In addition, the presence of cancer (tumor) cells that have migrated to epithelial-mesenchymal cells (hereinafter referred to as “epithelial-mesenchymal transition cells”) can be detected by using the above-mentioned relapse prognostic factor PLS3 as an index. In this way, as described above, for example, using peripheral blood as a test sample, it becomes possible to predict the degree of cancer progression (invasion or metastasis), the possibility of recurrence after treatment, and the prognosis, and as a tumor marker. Diagnosis of cancer as well as determination of therapeutic effects (including sensitivity to anti-cancer agents) may be possible.
　また本発明が提供する医薬組成物（癌進展抑制剤、再発予防剤または予後向上剤）は、固形がん、特に大腸がんや乳がんの患者について、癌進展を阻止するために、また術後の再発を予防し、予後を向上するために有効に使用することができる。 In addition, the pharmaceutical composition (cancer progression inhibitor, recurrence preventive agent or prognostic agent) provided by the present invention is used to prevent cancer progression in patients with solid cancer, particularly colorectal cancer or breast cancer, and postoperatively. Can be used effectively to prevent recurrence and improve prognosis.
　また本発明のスクリーニング方法によれば、固形がん、特に大腸がんまたは乳がんの患者について、癌進展（浸潤、転移）を抑制するための有効な成分、ならびに再発を予防し、また予後を向上させるために有効な成分を取得することが可能になる。すなわち当該スクリーニング方法は、固形がんの進展抑制、ならびに治療後の再発予防若しくは予後向上に有効な新規薬剤の開発に有効に利用することができる。 In addition, according to the screening method of the present invention, an effective component for suppressing cancer progression (invasion, metastasis) and prevention of recurrence and improvement of prognosis in patients with solid cancer, particularly colorectal cancer or breast cancer. Therefore, it is possible to obtain effective components. That is, the screening method can be effectively used for development of a new drug effective in suppressing the progression of solid cancer and preventing recurrence after treatment or improving the prognosis.
(a)は、PLS3発現LoVo細胞（LoVo-PLS3細胞）（右側）とコントロール用のLoVo細胞（LoVo-mock細胞）（左側）とを対比した画像である(×200、スケールバー：100μm)。(b)は、LoVo-PLS3細胞（下）とLoVo-mock細胞（上）のそれぞれについて、抗PLS3抗体（左側）および抗vimentin抗体（右側）でそれぞれ免疫染色したLoVo細胞の画像である(×400、スケールバー：50μm)。(c)は、LoVo-PLS3細胞（下）とLoVo-mock細胞（上）のそれぞれについて、抗E-cadherin抗体(赤)（左側）と抗F-actin抗体(緑)（右側）で免疫染色したLoVo細胞の画像である。なお、切片はすべて、DAPI(青)で核染色した（×63、スケールバー：20μm)。(a) is an image comparing PLS3-expressing LoVo cells (LoVo-PLS3 cells) (right side) with control LoVo cells (LoVo-mock cells) (left side) (× 200, scale bar: 100 μm). (b) Images of LoVo cells immunostained with anti-PLS3 antibody (left side) and anti-vimentin antibody (right side) for LoVo-PLS3 cells (bottom) and LoVo-mock cells (top), respectively (× 400, scale bar: 50 μm). (c) Immunostaining of anti-E-cadherin antibody (red) (left) and anti-F-actin antibody (green) (right) for LoVo-PLS3 cells (bottom) and LoVo-mock cells (top), respectively. This is an image of LoVo cells. All sections were stained with DAPI (blue) (× 63, scale bar: 20 μm). LoVo-PLS3細胞とLoVo-mock細胞のそれぞれについて、浸潤能（左図）および転移能（右図）を測定した結果を示す。縦軸は各細胞の蛍光強度（Fluoresce intensity）を示す。なお、データは平均値±s.d.を示す。The results of measuring invasive ability (left figure) and metastatic ability (right figure) of each of LoVo-PLS3 cells and LoVo-mock cells are shown. The vertical axis represents the fluorescence intensity (Fluoresce intensity) of each cell. The data shows the mean value ± s.d. LoVo-PLS3細胞とLoVo-mock細胞のそれぞれについて、LoVo細胞の増殖を、3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide（MMT） assayを用いて測定した結果を示す。横軸は細胞培養時間（hour）、縦軸は細胞増殖率（培養時間０hourのときの細胞増殖を１とした場合の相対比）を示す。データは平均±s.d.を表わす(*p<0.05)。Results of measuring the proliferation of LoVo cells for each of LoVo-PLS3 cells and LoVo-mock cells using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MMT) assay Indicates. The horizontal axis represents the cell culture time (hour), and the vertical axis represents the cell growth rate (relative ratio when the cell growth is 1 when the culture time is 0 hour). Data represent mean ± s.d. (* p <0.05). LoVo-PLS3細胞とLoVo-mock細胞のそれぞれについて、抗がん剤（5-FU）に対する感受性（耐性）を、3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide（MTT） assayを用いて測定した結果を示す。横軸は5-FU濃度（μg/ml）、縦軸は細胞増殖率（5-FU濃度が0μg/mlのときの細胞増殖を１とした場合の相対比）を示す。データは平均±s.d.を表わす(*p<0.05)。For each of LoVo-PLS3 cells and LoVo-mock cells, the sensitivity (resistance) to the anticancer drug (5-FU) was determined as 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide. (MTT) The result measured using assay is shown. The horizontal axis represents the 5-FU concentration (μg / ml), and the vertical axis represents the cell growth rate (relative ratio when the cell growth is 1 when the 5-FU concentration is 0 μg / ml). Data represent mean ± s.d. (* p <0.05). (a)は、PLS3発現LoVo細胞（LoVo-PLS3細胞）またはコントロール用のLoVo細胞（LoVo-mock細胞）におけるE-cadherin、TWIST、SNAIL、SMAD4、SLUGおよびZEB1をコードするmRNAの発現レベルを対比した図である。(b)は、PLS3発現LoVo細胞（LoVo-PLS3細胞）またはコントロール用のLoVo細胞（LoVo-mock細胞）におけるvimentin、FN1、N-cadherinおよびFOXC2のEMT関連遺伝子mRNAの発現レベルを対比した図である。(c)は、PLS3 siRNAで処理したCaR-1細胞（CaR-1 PLS3 siRNA）およびcontrol siRNAで処理したCaR-1細胞（CaR-1 control siRNA）におけるvimentin、E-cadherin、TWISTおよびSNAILのmRNAの発現レベルを対比した図である。すべてのデータは平均±s.d.で表わす。データはすべてGAPDH(*p<0.05、コントロールとの対比)に標準化したものである。(a) Compares the expression levels of mRNA encoding E-cadherin, TWIST, SNAIL, SMAD4, SLUG, and ZEB1 in PLS3-expressing LoVo cells (LoVo-PLS3 cells) or control LoVo cells (LoVo-mock cells) FIG. (b) is a diagram comparing the expression levels of EMT-related gene mRNAs of vimentin, FN1, N-cadherin and FOXC2 in PLS3-expressing LoVo cells (LoVo-PLS3 cells) or control LoVo cells (LoVo-mock cells). is there. (c) shows vimentin, E-cadherin, TWIST and SNAIL mRNA in CaR-1 cells treated with PLS3 siRNA (CaR-1 PLS3 siRNA) and CaR-1 cells treated with control siRNA (CaR-1 control siRNA) It is the figure which contrasted the expression level. All data are expressed as mean ± s.d. All data are normalized to GAPDH (* p <0.05, compared to control). LoVo細胞およびCaR-1細胞のRT-PCR（a）およびウェスタンブロット分析（b）の結果を示す。具体的には、(a)は、LoVo-PLS3細胞（左図）、LoVo-mock細胞（左図）、CaR-1 control siRNA細胞（右図）、およびCaR-1 PLS3 siRNA細胞（右図）中のPLS3 mRNAの発現レベルを示す。データは平均値±s.d.を示す。データはすべてGAPDHで標準化した(*-p<0.05)。(b)はLoVo-PLS3細胞（左図）、LoVo-mock細胞（左図）、CaR-1 control siRNA細胞（右図）およびCaR-1 PLS3 siRNA細胞（右図）中のPLS3のタンパク質レベルを示す。The results of RT-PCR (a) and Western blot analysis (b) of LoVo cells and CaR-1 cells are shown. Specifically, (a) shows LoVo-PLS3 cells (left), LoVo-mock cells (left), CaR-1 control siRNA cells (right), and CaR-1 PLS3 siRNA cells (right) The expression level of PLS3 mRNA is shown. Data show mean values ± s.d. All data were normalized with GAPDH (* -p <0.05). (b) PLS3 protein levels in LoVo-PLS3 cells (left), LoVo-mock cells (left), CaR-1Cacontrol siRNA cells (right) and CaR-1 PLS3 siRNA cells (right) Show. (a)リアルタイム定量RT-PCRによって測定した、結腸直腸がん患者の癌(T)組織(n=110)および非癌(N)組織(n=110)中のPLS3 mRNA発現レベルを示す。いずれも各組織中のGAPDH mRNAの発現量で標準化した値である（PLS3 mRNA/GAPDH mRNA）。(b)癌(T)組織中のPLS3 mRNA発現のレベルで分けた結腸直腸がん患者（高PLS3発現群(n=26)：PLS3(T)/GAPDH(T)≧2.0、低PLS3発現群(n=84)：PLS3(T)/GAPDH(T)<2.0）について、手術から５年間の生存率を示すKaplan-Meier overall survivalカーブである。(a) PLS3 mRNA expression levels in cancer (T) tissue (n = 110) and non-cancer (N) tissue (n = 110) of colorectal cancer patients as measured by real-time quantitative RT-PCR. All values are standardized by the expression level of GAPDH mRNA in each tissue (PLS3 mRNA / GAPDH mRNA). (b) Colorectal cancer patients (high PLS3 expression group (n = 26): PLS3 (T) / GAPDH (T) ≧ 2.0, low PLS3 expression group) divided by the level of PLS3 mRNA expression in cancer (T) tissue (n = 84): Kaplan-Meier overall survival curve showing survival rate for 5 years from surgery for PLS3 (T) / GAPDH (T) <2.0). 大腸がん患者（n=110）をDukes分類した各症例（Dukes A（n=31）、Dukes B（n=30）、Dukes C（n=38）、Dukes D（n=11））について、原発巣組織におけるPLS3 mRNA発現レベルを対比した結果を示す。PLS3 mRNA発現量はGAPDH mRNAの発現量で標準化した値として示す。For each case (Dukes A (n = 31), Dukes B (n = 30), Dukes C (n = 38), Dukes D (n = 11)) that classified Dukes classification of colon cancer patients (n = 110), The result of having compared the expression level of PLS3 (TM) mRNA in the primary focus tissue is shown. The expression level of PLS3 mRNA is shown as a value normalized by the expression level of GAPDH mRNA. （a）結腸直腸がん患者の癌(T)組織および非癌(N)組織を、抗PLS3抗体を用いて免疫組織化学染色した結果を示す。矢印は、PLS3を過剰発現した癌細胞を示す(×200、スケールバー：200μm)。N:非癌組織サイド、T: 癌組織サイド。(b)(a)の一部（癌細胞付近）を拡大した図である。矢印は、PLS3を過剰発現している癌細胞を示す(×200、スケールバー：50μm)。(A) shows the results of immunohistochemical staining of cancer (T) tissue and non-cancer (N) tissue of colorectal cancer patients using anti-PLS3 antibody. Arrows indicate cancer cells overexpressing PLS3 (× 200, scale bar: 200 μm). N: Non-cancerous tissue side, T: Vaginal cancer tissue side. (b) It is the figure which expanded a part (near cancer cell) of (a). Arrows indicate cancer cells overexpressing PLS3 (× 200, scale bar: 50 μm). (a)腫瘍静脈循環血（VDT）中のPLS3 mRNA発現レベルで分類した結腸直腸がん患者（高PLS3発現群（図中、「PLS3 high」と表記する）：PLS3/GAPDH≧VDTのカットオフ値、低PLS3発現群（図中、「PLS3 low」と表記する）：PLS3/GAPDH< VDTのカットオフ値）の未病生存率(DFS)カーブ（左）と全生存率(OS)カーブ（右）(*p<0.0001)を示す。(b) 末梢血（PB）中のPLS3 mRNA発現のレベルで分類した結腸直腸がん患者（高PLS3発現群（図中、「PLS3 high」と表記する）：PLS3/GAPDH≧PBのカットオフ値、低PLS3発現群（図中、「PLS3 low」と表記する）：PLS3/GAPDH＜PBのカットオフ値）の未病生存率(DFS)カーブ（左）と全生存率(OS)カーブ（右）(*p<0.0001)を示す。(a) Colorectal cancer patients classified by PLS3 mRNA expression level in tumor venous circulation blood (VDT) (high PLS3 expression group (indicated as “PLS3 high” in the figure)): PLS3 / GAPDH ≧ VDT cutoff Value, low PLS3 expression group (indicated as “PLS3 low” in the figure): PLS3 / GAPDH <VDT cutoff value) non-disease survival rate (DFS) curve (left) and overall survival rate (OS) curve ( Right) (* p <0.0001). (b) Colorectal cancer patients classified by PLS3S mRNA expression level in peripheral blood (PB) (High PLS3 expression group (indicated as “PLS3 high” in the figure): PLS3 / GAPDH ≧ PB cutoff value , Low PLS3 expression group (indicated as “PLS3」 low ”in the figure): PLS3 / GAPDH <PB cutoff value) disease-free survival (DFS) curve (left) and overall survival (OS) curve (right) ) (* P <0.0001). 各種の正常組織（図中、左から副腎、骨髄、大脳、小脳、結腸、胎児脳、胎児肝臓、心臓、腎臓、肝臓、肺、肝、胎盤、末梢血、前立腺、唾液腺、骨格筋、小腸、脊髄、脾臓、胃、精巣、甲状腺、気管、子宮）、でのPLS3mRNAの発現分布を調べた結果を示す。PLS3 mRNA発現量はいずれも各組織中のGAPDH mRNAの発現量で標準化した値として示す（PLS3 mRNA/GAPDH mRNA）。骨髄と末梢血中で、殆どPLS3mRNA発現していないことがわかる。Various normal tissues (from left, adrenal gland, bone marrow, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, liver, placenta, peripheral blood, prostate, salivary gland, skeletal muscle, small intestine, The results of examining the distribution of PLS3 mRNA expression in the spinal cord, spleen, stomach, testis, thyroid, trachea, uterus) are shown. The expression level of PLS3 mRNA is shown as a value standardized by the expression level of GAPDH mRNA in each tissue (PLS3 mRNA / GAPDH mRNA). It can be seen that PLS3 mRNA is hardly expressed in bone marrow and peripheral blood. 末梢血（PB）中のPLS3 mRNA発現のレベルで分類した大腸がん患者（高PLS3発現群（n=33）：PLS3/GAPDH≧PBのカットオフ値、低PLS3発現群（n=118）：PLS3/GAPDH＜PBのカットオフ値）の未病生存率(DFS)カーブ（左）（P=0.004）と全生存率(OS)カーブ（右）（P<0.001）を示す。Colorectal cancer patients classified by the level of PLS3 mRNA expression in peripheral blood (PB) (high PLS3 expression group (n = 33): PLS3 / GAPDH ≧ PB cut-off value, low PLS3 expression group (n = 118): The disease free survival (DFS) curve (left) (P = 0.004) and the overall survival (OS) curve (right) (P <0.001) of PLS3 / GAPDH <PB cutoff value) are shown. 実験例４（3）において、大腸がんの患者から採取した末梢血から単離した単核球層中のCD45(-)細胞についてサイトケラチン染色とPLS3の蛍光2重免染を行った結果を示す。左図はサイトケラチン染色の結果を、中央図はPLS3染色の結果を、右図は両者を重ねた結果をそれぞれ示す。In Example 4 (3), the results of cytokeratin staining and PLS3 fluorescence double-immunization of CD45 (-) cells in the mononuclear cell layer isolated from peripheral blood collected from patients with colorectal cancer Show. The left figure shows the result of cytokeratin staining, the middle figure shows the result of PLS3 staining, and the right figure shows the result of overlaying both. (a)リアルタイム定量RT-PCRによって測定した、乳がん患者(n=34)の癌(T)組織および非癌(N)組織中のPLS3 mRNA発現レベルを示す。いずれも各組織中のGAPDH mRNAの発現量で標準化した値である（PLS3 mRNA/GAPDH mRNA）。(b)癌(T)組織中のPLS3 mRNA発現のレベルで分けた乳がん患者（高PLS3発現群(n=17)：PLS3(T)/GAPDH(T)＞4.1、低PLS3発現群(n=77)：PLS3(T)/GAPDH(T)≦4.1）について、手術から１０年間の生存率を示すKaplan-Meier overall survivalカーブである。(a) PLS3 mRNA expression level in cancer (T) tissue and non-cancer (N) tissue of breast cancer patients (n = 34) measured by real-time quantitative RT-PCR. All values are standardized by the expression level of GAPDH mRNA in each tissue (PLS3 mRNA / GAPDH mRNA). (b) Breast cancer patients (high PLS3 expression group (n = 17): PLS3 (T) / GAPDH (T)> 4.1, low PLS3 expression group (n = 77): Kaplan-Meier overall survival curve showing survival rate for 10 years from surgery for PLS3 (T) / GAPDH (T) ≦ 4.1). 末梢血中のPLS3 mRNA発現レベルで分類した乳がん患者（高PLS3発現群：PLS3/GAPDH＞0.0025、低PLS3発現群：PLS3/GAPDH≦0.0025）の未病生存率(DFS)カーブ（右）と全生存率(OS)カーブ（左）を示す(p<0.0001)。Breast cancer patients (high PLS3 expression group: PLS3 / GAPDH> 0.0025, low PLS3 expression group: PLS3 / GAPDH ≦ 0.0025) classified according to PLS3 mRNA expression level in peripheral blood Viability (OS) curve (left) is shown (p <0.0001). 骨髄中のPLS3 mRNA発現レベルで分類した乳がん患者（高PLS3発現群：PLS3/GAPDH＞0.105、低PLS3発現群：PLS3/GAPDH≦0.105）の未病生存率(DFS)カーブ（右）と全生存率(OS)カーブを示す(p<0.005)。Breast cancer patients (high PLS3 expression group: PLS3 / GAPDH> 0.105, low PLS3 expression group: PLS3 / GAPDH ≦ 0.105) breast cancer patients classified according to PLS3 mRNA expression level in bone marrow and overall survival Shows the rate (OS) curve (p <0.005).
１．本発明で使用する用語の定義
　本明細書における塩基配列（ヌクレオチド配列）、核酸などの略号による表示は、ＩＵＰＡＣ－ＩＵＢの規定〔IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)〕、「塩基配列又はアミノ酸配列を含む明細書等の作製のためのガイドライン」（特許庁編）及び当該分野における慣用記号に従うものとする。 1. Definition of terms used in the present invention Indications by abbreviations such as base sequences (nucleotide sequences) and nucleic acids in this specification are defined by IUPAC-IUB [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and conventional symbols in the field.
　本明細書において「PLS3タンパク」および「PLS3遺伝子」とは、細胞の遊走能に影響を与える細胞の形態および骨格構造を制御するアクチンに結合するタンパク質：Plastin 3およびその遺伝子をそれぞれ意味する。なお、本明細書において、両者を総称して単に「PLS3」と称する場合がある。 In the present specification, “PLS3 protein” and “PLS3 gene” mean a protein that binds to actin that controls cell morphology and skeletal structure that affect cell migration ability: Plastin 3 and its gene, respectively. In the present specification, both may be collectively referred to simply as “PLS3”.
　本明細書において「PLS3遺伝子」とは、基本的に配列番号１に示されるヒト由来のPLS3遺伝子を意味する。但し、これに限られず、ヒト、マウス、ラットなどの生物種間で保存されるオーソログ遺伝子など、ヒト由来のPLS3タンパクと生物学的機能が同等であるタンパク質（例えば同族体(ホモログやスプライスバリアントなど)、変異体及び誘導体）をコードする「遺伝子」も含まれる。例えばヒト由来のPLS3タンパクのホモログをコードする遺伝子としては、当該PLS3タンパクをコードするヒトPLS3遺伝子に対応するマウスやラットなど他生物種の遺伝子が例示できる。これらの遺伝子（ホモログ）は、HomoloGene（http://www.ncbi.nlm.nih.gov/HomoloGene/）により同定することができる。具体的には、特定ヒト遺伝子名（PLS3遺伝子）や塩基配列のアクセション番号（GenBank Accession No. NM_005032.4）をLocusLink（http://www.ncbi.nlm.nih.gov/LocusLink/）の検索にかけて該当するヒト遺伝子データを見いだす。そのデータのリンクメニューにあるHomoloGeneにアクセスして表示された他生物種遺伝子とヒト遺伝子との遺伝子ホモログの相関を示したリストから、ヒトPLS3遺伝子に対応するマウスやラットなど他生物種の遺伝子を遺伝子（ホモログ）として選抜することができる。具体的には、ヒトPLS3遺伝子(GenBank Accession No. NM_005032.4)（配列番号１）のマウスホモログとして、マウスPLS3遺伝子（GenBank Accession No. NM_145629.1）を例示することができる。 In the present specification, the “PLS3 gene” basically means the PLS3 gene derived from human shown in SEQ ID NO: 1. However, the present invention is not limited to this, and a protein having a biological function equivalent to a human-derived PLS3 protein, such as an ortholog gene conserved between species such as human, mouse, and rat (for example, a homolog (such as a homolog or a splice variant). ), Variants and derivatives) are also included. For example, as a gene encoding a homologue of human-derived PLS3 protein, genes of other species such as mouse and rat corresponding to the human PLS3 gene encoding the PLS3 protein can be exemplified. These genes (homologs) can be identified by HomoloGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, the specific human gene name (PLS3 gene) and the accession number (GenBank Accession No. NM_005032.4) of LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) Search for relevant human genetic data. From the list showing the correlation of gene homologs between other species and human genes displayed by accessing the homologene in the link menu of the data, select genes of other species such as mice and rats that correspond to the human PLS3 gene. It can be selected as a gene (homolog). Specifically, the mouse PLS3 gene (GenBank Accession No. NM_145629.1) can be exemplified as a mouse homologue of the human PLS3 gene (GenBank Accession No. NM_005032.4) (SEQ ID NO: 1).
　また、本明細書で「遺伝子」とは、特に言及しない限り、調節領域、コード領域、エクソン、及びイントロンを区別することなく示すものとする。また本明細書で「遺伝子発現」とは、遺伝情報に基づいて、ｍＲＮＡが転写合成されることを意味する。 In the present specification, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified. In the present specification, “gene expression” means that mRNA is transcribed and synthesized based on genetic information.
　本明細書中において「ＤＮＡ」は、特に言及しない限り、ヒトゲノムＤＮＡを含む２本鎖ＤＮＡ、及びｃＤＮＡや合成ＤＮＡを含む１本鎖ＤＮＡ（センス鎖）、並びに当該センス鎖と相補的な配列を有する１本鎖ＤＮＡ（アンチセンス鎖）、及びそれらの断片のいずれもが含まれる。 In the present specification, unless otherwise specified, “DNA” refers to double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand) including cDNA and synthetic DNA, and a sequence complementary to the sense strand. These include both single-stranded DNA (antisense strand) and fragments thereof.
　本明細書中において「ＲＮＡ」とは、特に言及しない限り、１本鎖ＲＮＡのみならず、それに相補的な配列を有する１本鎖ＲＮＡ、さらにはそれらから構成される２本鎖ＲＮＡを包含する趣旨で用いられる。当該ＲＮＡには、total ＲＮＡ、ｍＲＮＡ、ｒＲＮＡ、ｓｉＲＮＡなどの合成ＲＮＡが含まれる。 In this specification, “RNA” includes not only single-stranded RNA but also single-stranded RNA having a sequence complementary thereto, and further double-stranded RNA composed thereof, unless otherwise specified. Used for purposes. The RNA includes synthetic RNA such as total RNA, mRNA, rRNA, siRNA and the like.
　また、本明細書中において、「ヌクレオチド」、「オリゴヌクレオチド」及び「ポリヌクレオチド」は、核酸と同義であって、ＤＮＡおよびＲＮＡの両方を含むものとする。また、これらは２本鎖であっても１本鎖であってもよく、ある配列を有する「ヌクレオチド」（または「オリゴヌクレオチド」、「ポリヌクレオチド」）といった場合、特に言及しない限り、これに相補的な配列を有する「ヌクレオチド」（または「オリゴヌクレオチド」及び「ポリヌクレオチド」）も包括的に意味するものとする。さらに、「ヌクレオチド」（または「オリゴヌクレオチド」及び「ポリヌクレオチド」）がＲＮＡである場合、配列表に示される塩基記号「Ｔ」は「Ｕ」と読み替えられるものとする。 Further, in this specification, “nucleotide”, “oligonucleotide” and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.
　本明細書において「PLS3タンパク」とは、基本的に配列番号２に示されるヒト由来のPLS3タンパク（GenBank Accession No. NP_005023.2）を意味する。しかし、これに限られず、ヒト、マウス、ラットなどの生物種間で保存されるオーソログ遺伝子からの翻訳産物である「タンパク質」、ならびに当該ヒトPLS3タンパクと生物学的機能が同等であることを限度として、その同族体（ホモログやスプライスバリアント）、変異体、誘導体、およびアミノ酸修飾体なども包含される。ここでホモログとしては、ヒトのPLS3タンパク（配列番号２）に対応するマウスやラットなど他生物種のタンパク質が例示でき、これらはHomoloGene(http://www.ncbi.nlm.nih.gov/HomoloGene/)により同定された遺伝子の塩基配列から演繹的に同定することができる。また変異体には、天然に存在するアレル変異体、天然に存在しない変異体、および人為的に欠失、置換、付加および挿入されることによって改変されたアミノ酸配列を有する変異体が包含される。なお、上記変異体としては、変異のないタンパク質と、少なくとも70％、好ましくは80％、より好ましくは95％、さらにより好ましくは97％相同なものを挙げることができる。上記ヒトPLS3に対するマウスホモログとして、マウスPLS3（GenBank Accession No. NP_663604.1）を挙げることができる。 In the present specification, “PLS3 protein” basically means a human-derived PLS3 protein (GenBank Accession No. NP_005023.2) represented by SEQ ID NO: 2. However, the present invention is not limited to this, but it is limited to the "protein" that is a translation product from orthologous genes conserved among species such as humans, mice, and rats, and the biological function equivalent to the human PLS3 protein. And their homologues (homologs and splice variants), mutants, derivatives, amino acid modifications and the like. Here, examples of homologs include proteins of other species such as mouse and rat corresponding to the human PLS3 protein (SEQ ID NO: 2). These are homologous genes (http://www.ncbi.nlm.nih.gov/HomoloGene). It can be identified a priori from the base sequence of the gene identified by /). Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein having no mutation. As a mouse homologue to the above human PLS3, mouse PLS3 (GenBank Accession No. NP_663604.1) can be mentioned.
　本明細書でいう「抗体」には、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、一本鎖抗体、またはFabフラグメントやFab発現ライブラリーによって生成されるフラグメントなどのように抗原結合性を有する上記抗体の一部が包含される。 As used herein, the term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or an antibody having an antigen binding property such as a Fab fragment or a fragment generated by a Fab expression library. Some are included.
　本発明において「大腸がん」とは、大腸（盲腸、結腸、直腸）に発生するがん腫であり、通常、盲腸がん、結腸がん、および直腸がんが挙げられるが、肛門管に発生する癌も含まれる。また本発明において「乳がん」とは***組織に発生するがん腫である。当該乳がんには、湿潤性乳管がんおよび非湿潤性乳がんが含まれるが、好ましくは湿潤性乳管がんである。 In the present invention, “colon cancer” is a carcinoma that occurs in the large intestine (cecum, colon, rectum), and usually includes cecal cancer, colon cancer, and rectal cancer. Cancers that develop are also included. In the present invention, “breast cancer” is a carcinoma that occurs in breast tissue. Such breast cancer includes wet duct cancer and non-wet breast cancer, preferably wet breast cancer.
　また本発明において「遊離癌細胞（ITC:Isolated Tumor Cell）」とは、固形がんの原発巣または転移組織から遊離した癌細胞を意味し、骨髄や血中に循環する癌細胞（血中循環癌細胞）も含まれる。 In the present invention, the term “free cancer cell (ITC: Isolated Tumor Cell)” means a cancer cell released from the primary tumor or metastatic tissue of a solid cancer, and is a cancer cell circulating in the bone marrow or blood (blood circulation). Cancer cells).
　本発明において「再発」とは、治癒切除や放射線療法などの癌根治療法後に、再び発生した癌細胞または潜在的に存在していた癌細胞が、検査で再び発見される大きさまで成長することをいう。当該再発には、局所再発、領域再発、遠隔転移（臓器転移）のいずれもが含まれる。 In the present invention, “recurrence” means that cancer cells that have recurred or potentially existed after a radical treatment such as curative resection or radiotherapy grow to a size that can be found again by examination. Say. The recurrence includes any of local recurrence, regional recurrence, and distant metastasis (organ metastasis).
　発明において「予後」とは、治癒切除や放射線療法などの癌根治療法後に、患者がどのような回復経過をとるか、特に、どのくらい生存できる（生命予後）についての見通しを意味する。なお、予後には機能予後と生命予後が含まれる。生命予後には、癌を再発することなく生存する場合（未病生存率）と、癌を再発しながらも生存する場合（再発生存率）の両方が含まれる。また「予後予測」とは、上記生命予後を予測することを意味する。本発明において「予後不良」とは、癌根治療後から５年以内に癌再発を理由として死亡する確率が相対的に高いことを意味する。逆に、「予後良好」とは、癌根治療後から５年以内に癌再発を理由として死亡する確率が相対的に低いことを意味する。 In the present invention, “prognosis” means the prospect of how a patient will recover after cancer radical treatment such as curative resection or radiotherapy, in particular, how long he can survive (life prognosis). The prognosis includes functional prognosis and life prognosis. The life prognosis includes both a case where the cancer survives without recurrence (non-disease survival rate) and a case where the cancer survives while recurrence (relapse survival rate). The “prognosis prediction” means predicting the life prognosis. In the present invention, “poor prognosis” means that the probability of death due to cancer recurrence is relatively high within 5 years after cancer root treatment. Conversely, “good prognosis” means that the probability of death due to cancer recurrence within 5 years after cancer root treatment is relatively low.
　さらに本明細書において「検査薬」とは、固形がん、特に大腸がんまたは乳がんの癌進展度（浸潤、転移）、または固形がん、特に大腸がんまたは乳がんの患者における再発可能性または予後を予測するために、直接または間接的に利用されるものをいう。また、当該「検査薬」は、固形がん、特に大腸がんまたは乳がんの癌進展度（浸潤、転移）を抑制したり、再発を抑制しまた予後を改善するために有効な成分（癌進展抑制剤、再発予防剤または予後向上剤の候補物質）をスクリーニングするためのツールとして、直接または間接的に利用されるものでもある。これには、固形がんに関連して、当該癌の原発巣、腫瘍還流静脈血および末梢血において発現が上昇するPLS3 mRNAまたはその発現産物（PLS3タンパク）を特異的に認識し、また結合することのできる（ポリ）（オリゴ）ヌクレオチドまたは抗体が包含される。これらの（ポリ）（オリゴ）ヌクレオチドおよび抗体は、上記性質に基づいて、上記生体内で発現した上記PLS3 mRNA及びPLS3タンパクを、生体内外で検出するためのプローブとして、また（ポリ）（オリゴ）ヌクレオチドは生体内で発現した上記PLS3遺伝子を増幅するためのプライマーとして有効に利用することができる。 Further, in the present specification, the “test agent” refers to the degree of cancer progression (infiltration, metastasis) of solid cancer, particularly colorectal cancer or breast cancer, or the possibility of recurrence in patients with solid cancer, particularly colorectal cancer or breast cancer, or It is used directly or indirectly to predict prognosis. In addition, the "test drug" is an effective component (cancer progression) that suppresses the degree of cancer progression (invasion, metastasis) of solid cancer, particularly colon cancer or breast cancer, suppresses recurrence, and improves the prognosis. It is also used directly or indirectly as a tool for screening a candidate substance for a suppressor, a preventive agent for recurrence, or a prognostic agent. It specifically recognizes and binds to PLS3 mRNA or its expression product (PLS3 protein) whose expression is elevated in the primary tumor, tumor reflux venous blood and peripheral blood in relation to solid cancer Possible (poly) (oligo) nucleotides or antibodies are included. Based on the above properties, these (poly) (oligo) nucleotides and antibodies are used as probes for detecting the PLS3 mRNA and PLS3 protein expressed in vivo in vivo and in vivo, and (poly) (oligo) Nucleotides can be used effectively as primers for amplifying the PLS3 gene expressed in vivo.
　本明細書において診断対象となる「生体組織」および「生体試料」には、固形がん、特に大腸がんまたは乳がんに伴い、PLS3 mRNAの発現が上昇する組織、および当該遺伝子の発現産物（PLS3タンパク）が生成するか若しくは分泌される組織（血液を含む）が含まれる。かかる組織としては、固形がんの癌組織（原発巣）、骨髄ならびに血液（門脈血や末梢末梢血）を挙げることができる。好ましくは末梢血である。 In this specification, “biological tissue” and “biological sample” to be diagnosed include tissues in which expression of PLS3 mRNA increases in association with solid cancer, particularly colon cancer or breast cancer, and expression products (PLS3 Proteins are produced or secreted (including blood). Examples of such tissues include solid cancer cancer tissues (primary lesions), bone marrow, and blood (portal vein blood and peripheral peripheral blood). Peripheral blood is preferable.
　本明細書において「対照者」とは、固形がん、特に大腸がんまたは乳がんに罹患しているがん患者（担癌者）に対抗する用語として使用される。具体的には、固形がんに罹患していない非担癌者を意味する。かかる対照者には、健常者のほか、癌以外の良性疾患（例えばヘルニアや胆石など）に罹患している者も含まれる。 In the present specification, the term “control person” is used as a term that opposes cancer patients (cancer carriers) suffering from solid cancer, particularly colorectal cancer or breast cancer. Specifically, it means a non-cancer-bearing person who does not suffer from solid cancer. Such controls include healthy individuals as well as those suffering from benign diseases other than cancer (such as hernias and gallstones).
　以下、PLS3遺伝子（ポリヌクレオチド）、並びにこの発現産物（PLS3）（タンパク質）およびそれらの派生物について、具体的な用途を説明する。 Hereinafter, specific uses of the PLS3 gene (polynucleotide), the expression product (PLS3) (protein) and their derivatives will be described.
　２．固形がんの再発予後因子を測定するための検査薬
　本発明は、固形がん、特に大腸がんまたは乳がんの患者について癌治療後の再発または予後を予測するために使用される検査薬に関する。当該検査薬は、再発予後因子PLS3を指標として、骨髄や血液、好ましくは末梢血中のPLS3遺伝子の発現（mRNA）またはその発現産物であるPLS3タンパクを検出し定量するために使用される。当該検査薬は、後述するスクリーニング方法において、固形がんの患者について、術後の再発を抑制し、また予後を向上させるための有効成分（再発予防剤や予後向上剤の候補物質）を探索するためにも使用される。 2. Test agent present invention for measuring the recurrence prognostic factors solid cancer, solid cancers, in particular to test agents that are used to predict recurrence or prognosis after cancer therapy for patients with colon cancer or breast cancer. The test agent is used for detecting and quantifying the expression (mRNA) of PLS3 gene or its expression product PLS3 protein in bone marrow and blood, preferably peripheral blood, using relapse prognosis factor PLS3 as an index. In the screening method described below, the test drug searches for active ingredients (candidate substances for recurrence prevention or prognosis improvement) for patients with solid cancer to suppress postoperative recurrence and improve prognosis. Also used for.
　さらに当該試薬は、固形がんの癌進展度（浸潤、転移）を調べるためにも、また後述するスクリーニング方法において固形がんの癌進展を抑制するための有効成分（癌進展抑制剤の候補物質）を探索するためにも使用される。 Further, the reagent is an active ingredient (candidate substance for cancer progression inhibitor) for examining the degree of cancer progression (invasion, metastasis) of solid cancer, and for suppressing cancer progression of solid cancer in the screening method described later. ) Is also used for searching.
　（1）ポリ（オリゴ）ヌクレオチド
　本発明において再発予後予測は、被験者から採取される生体試料（骨髄や血液）、好ましくは末梢血におけるPLS3遺伝子の発現上昇の有無または発現レベル（mRNA発現量）を評価することによって行われる。この場合、本発明の検査薬は、上記遺伝子の発現によって生じたＲＮＡまたはそれに由来するポリヌクレオチドを特異的に認識し増幅するためのプライマーとして、または該ＲＮＡまたはそれに由来するポリヌクレオチドを特異的に検出するためのプローブとして使用される。 (1) Poly (oligo) nucleotide In the present invention, the prognosis of recurrence is based on the presence or absence of increased expression of PLS3 gene or the expression level (mRNA expression level) in a biological sample (bone marrow or blood) collected from a subject, preferably peripheral blood. Done by evaluating. In this case, the test agent of the present invention specifically serves as a primer for specifically recognizing and amplifying the RNA produced by the expression of the gene or a polynucleotide derived therefrom, or specifically the polynucleotide derived therefrom. Used as a probe for detection.
　本発明の検査薬は、PLS3 mRNAもしくは当該mRNAに由来するポリヌクレオチド（cDNA）を選択的に（特異的に）認識するものであればよく、PLS3遺伝子の塩基配列（全長配列）からなるポリヌクレオチドであってもよいし、その相補配列からなるポリヌクレオチドであってもよい。また、上記全長配列またはその相補配列の部分配列からなるポリヌクレオチドであってもよい。この場合、部分配列としては、上記全長配列またはその相補配列の塩基配列から任意に選択される少なくとも15個の連続した塩基長を有するポリヌクレオチドを挙げることができる。 The test agent of the present invention only needs to selectively (specifically) recognize PLS3 mRNA or a polynucleotide (cDNA) derived from the mRNA, and is a polynucleotide comprising the base sequence (full-length sequence) of the PLS3 gene. It may be a polynucleotide consisting of its complementary sequence. Moreover, the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.
　なお、ここで「選択的に（特異的に）認識する」とは、例えばノーザンブロット法においては、PLS3 mRNAまたはこれに由来するポリヌクレオチド(cDNA)が特異的に検出できること、またRT-PCR法においてはPLS3 mRNAまたはこれに由来するポリヌクレオチド(cDNA)が特異的に生成されることを意味するが、それに限定されることなく、当業者が上記検出物または生成物がPLS3 mRNAに由来するものであると判断できるものであればよい。 Here, “selectively (specifically) recognize” means that, for example, in Northern blotting, PLS3 mRNA or a polynucleotide (cDNA) derived therefrom can be specifically detected, and RT-PCR method. Means that PLS3 mRNA or a polynucleotide (cDNA) derived therefrom is specifically produced, but the present invention is not limited thereto, and those skilled in the art can detect the product or product derived from PLS3 mRNA. Anything that can be determined to be.
　すなわち、本発明の検査薬は、PLS3遺伝子の塩基配列において、連続する少なくとも15塩基を有するポリヌクレオチド及び／またはそれに相補的なポリヌクレオチドからなることを特徴とする。ここで相補的なポリヌクレオチド（相補鎖、逆鎖）とは、PLS3遺伝子の塩基配列からなるポリヌクレオチドの全長配列、または該塩基配列において少なくとも連続した15塩基長の塩基配列を有するその部分配列（ここでは便宜上、これらを「正鎖」ともいう）に対して、A:TおよびG:Cといった塩基対関係に基づいて、塩基的に相補的な関係にあるポリヌクレオチドを意味するものである。ただし、かかる相補鎖は、対象とする正鎖の塩基配列と完全に相補配列を形成する場合に限らず、対象とする正鎖とストリンジェントな条件でハイブリダイスすることができる程度の相補関係を有するものであってもよい。なお、ここでストリンジェントな条件は、Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA) に教示されるように、複合体或いはプローブを結合する核酸の融解温度(Tm)に基づいて決定することができる。例えばハイブリダイズ後の洗浄条件として、通常「１×SSC、0.1%SDS、37℃」程度の条件を挙げることができる。相補鎖はかかる条件で洗浄しても対象とする正鎖とハイブリダイズ状態を維持するものであることが好ましい。特に制限されないが、より厳しいハイブリダイズ条件として「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件として「0.1×SSC、0.1%SDS、65℃」程度の洗浄条件を挙げることができる。具体的には、このような相補鎖として、対象の正鎖の塩基配列と完全に相補的な関係にある塩基配列からなる鎖、並びに該鎖と少なくとも90％、好ましくは95％の相同性を有する塩基配列からなる鎖を例示することができる。 That is, the test agent of the present invention is characterized by comprising a polynucleotide having at least 15 consecutive bases and / or a polynucleotide complementary thereto in the base sequence of the PLS3 gene. Here, the complementary polynucleotide (complementary strand, reverse strand) is the full-length sequence of a polynucleotide consisting of the base sequence of the PLS3 gene, or a partial sequence thereof having a base sequence of at least 15 bases in length in the base sequence ( Here, for the sake of convenience, these are also referred to as “positive strands”), and refer to polynucleotides that are in a base-complementary relationship based on base pair relationships such as A: T and G: C. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. Note that stringent conditions here combine complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand. An example is a chain composed of a base sequence having the same.
　本発明の検査薬は、例えば配列番号１で示されるヒト由来のPLS3遺伝子の塩基配列をもとに、例えばベクターNTI（Infomax社製）を利用して設計することができる。具体的には前記PLS3遺伝子の塩基配列（配列番号１）をベクターNTIのソフトウエアにかけて得られる、プライマーまたはプローブの候補配列、若しくは少なくとも該配列を一部に含む配列を、プライマーまたはプローブとして使用することができる。 The test agent of the present invention can be designed using, for example, the vector NTI (manufactured by Infomax) based on the base sequence of the human-derived PLS3 gene represented by SEQ ID NO: 1. Specifically, a primer or probe candidate sequence obtained by applying the base sequence of the PLS3 gene (SEQ ID NO: 1) to the vector NTI software, or a sequence partially including the sequence is used as a primer or probe. be able to.
　本発明の検査薬は、上述するように連続する少なくとも15塩基の長さを有するポリヌクレオチドであればよいが、具体的にはその用途に応じて、長さを適宜選択し設定することができる。 The test agent of the present invention may be any polynucleotide having a length of at least 15 consecutive bases as described above, and specifically, the length can be appropriately selected and set according to the application. .
　本発明の検査薬は、ノーザンブロット法、RT-PCR法、in situハイブリダーゼーション法などといった、特定遺伝子の発現を特異的に検出する公知の方法において、常法に従ってプライマーまたはプローブとして利用することができる。該利用によって被験者から採取された試料中のPLS3遺伝子の発現上昇の有無または発現レベル（発現量）を評価することができる。 The test agent of the present invention should be used as a primer or probe according to a conventional method in known methods for specifically detecting the expression of a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, etc. Can do. By using this, it is possible to evaluate the presence or absence of expression increase or expression level (expression level) of PLS3 gene in a sample collected from a subject.
　測定対象試料（生体試料）としては、使用する検出方法の種類に応じて、被験者の固形がんの癌組織または骨髄の一部をバイオプシ等で採取するか、または血液（門脈血や末梢血液）等を採取し、そこから常法に従って調製したtotal RNAを用いてもよいし、さらに該RNAをもとにして調製される各種のポリヌクレオチド（cDNA）を用いてもよい。 Depending on the type of detection method used, the sample to be measured (biological sample) can be obtained by collecting a part of cancer tissue or bone marrow of a subject's solid cancer with a biopsy or the like, or blood (portal blood or peripheral blood) ) And the like, and total RNA prepared therefrom according to a conventional method may be used, or various polynucleotides (cDNA) prepared based on the RNA may be used.
　本発明の検査薬を、再発予後因子PLS3の測定に際してプライマーとして用いる場合には、PLS3遺伝子の塩基配列（配列番号１）またはそれに相補的な塩基配列にストリンジェントな条件でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチドとして、通常15bp～100bp、好ましくは15bp～50bp、より好ましくは15bp～35bpの塩基長を有するものが例示できる。また検出プローブとして用いる場合には、通常15bp～全配列の塩基数、好ましくは15bp～1kb、より好ましくは100bp～1kbの塩基長を有するものが例示できる。なお、本発明で使用するプライマーの一例として、下記配列を有するポリヌクレオチドを挙げることができる。
　PLS3 F プライマー：ccttccgtaactggatgaactc（配列番号３） 
　PLS3 R プライマー：ggatgcttccctaattcaacag（配列番号４） 。 When the test agent of the present invention is used as a primer for the measurement of the recurrence prognosis factor PLS3, it hybridizes under stringent conditions to the base sequence of the PLS3 gene (SEQ ID NO: 1) or a base sequence complementary thereto, at least 15 Examples of the polynucleotide having a base include those having a base length of usually 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp. When used as a detection probe, those having a base length of usually 15 bp to the entire sequence, preferably 15 bp to 1 kb, more preferably 100 bp to 1 kb can be exemplified. An example of a primer used in the present invention is a polynucleotide having the following sequence.
PLS3 F primer: ccttccgtaactggatgaactc (SEQ ID NO: 3)
PLS3 R primer: ggatgcttccctaattcaacag (SEQ ID NO: 4).
　本発明の検査薬（プローブまたはプライマー）には、PLS3 mRNAまたはその派生物（cDNA）を検出するために適当な標識物、例えば蛍光色素、酵素、タンパク質、放射性同位体、化学発光物質、ビオチン等が付加されたものが含まれる。 The test agent (probe or primer) of the present invention includes a label suitable for detecting PLS3 mRNA or its derivative (cDNA), such as a fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance, biotin, etc. Is included.
　本発明において用いられる蛍光色素としては、一般にヌクレオチドを標識して、核酸の検出や定量に用いられるものが好適に使用でき、例えば、ＨＥＸ（4,7,2’,4’,5’,7’-hexachloro-6-carboxylfluorescein、緑色蛍光色素）、フルオレセイン（fluorescein）、ＮＥＤ（商品名、アプライドバイオシステムズ社製、黄色蛍光色素）、あるいは、６－ＦＡＭ（商品名、アプライドバイオシステムズ社製、黄緑色蛍光色素）、ローダミン（rhodamin）またはその誘導体〔例えば、テトラメチルローダミン（TMR）〕を挙げることができるが、これらに限定されない。蛍光色素でヌクレオチドを標識する方法は、公知の標識法のうち適当なものを使用することができる〔Nature Biotechnology, 14, 303-308 (1996)参照〕。また、市販の蛍光標識キットを使用することもできる（例えば、アマシャム・ファルマシア社製、オリゴヌクレオチドＥＣＬ　３’－オリゴラベリングシステム等）。 As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto. As a method for labeling a nucleotide with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system, manufactured by Amersham Pharmacia).
　またプローブ（オリゴまたはポリヌクレオチド）は任意の固相に固定化して用いることもできる。このため本発明の検査薬は、上記プローブ（オリゴまたはポリヌクレオチド）を固定化プローブ（例えばプローブを固定化したDNAチップ、cDNAマイクロアレイ、オリゴDNAアレイ、メンブレンフィルター等。以下「DNAチップ等」と総称する。）の形態として提供することができる。 Also, the probe (oligo or polynucleotide) can be immobilized on an arbitrary solid phase and used. For this reason, the test agent of the present invention is a probe in which the above probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).
　固定化に使用される固相は、オリゴまたはポリヌクレオチドを固定化できるものであれば特に制限されることなく、例えばガラス板、ナイロンメンブレン、マイクロビーズ、シリコンチップ、キャピラリーまたはその他の基板等を挙げることができる。固相へのオリゴまたはポリヌクレオチドの固定は、予め合成したオリゴまたはポリヌクレオチドを固相上に載せる方法であっても、また目的とするオリゴまたはポリヌクレオチドを固相上で合成する方法であってもよい。固定方法は、例えばDNAマイクロアレイであれば、市販のスポッター（Amersham社製など）を利用するなど、固定化プローブの種類に応じて当該技術分野で周知である〔例えば、photolithographic技術(Affymetrix社)、インクジェット技術(Rosetta Inpharmatics社)によるオリゴヌクレオチドのin situ合成等〕。 The solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to. The immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
　本発明の検査薬（プローブ）を固定化したDNAチップ等によれば、生体組織から採取したRNAをもとに調製される標識DNAまたはRNAとハイブリダイズさせ、該ハイブリダイズによって形成された上記プローブ（本発明検査薬）と標識DNAまたはRNAとの複合体を、該標識DNAまたはRNAの標識を指標として検出することにより、生体組織中でのPLS3遺伝子の発現の有無または発現レベル（発現量）を評価することができる。 According to the DNA chip or the like on which the test agent (probe) of the present invention is immobilized, the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from biological tissue Presence / absence or expression level (expression level) of the PLS3 gene in living tissue by detecting a complex of the (inspection drug of the present invention) and labeled DNA or RNA using the label of the labeled DNA or RNA as an indicator Can be evaluated.
　上記DNAチップ等は、PLS3 mRNAまたはその派生物（cDNA）と結合し得る1種または2種以上のプローブ（検査薬）を含んでいれば良い。複数の検査薬（プローブ）を含むDNAチップ等の利用によれば、ひとつの生体試料について、同時に複数の遺伝子の発現の有無または発現レベルの評価が可能である。 The above-mentioned DNA chip or the like only needs to contain one or more types of probes (test agents) that can bind to PLS3 mRNA or its derivative (cDNA). By using a DNA chip or the like containing a plurality of test agents (probes), it is possible to simultaneously evaluate the presence or absence of expression of a plurality of genes or the expression level of one biological sample.
　本発明の検査薬は、固形がんの癌進展度（浸潤、転移）を評価したり、固形がんの患者について、治療後の再発可能性や予後を予測するのに有用である。具体的には、該検査薬を利用した癌進展度の評価ならびに再発予後の予測は、固形がん患者の生体試料と対照者（非担癌患者）の生体試料におけるPLS3遺伝子の発現レベルの違いを判定することによって行うことができる。この場合、具体的にはPLS3遺伝子の発現は、固形がんの原発巣、骨髄、血液（門脈血、末梢血）中で上昇しているので、当該発現量が対照者（非担癌者）に関する対応する組織、骨髄または血液中の発現量と比べて高ければ、当該被験者について、癌進展度（浸潤、転移）が高い、術後再発の危険性がある、また予後が不良である可能性が高くなる。具体的には、大腸がん患者については、原発巣におけるPLS3発現量が内部コントロールであるGAPDHの発現量に対して2より大きい場合、また末梢血や門脈血におけるPLS3発現量が対照者における各発現量の「平均値±2 s.d.」より大きい場合、PLS3高発現者として上記可能性が高くなる。また乳がん患者については、対照者の95％信頼区間上限がcut off値になり、当該値よりPLS3発現量が高い場合は、PLS3高発現者として上記可能性が高くなる。 The test agent of the present invention is useful for evaluating the degree of cancer progression (infiltration and metastasis) of solid cancer and predicting the possibility of recurrence and prognosis after treatment for patients with solid cancer. Specifically, the evaluation of the degree of cancer progression and the prediction of the prognosis of recurrence using the test drug are based on the difference in the expression level of the PLS3 gene in the biological sample of the solid cancer patient and the biological sample of the control person (non-bearing patient). This can be done by determining. In this case, specifically, the expression of the PLS3 gene is elevated in the solid tumor primary lesion, bone marrow, and blood (portal vein blood, peripheral blood). ) Is higher than the corresponding tissue, bone marrow or blood expression level, the subject may have a high degree of cancer progression (invasion, metastasis), risk of recurrence after surgery, and poor prognosis Increases nature. Specifically, for colorectal cancer patients, when the PLS3 expression level in the primary lesion is greater than 2 compared to the internal control level of GAPDH, the PLS3 expression level in peripheral blood and portal vein blood When the expression level is larger than the “average value ± 2 sd”, the above possibility increases as a PLS3 high expression person. For breast cancer patients, the upper limit of the 95% confidence interval of the control person is the cut off value, and if the expression level of PLS3 is higher than that value, the above possibility becomes high as a PLS3 high expression person.
　（2）抗体
　本発明は、検査薬として、PLS3タンパクを特異的に認識することのできる抗体を提供する。当該抗体として、具体的には、配列番号２に記載のアミノ酸配列を有するヒト由来のPLS3タンパクを特異的に認識することのできる抗体を挙げることができる。 (2) Antibody The present invention provides an antibody capable of specifically recognizing PLS3 protein as a test agent. Specific examples of the antibody include an antibody capable of specifically recognizing a human-derived PLS3 protein having the amino acid sequence set forth in SEQ ID NO: 2.
　本発明は、前述するように、PLS3遺伝子の発現がEMS誘導に関係していること、固形がん、特に大腸がんまたは乳がんの原発巣、骨髄、血液（門脈血または末梢血）におけるPLS3遺伝子の発現の高さが、癌進展度（浸潤、転移）、術後の再発および予後不良に深く関係しているという知見をもとに、PLS3タンパク産生増加の有無やその程度を検出することによって、固形がんの癌進展度（浸潤、転移）を評価したり、術後の再発の危険性や予後を予測することができるとの発想に基づくものである。 As described above, the present invention relates to the fact that the expression of the PLS3 gene is related to EMS induction, PLS3 in solid tumors, particularly the primary lesions of colon cancer or breast cancer, bone marrow, blood (portal blood or peripheral blood). To detect the presence or extent of increased PLS3 protein production based on the knowledge that high gene expression is closely related to cancer progression (invasion, metastasis), postoperative recurrence, and poor prognosis Is based on the idea that the degree of cancer progression (invasion, metastasis) of solid cancer can be evaluated, and the risk and prognosis of postoperative recurrence can be predicted.
　従って、上記抗体は、固形がんの患者における上記PLS3タンパク産生増加の有無またはその程度を検出することによって、当該患者の癌進展度を評価したり、術後の再発の有無や予後を予測するための検査薬として使用される。 Therefore, the antibody detects the presence or extent of the above-mentioned increase in PLS3 protein production in patients with solid cancer, thereby assessing the degree of cancer progression in the patient or predicting the presence or prognosis of postoperative recurrence Used as a testing agent for.
　本発明の抗体は、その形態に特に制限はなく、PLS3タンパクを免疫抗原とするポリクローナル抗体であっても、またそのモノクローナル抗体であってもよい。さらに当該PLS3タンパクのアミノ酸配列（配列番号２）のうち少なくとも連続する、通常8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体も、本発明の抗体に含まれる。 The form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using PLS3 protein as an immunizing antigen or a monoclonal antibody thereof. Furthermore, an antibody having an antigen-binding property to a polypeptide consisting of at least continuous amino acids of the PLS3 protein (SEQ ID NO: 2), usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, is also of the present invention. Included in antibodies.
　これらの抗体の製造方法は、すでに周知であり、本発明の抗体もこれらの常法に従って製造することができる（Current protocols in Molecular Biology , Chapter 11.12～11.13(2000)）。具体的には、本発明の抗体がポリクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したPLS3タンパクを用いて、あるいは常法に従って当該PLS3タンパクの部分アミノ酸配列を有するオリゴペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、モノクローナル抗体の場合には、常法に従って大腸菌等で発現し精製したPLS3タンパク、あるいはタンパク質の部分アミノ酸配列を有するオリゴペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞の中から得ることができる（Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4～11.11）。 The methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the PLS3 protein is synthesized using a PLS3 protein expressed and purified in Escherichia coli according to a conventional method, or according to a conventional method. Thus, it is possible to immunize a non-human animal such as a rabbit and obtain it from the serum of the immunized animal according to a conventional method. On the other hand, in the case of a monoclonal antibody, spleen cells and bone marrow obtained by immunizing a non-human animal such as a mouse with PLS3 protein expressed and purified in Escherichia coli or the like according to a conventional method or an oligopeptide having a partial amino acid sequence of the protein are obtained. It can be obtained from hybridoma cells prepared by cell fusion with tumor cells (Current protocol, in molecular molecular biology, edit. Ausubel et al. (1987) Publishing, John Wiley, and Sons, Section 11.1-11.11).
　抗体の作製に免疫抗原として使用されるPLS3タンパクは、本発明により提供される遺伝子の配列情報（例えば配列番号１）に基づいて、ＤＮＡクローニング、各プラスミドの構築、宿主へのトランスフェクション、形質転換体の培養および培養物からのタンパク質の回収の操作により得ることができる。これらの操作は、当業者に既知の方法、あるいは文献記載の方法（Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)）などに準じて行うことができる。 The PLS3 protein used as an immunizing antigen for antibody production is based on DNA sequence information (for example, SEQ ID NO: 1) provided by the present invention, DNA cloning, construction of each plasmid, transfection into a host, transformation It can be obtained by manipulation of body culture and protein recovery from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
　具体的には、PLS3タンパクをコードする遺伝子が所望の宿主細胞中で発現できる組み換えDNA（発現ベクター）を作製し、これを宿主細胞に導入して形質転換し、該形質転換体を培養して、得られる培養物から、目的タンパク質を回収することによって、本発明抗体の製造のための免疫抗原としてのタンパク質を得ることができる。またPLS3タンパクの部分ペプチドは、本発明により提供されるアミノ酸配列の情報（配列番号２）に従って、一般的な化学合成法（ペプチド合成）によって製造することもできる。 Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding PLS3 protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. The protein as the immunizing antigen for producing the antibody of the present invention can be obtained by recovering the target protein from the obtained culture. The partial peptide of PLS3 protein can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information (SEQ ID NO: 2) provided by the present invention.
　なお、本発明のPLS3タンパクには、配列番号２に示すアミノ酸配列に関わるタンパク質のみならず、その相同物も包含される。該相同物としては、上記配列番号２で示されるアミノ酸配列において、１若しくは複数のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、且つ配列番号２で示されるタンパク質と免疫学的に同等の活性を有するタンパク質を挙げることができる。 The PLS3 protein of the present invention includes not only the protein related to the amino acid sequence shown in SEQ ID NO: 2, but also homologues thereof. The homologue consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and is immunologically equivalent to the protein shown in SEQ ID NO: 2. Can be mentioned.
　ここで同等の免疫学的活性を有するタンパク質としては、適当な動物あるいはその細胞において特定の免疫反応を誘発し、かつPLS3タンパクに対する抗体と特異的に結合する能力を有するタンパク質を挙げることができる。 Here, examples of the protein having equivalent immunological activity include a protein having an ability to induce a specific immune reaction in an appropriate animal or its cells and specifically bind to an antibody against PLS3 protein.
　また本発明の抗体は、PLS3タンパクの部分アミノ酸配列を有するオリゴペプチドを用いて調製されるものであってよい。かかる抗体の製造のために用いられるオリゴ（ポリ）ペプチドは、機能的な生物活性を有することは要しないが、PLS3タンパクと同様な免疫原特性を有するものであることが望ましい。好ましくはこの免疫原特性を有し、且つPLS3タンパクのアミノ酸配列において少なくとも連続する8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるオリゴ（ポリ）ペプチドを例示することができる。 The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of PLS3 protein. The oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as PLS3 protein. An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the PLS3 protein can be exemplified.
　かかるオリゴ（ポリ）ペプチドに対する抗体の製造は、宿主に応じて種々のアジュバントを用いて免疫学的反応を高めることによって行うこともできる。限定はされないが、そのようなアジュバントには、フロイントアジュバント、水酸化アルミニウムのようなミネラルゲル、並びにリゾレシチン、プルロニックポリオル、ポリアニオン、ペプチド、油乳剤、キーホールリンペットヘモシアニン及びジニトロフェノールのような表面活性物質、ＢＣＧ（カルメット－ゲラン桿菌）やコリネバクテリウム-パルヴムなどのヒトアジュバントが含まれる。 Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
　なお、PLS3タンパクの抗体は、簡便には、例えば「Santa Cruz Biotechnology Inc., CA, USA」などから商業的に入手することができる。 In addition, the antibody of PLS3 protein can be obtained commercially, for example, from “Santa Cruz Biotechnology Inc., CA, USA”.
　本発明の抗体はPLS3タンパクに特異的に結合する性質を有することから、該抗体を利用することによって、大腸がんまたは乳がん患者の組織内で発現産生されたPLS3タンパクまたは血液中に分泌されたPLS3タンパクを特異的に検出することができる。即ち、当該抗体は大腸がんまたは乳がん患者の生体組織または生体試料（血液を含む）中のPLS3タンパクの有無およびその程度を検出するためのプローブとして有用である。 Since the antibody of the present invention has the property of specifically binding to PLS3 protein, it was secreted into PLS3 protein or blood expressed and produced in the tissues of colon cancer patients or breast cancer patients by using the antibody. PLS3 protein can be specifically detected. That is, the antibody is useful as a probe for detecting the presence and level of PLS3 protein in a biological tissue or biological sample (including blood) of a colon cancer or breast cancer patient.
　具体的には、固形がんの癌組織の一部または骨髄をバイオプシ等で採取するか、もしくは血液（末梢血、門脈血、腫瘍還流静脈血）などを採取し、そこから常法に従って調製した抽出物やタンパク質を用いて、例えばウェスタンブロット法、ELISA法など公知の検出方法において、本発明抗体を常法に従ってプローブとして使用することによって、PLS3タンパクを検出し定量することができる。 Specifically, a portion of solid cancer tissue or bone marrow is collected with a biopsy, etc., or blood (peripheral blood, portal vein blood, tumor reflux venous blood), etc. is collected and prepared according to a conventional method. The PLS3 protein can be detected and quantified using the extracted extract or protein by using the antibody of the present invention as a probe in a known detection method such as Western blotting or ELISA.
　PLS3タンパクの測定に際しては、固形がん患者の生体試料におけるPLS3タンパクと対照者（非担癌者）の生体試料におけるPLS3タンパクとの量の違いを判定すればよい。この場合、具体的には、PLS3タンパクは、固形がん患者の原発巣、骨髄または血液（末梢血、腫瘍還流静脈血、門脈血）中において、対照者のそれらと比較して産生量が上昇している。このため、固形がん患者の原発巣、骨髄、または血液中のPLS3タンパクの量が、対照者について対応する組織、骨髄または血液中のPLS3タンパク量と比べて相対的に上昇していれば、被験者について、癌進展度（浸潤、転移）が高いこと、術後再発の危険性があること、予後が不良である可能性が高くなる。 In the measurement of PLS3 protein, the difference in the amount of PLS3 protein in the biological sample of the solid cancer patient and the amount of PLS3 protein in the biological sample of the control person (non-cancer-bearing person) may be determined. In this case, specifically, PLS3 protein is produced in solid tumor patients' primary lesions, bone marrow or blood (peripheral blood, tumor reflux venous blood, portal vein blood) in comparison with those of controls. It is rising. Therefore, if the amount of PLS3 protein in the primary tumor, bone marrow, or blood of a solid cancer patient is relatively increased compared to the amount of PLS3 protein in the corresponding tissue, bone marrow, or blood for the control, The subject has a high degree of cancer progression (invasion, metastasis), a risk of recurrence after surgery, and a poor prognosis.
　（3）検査薬キット
　本発明は、上記検査薬を含む、固形がんの癌進展度の評価、または固形がん患者について術後の再発可能性や予後予測に使用するための検査薬キットにも関する。当該検査薬キットは、上記プローブまたはプライマーとして用いられるオリゴまたはポリヌクレオチド（なお、これらは標識されていても、また固相に固定化されていてもよい）または抗体を少なくとも１つ含むものである。本発明の試薬キットは、本発明の検査薬として前述するプローブまたはプライマーの他、必要に応じてハイブリダイゼーション用の試薬、プローブの標識、ラベル体の検出剤、緩衝液など、本発明の方法の実施に必要な他の試薬、器具などを適宜含んでいてもよい。 (3) Test drug kit The present invention provides a test drug kit for use in the evaluation of the degree of cancer progression of solid cancer, or the possibility of postoperative recurrence and prognosis for patients with solid cancer, including the above test drugs. Also related. The test drug kit contains at least one oligo or polynucleotide (which may be labeled or immobilized on a solid phase) or antibody used as the probe or primer. The reagent kit of the present invention includes, in addition to the probe or primer described above as the test agent of the present invention, a hybridization reagent, a probe label, a label detection agent, a buffer solution, etc., if necessary. Other reagents and instruments necessary for the implementation may be included as appropriate.
　３．固形がん患者の再発予後予測方法
　本発明は、前述する本発明の検査薬を利用した固形がん、特に大腸がんまたは乳がんの患者を対象とした再発予後予測方法を提供する。 3. Method for Predicting Recurrence Prognosis of Solid Cancer Patient The present invention provides a method for predicting recurrence prognosis for patients with solid cancer, particularly colorectal cancer or breast cancer, using the above-described test agent of the present invention.
　具体的には、本発明の再発予後予測方法は、被験者から採取した生体試料に含まれるPLS3遺伝子の発現レベル、およびこの遺伝子に由来するタンパク質（PLS3タンパク）を検出し、その発現量またはそのタンパク質量を測定することにより、被験者について再発予後を予測するものである。 Specifically, the recurrence prognosis prediction method of the present invention detects the expression level of a PLS3 gene and a protein derived from this gene (PLS3 protein) contained in a biological sample collected from a subject, and the expression level or the protein thereof. By measuring the amount, the prognosis for recurrence is predicted for the subject.
　本発明の再発予後予測方法は次の（１）および（２）の工程を含む：
（１）固形がんの患者から採取した生体試料（被験試料）におけるPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程、
（２）上記で測定したPLS3遺伝子発現量（被験発現量）またはPLS3タンパク産生量（被験産生量）を、対照者から採取した生体試料（対照試料）におけるPLS3遺伝子発現量（対照発現量）またはPLS3タンパク産生量（対照産生量）と対比する工程。 The recurrence prognosis prediction method of the present invention includes the following steps (1) and (2):
(1) measuring a PLS3 gene expression level or PLS3 protein production level in a biological sample (test sample) collected from a patient with solid cancer;
(2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured as described above is the PLS3 gene expression level (control expression level) in a biological sample (control sample) collected from a control person or The process of comparing with PLS3 protein production (control production).
　かかる方法において得られた対照発現量または対照産生量に比して被験発現量または被験発現量が高い場合には、被験者について術後に再発する可能性が高いか、予後が不良と決定することができる。 If the test expression level or test expression level is high compared to the control expression level or control production level obtained by such a method, the subject is likely to relapse after surgery or the prognosis is determined to be poor. Can do.
　ここで用いられる生体試料としては、被験者については癌原発巣、骨髄、または血液（腫瘍還流静脈、末梢血）から調製される試料（被験試料）を、また対照者については当該被験試料に対応する生体組織、骨髄、血液（門脈血、末梢血）を挙げることができる。好ましくは末梢血である。また被験試料として、骨髄や血液中から単離、回収した遊離癌細胞を用いることもできる。具体的には、該組織、骨髄または血液から調製されるRNA含有試料、若しくはそれからさらに調製されるポリヌクレオチドを含む試料、または上記組織から調製されるタンパク質を含む試料を挙げることができる。かかるRNA、ポリヌクレオチドまたはタンパク質を含む試料は、被験者の癌組織の一部または骨髄をバイオプシ等で採取するか、もしくは血液を採取し、そこから常法に従って調製することができる。 The biological sample used here corresponds to the sample (test sample) prepared from the primary tumor, bone marrow, or blood (tumor reflux vein, peripheral blood) for the subject, and to the test sample for the control. Biological tissue, bone marrow, blood (portal blood, peripheral blood) can be mentioned. Peripheral blood is preferable. In addition, free cancer cells isolated and recovered from bone marrow or blood can also be used as test samples. Specifically, an RNA-containing sample prepared from the tissue, bone marrow or blood, a sample containing a polynucleotide further prepared therefrom, or a sample containing a protein prepared from the tissue can be mentioned. A sample containing such RNA, polynucleotide or protein can be prepared according to a conventional method by collecting a part of cancer tissue or bone marrow of a subject with a biopsy or the like, or collecting blood therefrom.
　本発明の方法は、測定対象として用いる生体試料の種類に応じて、具体的には下記のようにして実施される。 The method of the present invention is specifically performed as follows according to the type of biological sample used as a measurement target.
　(1) 測定対象の生体試料としてRNAを利用する場合
　測定対象物としてRNAを利用する場合、再発予後予測方法は、具体的には下記の工程(a)、(b)及び(c)を含む方法によって実施することができる：
(a)被験者から採取された生体試料（被験試料）から調製されたRNAまたはそれから転写された相補的ポリヌクレオチドと、前記本発明の検査薬（ポリ（オリゴ）ヌクレオチド）とを結合させる工程、
(b)該検査薬に結合した被験試料由来のRNAまたは該RNAから転写された相補的ポリヌクレオチドを、上記検査薬を指標として測定する工程、
(c)上記(b)の測定結果（被験結果）を、対照者（非担癌者）由来の生体試料（対照試料）に対して同様に(a)および(b)の工程から得られる結果（対照結果）と対比する工程。 (1) When the biological sample to be measured utilizing RNA as when the measurement object using the RNA, relapse prognosis method specifically includes the following steps (a), (b) and (c) Can be implemented by:
(a) a step of binding RNA prepared from a biological sample collected from a subject (test sample) or a complementary polynucleotide transcribed therefrom and the test agent (poly (oligo) nucleotide) of the present invention,
(b) a step of measuring RNA derived from a test sample bound to the test agent or a complementary polynucleotide transcribed from the RNA using the test agent as an index,
(c) Results obtained from the steps (a) and (b) for the biological sample (control sample) derived from the control person (non-cancer-bearing person) in the same manner as the measurement result (test result) in (b) above. Step to contrast with (control result).
　当該方法は、該RNA中のPLS3遺伝子の発現レベルを検出し、測定することによって実施される。具体的には、前述のポリ（オリゴ）ヌクレオチドからなる本発明の検査薬をプライマーまたはプローブとして用いて、ノーザンブロット法、ＲＴ-ＰＣＲ法、ＤＮＡチップ解析法、in situハイブリダイゼーション解析法などの公知の方法を行うことにより実施できる。 This method is carried out by detecting and measuring the expression level of the PLS3 gene in the RNA. Specifically, known methods such as Northern blot method, RT-PCR method, DNA chip analysis method, in-situ hybridization analysis method, etc. using the above-described test agent of the present invention comprising poly (oligo) nucleotides as a primer or probe. It can implement by performing the method of.
　ノーザンブロット法を利用する場合は、本発明の上記検査薬をプローブとして用いることによって、RNA中のPLS3遺伝子の発現の有無やその発現レベルを検出、測定することができる。具体的には、本発明の検査薬（相補鎖）を放射性同位元素（32P、33Pなど：RI）や蛍光物質などで標識し、それを、常法に従ってナイロンメンブレン等にトランスファーした被験者の生体組織由来のRNAとハイブリダイズさせた後、形成された検査薬（DNA）とRNAとの二重鎖を、検査薬の標識物（RI若しくは蛍光物質などの標識物質）に由来するシグナルを放射線検出器（BAS-1800II、富士フィルム社製）または蛍光検出器などで検出、測定する方法を例示することができる。また、AlkPhos Direct Labelling and Detection System (Amersham Pharmacia Biotech社製)を用いて、該プロトコールに従って検査薬（プローブDNA）を標識し、被験者の生体試料由来のRNAとハイブリダイズさせた後、検査薬の標識物に由来するシグナルをマルチバイオイメージャーSTORM860（Amersham Pharmacia Biotech社製）で検出、測定する方法を使用することもできる。 When using the Northern blot method, the presence or absence of the expression of the PLS3 gene in RNA and its expression level can be detected and measured by using the above-described test agent of the present invention as a probe. Specifically, the test agent of the invention (complementary strand) radioisotopes (such as 32 P, 33 P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method After hybridization with biological tissue-derived RNA, the formed diagnostic agent (DNA) and RNA duplex is radiated to a signal derived from the label of the diagnostic agent (labeled substance such as RI or fluorescent substance). A method of detecting and measuring with a detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector can be exemplified. In addition, using AlkPhos Direct Labeling and Detection System (Amersham Pharmacia Biotech), the test drug (probe DNA) is labeled according to the protocol and hybridized with RNA derived from the biological sample of the subject, and then labeled with the test drug. It is also possible to use a method in which a signal derived from a product is detected and measured with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech).
　RT-PCR法を利用する場合は、本発明の上記検査薬をプライマーとして用いることによって、RNA中のPLS3遺伝子の発現の有無や発現レベルを検出、測定することができる。具体的には、被験者の生体試料由来のRNAから常法に従ってcDNAを調製して、これを鋳型として標的のPLS3遺伝子の領域が増幅できるように、本発明の検査薬から調製した一対のプライマー（上記cDNA（－鎖）に結合する正鎖、＋鎖に結合する逆鎖）をこれとハイブリダイズさせて、常法に従ってPCR法を行い、得られた増幅二本鎖DNAを検出する方法を例示することができる。なお、増幅された二本鎖DNAの検出は、上記PCRを予めRIや蛍光物質で標識しておいたプライマーを用いて行うことによって産生される標識二本鎖DNAを検出する方法、産生された二本鎖DNAを常法に従ってナイロンメンブレン等にトランスファーさせて、標識した検査薬をプローブとして使用してこれとハイブリダイズさせて検出する方法などを用いることができる。なお、生成された標識二本鎖DNA産物はアジレント2100バイオアナライザ（横河アナリティカルシステムズ社製）などで測定することができる。また、SYBR Green RT-PCR Reagents (Applied Biosystems 社製)で該プロトコールに従ってRT-PCR反応液を調製し、ABI PRISM 7700 Sequence Detection System (Applied Biosystems 社製)で反応させて、該反応物を検出することもできる。 When using the RT-PCR method, the presence or absence and expression level of the PLS3 gene in RNA can be detected and measured by using the above-described test agent of the present invention as a primer. Specifically, a pair of primers prepared from the test agent of the present invention is prepared so that cDNA can be prepared from RNA derived from a biological sample of a subject according to a conventional method, and the target PLS3 gene region can be amplified using this as a template ( An example of a method for detecting the amplified double-stranded DNA obtained by hybridizing the above cDNA (-strand) to the positive strand and the reverse strand binding to the + strand and performing PCR according to a conventional method. can do. The detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and a labeled test agent is used as a probe and hybridized with this to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). Also, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems), and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.
　DNAチップ解析を利用する場合は、本発明の上記検査薬をDNAプローブ（１本鎖または２本鎖）として貼り付けたDNAチップを用意し、これに被験者の生体組織由来のRNAから常法によって調製されたcRNAとハイブリダイズさせて、形成されたDNAとcRNAとの二本鎖を、本発明の検査薬から調製される標識プローブと結合させて検出する方法を挙げることができる。また、上記DNAチップとして、PLS3遺伝子の遺伝子発現レベルの検出、測定が可能なDNAチップを用いることもできる。 When using DNA chip analysis, prepare a DNA chip to which the above-mentioned test agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and then apply RNA from RNA derived from the biological tissue of the subject by a conventional method. A method of detecting a double strand of DNA and cRNA formed by hybridization with the prepared cRNA by binding with a labeled probe prepared from the test agent of the present invention can be mentioned. In addition, a DNA chip that can detect and measure the gene expression level of the PLS3 gene can also be used as the DNA chip.
　 (2) 測定対象の生体試料としてタンパク質を用いる場合
　測定対象物としてタンパク質を用いる場合は、本発明の再発予後予測方法は、生体試料中のPLS3タンパクを検出し、その量を測定することによって実施される。具体的には下記の工程(a)、(b)及び(c)を含む方法によって実施することができる：
(a)被験者から採取した生体試料（被験試料）中のPLS3タンパクを、抗体に関する本発明の検査薬（PLS3タンパクを認識する抗体）と結合させる工程、
(b)該検査薬に結合した被験試料中のPLS3タンパクを、上記検査薬を指標として測定する工程、
(c)上記(b)の測定結果（被験結果）を、対照者から採取した生体試料（対照試料）に対して同様に(a)および(b)の工程から得られる結果（対照結果）と対比する工程。 (2) When protein is used as the biological sample to be measured When protein is used as the measurement target, the method for predicting recurrence of the present invention is carried out by detecting PLS3 protein in the biological sample and measuring the amount thereof. Is done. Specifically, it can be carried out by a method comprising the following steps (a), (b) and (c):
(a) a step of binding PLS3 protein in a biological sample collected from a subject (test sample) with the test agent of the present invention relating to an antibody (an antibody recognizing PLS3 protein),
(b) measuring the PLS3 protein in the test sample bound to the test agent using the test agent as an index,
(c) The measurement result (test result) in (b) above is the same as the result (control result) obtained from the steps (a) and (b) for the biological sample (control sample) collected from the control person. Contrast process.
　より具体的には、本発明の検査薬として抗体を用いて、ウエスタンブロット法などの公知方法で、PLS3タンパクを検出、定量する方法を挙げることができる。 More specifically, a method for detecting and quantifying PLS3 protein by a known method such as Western blotting using an antibody as the test agent of the present invention can be mentioned.
　ウエスタンブロット法は、一次抗体として本発明の検査薬を用いた後、二次抗体として125Ｉなどの放射性同位元素、蛍光物質、ホースラディッシュペルオキシターゼ（HRP）などの酵素等で標識した標識抗体（一次抗体に結合する抗体）を用い、得られる標識化合物の放射性同位元素、蛍光物質などの標識物質に由来するシグナルを放射線測定器（BAS-1800II：富士フィルム社製など）、蛍光検出器などで検出し、測定することによって実施できる。また、一次抗体として本発明の試薬を用いた後、ECL Plus Western Blotting Detction System (アマシャム ファルマシアバイオテク社製)を用いて、該プロトコールに従って検出し、マルチバイオイメージャーSTORM860(アマシャム ファルマシアバイオテク社製)で測定することもできる。 Western blotting uses a test antibody of the present invention as a primary antibody, followed by a labeled antibody (primary antibody labeled with a radioisotope such as 125 I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), etc. as a secondary antibody. Using a radiometer (BAS-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, etc., detect the signal derived from the labeling substance such as a radioisotope of the obtained labeled compound or a fluorescent substance. And can be implemented by measuring. In addition, after using the reagent of the present invention as a primary antibody, it was detected according to the protocol using ECL Plus Western Blotting Detction System (Amersham Pharmacia Biotech), and measured with a multi-bioimager STORM860 (Amersham Pharmacia Biotech) You can also
　なお、上記において測定対象とするPLS3タンパクの機能または活性は、当該タンパク質の量と正の相関関係を有している。従って、上記タンパク質の量の測定に代えて、該タンパク質の機能または活性の測定を行うことによっても、上記本発明の方法を実施することができる。すなわち本発明の方法には、PLS3の機能または活性を指標として、本願明細書に開示の方法に従って測定、評価することからなる、再発予後予測方法も含まれる。なお、PLS3はアクチン繊維を束ねる機能を有することが知られており、その測定方法も公知である（Giganti, A., et al. Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement. J Cell Sci 118, 1255-1265 (2005)；Oprea, G.E., et al. Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy. Science 320, 524-527 (2008)）。 In addition, the function or activity of the PLS3 protein to be measured in the above has a positive correlation with the amount of the protein. Therefore, instead of measuring the amount of the protein, the method of the present invention can also be carried out by measuring the function or activity of the protein. That is, the method of the present invention also includes a method for predicting recurrence, comprising measuring and evaluating according to the method disclosed in the present specification using the function or activity of PLS3 as an index. PLS3 is known to have a function of binding actin fibers, and its measurement method is also known (Giganti, A., et al. Actin-filament cross-linking protein T-plastin increases Arp2 / 3-mediated actin-based movement. J Cell Sci 118, 1255-1265 (2005); Oprea, GE, et al. Plastin 3 is a protective modifier of autosomal recessive spinal muscular atrophy. Science 320, 524-527 (2008)).
　(3)再発可能性、予後の予測
　固形がんの患者（被験者）について、術後の再発可能性および予後の予測は、被験者から採取した生体試料（原発巣組織、骨髄または血液など）（被験試料）におけるPLS3遺伝子の発現レベル、もしくはこの遺伝子の発現産物であるPLS3タンパクの量、機能若しくは活性（以下これらを合わせて「タンパク質レベル」ということがある）を、対照者（非担癌者）から採取した生体試料（被験試料に対応する組織、骨髄または血液など）における当該遺伝子発現レベルまたは当該タンパク質レベルと比較し、両者の違いを判定することによって行うことができる。生体試料として、好ましくは採取の容易性から末梢血を挙げることができる。 (3) Probability of recurrence and prognosis For patients with solid cancer (subject), the possibility of recurrence and prognosis after surgery is determined by biological samples collected from the subject (primary tissue, bone marrow, blood, etc.) The expression level of the PLS3 gene in the sample) or the amount, function, or activity of the PLS3 protein that is the expression product of this gene (hereinafter, these may be collectively referred to as “protein level”) It can be performed by comparing the gene expression level or the protein level in a biological sample (tissue, bone marrow, blood or the like corresponding to the test sample) and determining the difference between the two. The biological sample is preferably peripheral blood because of its ease of collection.
　被験者の生体試料（被験試料）と対照者の生体試料（対照試料）とのPLS3遺伝子発現量（レベル）またはPLS3タンパクの量（レベル）の比較は、被験試料と対照試料を対象とした測定を並行して行うことで実施できる。並行して行わない場合は、複数（少なくとも２つ、好ましくは３以上、より好ましくは５以上）の対照試料を用いて均一な測定条件で測定して得られたPLS3遺伝子の発現レベル、若しくは当該遺伝子の発現産物であるPLS3タンパクの量（レベル）の平均値または統計的中間値を、対照者の遺伝子発現レベル若しくはタンパク質の量として、比較に用いることができる。 Comparison of PLS3 gene expression level (level) or PLS3 protein level (level) between the biological sample of the subject (test sample) and the biological sample of the control (control sample) is based on the measurement of the test sample and the control sample. This can be done in parallel. If not performed in parallel, the expression level of the PLS3 gene obtained by measurement under uniform measurement conditions using a plurality (at least 2, preferably 3 or more, more preferably 5 or more) of control samples, or The average value or statistical intermediate value of the amount (level) of PLS3 protein, which is the gene expression product, can be used for comparison as the gene expression level or protein amount of the control.
　被験者が、術後再発可能性が高いか、予後不良であるか否かの判断は、被験試料におけるPLS3遺伝子の発現レベル、またはその発現産物であるPLS3タンパクのレベルが、上記被験試料に対応する対照試料のレベルと比較して有意に多いことを指標にして行うことができ、この場合、当該被験者は、術後再発可能性が高いか、予後不良であると判断することができる。 Whether a subject has a high possibility of recurrence after surgery or has a poor prognosis depends on whether the expression level of the PLS3 gene in the test sample or the level of the PLS3 protein, which is the expression product, corresponds to the test sample. It can be performed using as an index that the amount is significantly higher than the level of the control sample. In this case, the subject can be determined to have a high possibility of recurrence after surgery or a poor prognosis.
　４．固形がんの癌進展抑制剤、再発予防剤又は予後向上剤（本発明薬剤）
　後述する実験例に示すように、下記に示すPLS3遺伝子のsiRNAによると、PLS3遺伝子を発現する固形がん細胞におけるPLS3 mRNAの発現が抑制される。
sc-43215 A：
CCACGGAUAGAUAUUAACAtt（配列番号５）
UGUUAAUAUCUAUCCGUGGtt（配列番号６）
sc-43215 B
GAACUCUCUUGGUGUCAAUtt（配列番号７）
AUUGACACCAAGAGAGUUCtt（配列番号８）
sc-43215 C
GAAGAGUGGCAAUCUAACAtt（配列番号９）
UGUUAGAUUGCCACUCUUCtt（配列番号１０）。 4). Cancer progression inhibitor, recurrence preventive or prognostic improver for solid cancer (drug of the present invention)
As shown in the experimental examples to be described later, the PLS3 gene siRNA shown below suppresses the expression of PLS3 mRNA in solid cancer cells expressing the PLS3 gene.
sc-43215 A:
CCACGGAUAGAUAUUAACAtt (SEQ ID NO: 5)
UGUUAAUAUCUAUCCGUGGtt (SEQ ID NO: 6)
sc-43215 B
GAACUCUCUUGGUGUCAAUtt (SEQ ID NO: 7)
AUUGACACCAAGAGAGUUCtt (SEQ ID NO: 8)
sc-43215 C
GAAGAGUGGCAAUCUAACAtt (SEQ ID NO: 9)
UGUUAGAUUGCCACUCUUCtt (SEQ ID NO: 10).
　従って、当該siRNAは、PLS3 mRNA発現によるEMS誘導を阻止することにより、固形がんの癌進展を抑制する薬剤（癌進展抑制剤）の有効成分として使用することができる。さらに、当該siRNAは、PLS3 mRNA発現を抑制することにより、固形がん、特に大腸がんや乳がんの患者について、術後の再発を予防する薬剤（再発予防剤）または予後を向上する薬剤（予後向上剤）の有効成分として使用することができる。以下、これらの薬剤を「本発明薬剤」と総称する。 Therefore, the siRNA can be used as an active ingredient of a drug that suppresses cancer progression of a solid cancer (cancer progression inhibitor) by blocking EMS induction by PLS3 mRNA expression. In addition, the siRNA suppresses PLS3 mRNA expression to prevent postoperative recurrence (relapse prevention agent) or improve prognosis (prognosis) for patients with solid cancer, especially colorectal cancer and breast cancer. It can be used as an active ingredient of an improver. Hereinafter, these drugs are collectively referred to as “the drug of the present invention”.
　ゆえに本発明は、PLS3遺伝子の発現を抑制するsiRNA を有効成分とする上記本発明薬剤を提供する。 Therefore, the present invention provides the above-mentioned agent of the present invention comprising siRNAsi that suppresses the expression of PLS3 gene as an active ingredient.
　本発明が対象とするsiRNA は、二本鎖であり、PLS3遺伝子の標的配列に相補的な配列であるセンス鎖と該センス鎖に相補的な配列であるアンチセンス鎖がハイブリダイズしてなっている。かかるsiRNAは、PLS3遺伝子の発現を抑制するものであれば特に制限されないが、好ましくは、前述するsiRNA（配列番号５～１０）を挙げることができる。 The siRNA RNA targeted by the present invention is double-stranded, and is formed by hybridizing a sense strand that is complementary to the target sequence of the PLS3 gene and an antisense strand that is complementary to the sense strand. Yes. Such siRNA is not particularly limited as long as it suppresses the expression of the PLS3 gene, and preferred examples include siRNA (SEQ ID NOs: 5 to 10) described above.
　またPLS3遺伝子の発現を抑制するsiRNAに限らず、PLS3遺伝子の発現を抑制する作用を有するものも本発明薬剤の有効成分として使用することができる。かかる作用を有するものとして、アンチセンスポリヌクレオチドを挙げることができる。当該アンチセンスポリヌクレオチドは、PLS3遺伝子の塩基配列に相補的な、または実質的に相補的な塩基配列またはその一部を有するものであり、PLS3遺伝子のＲＮＡとハイブリダイズすることによって、該ＲＮＡの合成または機能を阻害するか、あるいは該ＲＮＡとの相互作用を介してPLS3遺伝子の発現を調節・制御する作用を有するものである。アンチセンスポリヌクレオチドには、上記作用を有するものであれば、いずれのアンチセンスポリヌクレオチドであってもよく、アンチセンスＲＮＡ、アンチセンスＤＮＡなどが含まれる。 Further, not only siRNA that suppresses the expression of the PLS3 gene, but also those having an action of suppressing the expression of the PLS3 gene can be used as the active ingredient of the drug of the present invention. Antisense polynucleotide can be mentioned as what has this effect | action. The antisense polynucleotide has a base sequence complementary to or substantially complementary to the base sequence of the PLS3 gene, or a part thereof, and hybridizes with the RNA of the PLS3 gene to thereby It has an effect of inhibiting synthesis or function, or regulating / controlling the expression of PLS3 gene through interaction with the RNA. The antisense polynucleotide may be any antisense polynucleotide as long as it has the above action, and includes antisense RNA, antisense DNA, and the like.
　アンチセンスポリヌクレオチドは通常、10～1000個程度、好ましくは15～500個程度、更に好ましくは16～30個程度の塩基から構成される。ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンスＤＮＡを構成する各ヌクレオチドのリン酸残基（ホスフェート）は、例えば、ホスホロチオエート、メチルホスホネート、ホスホロジチオネートなどの化学修飾リン酸残基に置換されていてもよい。これらのアンチセンスポリヌクレオチドは、公知のＤＮＡ合成装置などを用いて製造することができる。 The antisense polynucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases. In order to prevent degradation by a hydrolase such as nuclease, phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are converted to chemically modified phosphate residues such as phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer.
　さらにPLS3遺伝子の発現を抑制するsiRNAに限らず、PLS3の機能を抑制する作用を有するものも本発明薬剤の有効成分として使用することができる。かかる作用を有するものとして、PLS3に対する抗体、特に中和抗体を挙げることができる。なお、中和抗体とは、抗原に結合することによりその抗原が本来有していた機能又は活性を阻害する性質を有する抗体を意味する。PLS3に対する中和抗体とは、PLS3に結合することによりPLS3が有する機能又は活性を阻害する性質を有する抗体を言う。 Furthermore, not only siRNA that suppresses the expression of the PLS3 gene, but also those having an action of suppressing the function of PLS3 can be used as the active ingredient of the drug of the present invention. Examples of such an action include antibodies against PLS3, particularly neutralizing antibodies. The neutralizing antibody means an antibody having the property of inhibiting the function or activity originally possessed by the antigen by binding to the antigen. The neutralizing antibody against PLS3 refers to an antibody having a property of inhibiting the function or activity of PLS3 by binding to PLS3.
　本発明薬剤は、上記のPLS3遺伝子の発現を抑制したりまたその機能を抑制する有効成分（siRNA、アンチセンスポリヌクレオチド、抗体など） に加え、任意の担体や添加剤、例えば医薬上許容される担体および添加剤を含むことができる。 The agent of the present invention is an active ingredient (siRNA, antisense polynucleotide, antibody, etc.) that suppresses the expression of PLS3 gene or suppresses its function, and any carrier or additive, for example, pharmaceutically acceptable Carriers and additives can be included.
　医薬上許容される担体および添加剤としては、例えば、ショ糖、デンプン等の賦形剤；セルロース、メチルセルロース等の結合剤；デンプン、カルボキシメチルセルロース等の崩壊剤；ステアリン酸マグネシウム、エアロジル等の滑剤；クエン酸、メントール等の芳香剤；安息香酸ナトリウム、亜硫酸水素ナトリウム等の保存剤；クエン酸、クエン酸ナトリウム等の安定剤；メチルセルロース、ポリビニルピロリド等の懸濁剤；界面活性剤等の分散剤；水、生理食塩水等の希釈剤；ベースワックス等が挙げられるが、それらに限定されるものではない。 Examples of pharmaceutically acceptable carriers and additives include excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil; Fragrances such as citric acid and menthol; preservatives such as sodium benzoate and sodium bisulfite; stabilizers such as citric acid and sodium citrate; suspending agents such as methylcellulose and polyvinylpyrrolide; dispersants such as surfactants Diluents such as water and physiological saline; base wax and the like, but are not limited thereto.
　経口投与に好適な製剤としては、液剤、カプセル剤、サッシェ剤、錠剤、懸濁液剤、乳剤等を挙げることができる。非経口的な投与（例えば、皮下注射、筋肉注射、局所注入、腹腔内投与など）に好適な製剤としては、水性および非水性の等張な無菌の注射液剤があり、これには抗酸化剤、緩衝液、制菌剤、等張化剤等が含まれていてもよい。また、非経口的な投与製剤としては、他に水性および非水性の無菌の懸濁液剤が挙げられ、これには懸濁剤、可溶化剤、増粘剤、安定化剤、防腐剤等が含まれていてもよい。 Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. In addition, parenteral administration preparations include aqueous and non-aqueous sterile suspensions, including suspensions, solubilizers, thickeners, stabilizers, preservatives, and the like. It may be included.
　本発明薬剤の投与量は、投与対象となる被験者の体重や年齢、並びに病気の重篤度等によって異なり、一概に設定することはできないが、例えば、成人１日あたり有効成分量として数mg～数十mg／kg体重を挙げることができ（例えば、0.1～90mg/kg体重の範囲が例示される）、これを１日１回～数回に分けて投与することができる。 The dose of the drug of the present invention varies depending on the body weight and age of the subject to be administered, the severity of the disease, etc., and cannot be set unconditionally. For example, the amount of the active ingredient per day for an adult is several mg to Several tens mg / kg body weight can be mentioned (for example, the range of 0.1 to 90 mg / kg body weight is exemplified), and this can be administered once to several times a day.
　上記の有効成分がDNAによりコードされるものである場合は、該DNAを遺伝子治療用ベクターに組込み、遺伝子治療を行うことも考えられる。更に、上記有効成分がアンチセンスポリヌクレオチドの場合は、そのままもしくは遺伝子治療用ベクターにこれを組込むことにより、遺伝子治療を行うこともできる。これらの場合も、遺伝子治療用組成物の投与量、投与方法は患者の体重、年齢、症状などにより変動し、当業者であれば適宜選択することが可能である。 In the case where the above active ingredient is encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. Furthermore, when the active ingredient is an antisense polynucleotide, gene therapy can be performed as it is or by incorporating it into a gene therapy vector. Also in these cases, the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
　５．癌進展抑制、再発抑制または予後向上に有効な成分をスクリーニングする方法
　後述する実験例で示すように、本発明者らは、固形がん、特に大腸がんまたは乳がんの原発巣、骨髄、または血液（末梢血、腫瘍還流静脈血、門脈血）において特異的にPLS3遺伝子の発現が上昇していること、PLS3遺伝子の発現によりEMSが誘導されることから、PLS3の高発現が、固形がんの癌進展（浸潤、転移）に深く関わっていることを見出した。また本発明者らは、固形がん、特に大腸がんまたは乳がんの原発巣、骨髄または血液（末梢血、腫瘍還流静脈血、門脈血）におけるPLS3遺伝子の高発現と、当該患者の術後の再発および予後不良とが正の相関関係にあることから、PLS3の高発現が、固形がん患者の術後の再発や予後不良に深く関わっていることを見出した。 5). Method for Screening Components Effective for Suppressing Cancer Progression, Relapse Suppression, or Prognosis Improvement As shown in the experimental examples described later, the present inventors have developed solid tumors, particularly colon cancer or breast cancer primary lesions, bone marrow, or blood. PLS3 gene expression is specifically increased in (peripheral blood, tumor reflux venous blood, portal vein blood), and EMS is induced by the expression of PLS3 gene. Was found to be deeply involved in cancer progression (invasion, metastasis). In addition, the present inventors also showed high expression of the PLS3 gene in solid tumors, particularly colon cancer or breast cancer primary lesions, bone marrow or blood (peripheral blood, tumor reflux venous blood, portal vein blood), and postoperative patients. Therefore, we found that high expression of PLS3 is deeply related to postoperative recurrence and poor prognosis in solid cancer patients.
　これらのことから、PLS3遺伝子の発現（mRNA発現）やPLS3タンパクの機能を抑制しえる物質は、固形がんの癌進展を抑制する有効成分として、また術後の再発を予防し、また予後を向上する有効成分として有用であると考えられる。 Therefore, substances that can suppress the expression of PLS3 gene (mRNA expression) and PLS3 protein function as active ingredients that suppress the progression of solid cancer, prevent recurrence after surgery, and prognosis. It is considered useful as an active ingredient that improves.
　下記に説明する本発明のスクリーニング方法は、被験物質の中から、（3-1）PLS3遺伝子の発現抑制、（3-2）PLS3タンパクの産生量の低下、または（3-3）PLS3タンパクが有する機能の低下、を指標として、これらの作用を示す物質を探索することによって、固形がんの癌進展抑制剤、または固形がんの患者について術後の再発を抑制したり、予後を向上するための薬剤（再発抑制剤、予後向上剤）（以下、これらを総称して「本発明薬剤」ともいう）の有効成分を取得しようとするものである。 The screening method of the present invention described below includes: (3-1) suppression of PLS3 gene expression, (3-2) decrease in PLS3 protein production, or (3-3) PLS3 protein among test substances. By searching for substances that exhibit these effects using the decrease in function as an indicator, it is possible to suppress postoperative recurrence or improve prognosis for cancer progression inhibitors of solid cancer or patients with solid cancer Therefore, it is intended to obtain an active ingredient of a drug (relapse inhibitor, prognosis improver) (hereinafter collectively referred to as “the drug of the present invention”).
　なお、本発明薬剤の有効成分となりえる候補物質としては、核酸、ペプチド、タンパク質、有機化合物（低分子化合物、高分子化合物を含む）、無機化合物などを挙げることができる。本発明のスクリーニング方法は、これらの候補物質を含む試料を対象として実施することができる（これらを総称して「被験物質」という）。ここで、候補物質を含む試料には、細胞抽出物、遺伝子ライブラリーの発現産物、微生物培養上清、および菌体成分などが含まれる。 In addition, examples of candidate substances that can be active ingredients of the drug of the present invention include nucleic acids, peptides, proteins, organic compounds (including low molecular compounds and high molecular compounds), inorganic compounds, and the like. The screening method of the present invention can be carried out on samples containing these candidate substances (collectively referred to as “test substances”). Here, the sample containing the candidate substance includes a cell extract, an expression product of a gene library, a microorganism culture supernatant, a cell component, and the like.
　(i) PLS3遺伝子の発現を抑制する作用を有する物質のスクリーニング方法
　当該スクリーニングは、被験物質の中から、PLS3遺伝子の発現を抑制する作用を有する物質を、PLS3 mRNAの発現量を指標として探索し、本発明薬剤の有効成分の候補物質として取得する方法である。 (i) Method for screening a substance having an action of suppressing the expression of PLS3 gene In this screening, a substance having an action of suppressing the expression of PLS3 gene is searched from test substances using the expression level of PLS3 mRNA as an index. This is a method for obtaining a candidate substance for an active ingredient of the drug of the present invention.
　当該方法は、具体的には下記の工程（１）～（３）を行うことによって実施することができる。
（１）被験物質とPLS3遺伝子を発現可能な細胞とを接触させる工程、
（２）被験物質を接触させた細胞のPLS3遺伝子の発現量（被験発現量）を測定する工程、及び
（３）上記の被験発現量が、被験物質を接触させない対照細胞のPLS3遺伝子の発現量（対照発現量）よりも小さい場合に、当該被験物質を上記候補物質として選択する工程。 Specifically, this method can be carried out by performing the following steps (1) to (3).
(1) contacting the test substance with a cell capable of expressing the PLS3 gene;
(2) a step of measuring the expression level (test expression level) of the PLS3 gene in the cell contacted with the test substance, and (3) the expression level of the PLS3 gene in the control cell not contacting the test substance. A step of selecting the test substance as the candidate substance when it is smaller than (control expression level).
　かかるスクリーニングに用いられる細胞としては、内在性または外来性の別を問わず、PLS3遺伝子を発現し得る細胞であればよい。なおPLS3遺伝子の由来も特に制限されず、ヒト由来のPLS3遺伝子（配列番号１）であっても、またヒト以外のマウスなどの哺乳類やその他の生物種に由来するものであってもよい。好ましくはヒト由来のPLS3遺伝子である。かかる細胞として、具体的には、造血細胞以外のPLS3遺伝子を発現しえる細胞、すなわち固形組織細胞（ヒトおよびその他の生物種由来のものを含む）、およびそこから単離調製された当該組織細胞の初代培養細胞を挙げることができる。また、定法に従って、PLS3遺伝子のcDNAを有する発現ベクターを導入してPLS3 mRNAを発現可能な状態に調製された形質転換細胞を使用することもできる。なお、スクリーニングに用いられる細胞の範疇には、細胞の集合体である組織も含まれる。 The cells used for such screening may be cells that can express the PLS3 gene regardless of whether they are endogenous or exogenous. The origin of the PLS3 gene is not particularly limited, and may be a human-derived PLS3 gene (SEQ ID NO: 1), or may be derived from mammals such as mice other than humans or other biological species. PLS3 gene derived from human is preferable. As such cells, specifically, cells capable of expressing PLS3 gene other than hematopoietic cells, that is, solid tissue cells (including those derived from humans and other species), and the tissue cells isolated and prepared therefrom Of primary cultured cells. In addition, according to a standard method, a transformed cell prepared by introducing an expression vector having cDNA of PLS3 gene and capable of expressing PLS3 mRNA can be used. The category of cells used for screening includes tissues that are aggregates of cells.
　本発明のスクリーニング方法の工程（１）において、被験物質とPLS3遺伝子発現可能細胞とを接触させる条件は、特に制限されないが、当該細胞が死滅せず、且つPLS3遺伝子が発現し得る培養条件（温度、ｐＨ、培地組成など）を選択することが好ましい。 In the step (1) of the screening method of the present invention, the condition for bringing the test substance into contact with the cell capable of expressing the PLS3 gene is not particularly limited, but the culture condition (temperature) in which the cell does not die and the PLS3 gene can be expressed. , PH, medium composition, etc.).
　候補物質の選別は、例えば上記条件で被験物質とPLS3遺伝子発現可能細胞とを接触させて、PLS3遺伝子の発現を抑制させてそのmRNAの発現量を低下させる物質を探索することによって行うことができる。具体的には、被験物質存在下でPLS3遺伝子発現可能細胞を培養した場合のPLS3 mRNAの発現量が、被験物質非存在下で上記に対応するPLS3遺伝子発現可能細胞を培養した場合に得られるPLS3 mRNAの発現量（対照発現量）よりも小さいことを指標として、細胞と接触させた当該被験物質を候補物質として選別することができる。 Selection of candidate substances can be performed, for example, by contacting a test substance with a cell capable of expressing the PLS3 gene under the above conditions and searching for a substance that suppresses the expression of the PLS3 gene and reduces the expression level of the mRNA. . Specifically, the expression level of PLS3 mRNA when cultivating cells capable of expressing PLS3 gene in the presence of a test substance is PLS3 obtained when culturing cells capable of expressing PLS3 gene corresponding to the above in the absence of test substance The test substance that has been brought into contact with the cell can be selected as a candidate substance using as an index the expression level that is smaller than the expression level of mRNA (control expression level).
　PLS3 mRNAの発現量の測定（検出、定量）は、PLS3遺伝子発現可能細胞のPLS3 mRNAの発現量を、当該PLS3 mRNAの塩基配列と相補的な配列を有するオリゴヌクレオチド（前述する本発明の試薬）などを利用したノーザンブロット法やRT-PCR法、リアルタイム定量RT-PCR法などの公知の方法、またはDNAアレイを利用した測定方法を実施することなどにより行うことができる。 The measurement (detection and quantification) of the expression level of PLS3 mRNA is performed by measuring the expression level of PLS3 mRNA in a cell capable of expressing PLS3 gene with an oligonucleotide having a sequence complementary to the base sequence of the PLS3 mRNA (the reagent of the present invention described above) It can be carried out by carrying out a known method such as a Northern blot method using RT and the like, RT-PCR method, real-time quantitative RT-PCR method, or a measuring method using a DNA array.
　またPLS3遺伝子の発現レベルの検出及び定量は、PLS3遺伝子の発現を制御する遺伝子領域（発現制御領域）に、例えばルシフェラーゼ遺伝子などのマーカー遺伝子をつないだ融合遺伝子を導入した細胞株を用いて、マーカー遺伝子由来のタンパク質の活性を測定することによっても実施できる。本発明のPLS3遺伝子の発現制御物質のスクリーニング方法には、かかるマーカー遺伝子の発現量を指標として標的物質を探索する方法も包含されるものであり、この意味において前述する（V）の欄に記載する「PLS3遺伝子」の概念には、PLS3遺伝子の発現制御領域とマーカー遺伝子との融合遺伝子が含まれる。 In addition, the detection and quantification of the expression level of the PLS3 gene is performed using a cell line in which a fusion gene in which a marker gene such as a luciferase gene is connected to a gene region (expression control region) that controls the expression of the PLS3 gene is used. It can also be carried out by measuring the activity of a gene-derived protein. The screening method for a PLS3 gene expression control substance of the present invention includes a method for searching for a target substance using the expression level of the marker gene as an index. In this sense, the method described in the section (V) described above is included. The concept of “PLS3 gene” includes a fusion gene of an expression control region of the PLS3 gene and a marker gene.
　なお、上記マーカー遺伝子としては、発光反応や呈色反応を触媒する酵素の構造遺伝子が好ましい。具体的には、上記のルシフェラーゼ遺伝子のほか、アルカリフォスファターゼ遺伝子、クロラムフェニコール・アセチルトランスフェラーゼ遺伝子、βグルクロニダーゼ遺伝子、βガラクトシダーゼ遺伝子、及びエクオリン遺伝子などのレポーター遺伝子を例示できる。融合遺伝子の作成、およびマーカー遺伝子由来の活性測定は公知の方法で行うことができる。 The marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction. Specifically, reporter genes such as alkaline phosphatase gene, chloramphenicol acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, and aequorin gene can be exemplified in addition to the above luciferase gene. Creation of a fusion gene and measurement of activity derived from a marker gene can be performed by known methods.
　かかる本発明のスクリーニング方法により選別される物質は、PLS3遺伝子の遺伝子発現抑制剤として位置づけることができる。これらの物質は、PLS3遺伝子の発現を抑制することによって、EM誘導を阻止して、浸潤や転移などの癌進展を抑制する薬物、また術後の再発を予防する薬物、予後を向上する薬物の有効成分となりえる候補物質となる。 The substance selected by the screening method of the present invention can be positioned as a gene expression inhibitor for the PLS3 gene. These substances are drugs that inhibit EM induction by suppressing the expression of the PLS3 gene and suppress cancer progression such as invasion and metastasis, drugs that prevent postoperative recurrence, and drugs that improve prognosis. Candidate substances that can be active ingredients.
　(ii) PLS3タンパクの産生を低下させる作用を指標とした物質のスクリーニング方法
　当該スクリーニングは、被験物質の中から、PLS3タンパクの産生を低下させる作用を有する物質を、PLS3タンパクの産生量の低下を指標として探索し、本発明薬剤 の有効成分として取得する方法である。 (ii) Method for screening substances using the action of reducing the production of PLS3 protein as an indicator The screening is to reduce the production of PLS3 protein from substances to be tested that have the action of reducing the production of PLS3 protein. It is a method of searching as an index and obtaining it as an active ingredient of the drug of the present invention.
　当該方法は、具体的には下記の工程（1’）～（3’）を行うことによって実施することができる。
（1’）被験物質とPLS3タンパクを産生可能な細胞またはこの細胞から調製した細胞画分とを接触させる工程、
（2’）被験物質を接触させた細胞または細胞画分のPLS3タンパクの産生量（被験産生量）を測定する工程、及び
（3’）上記の被験産生量が、被験物質を接触させない対照細胞（PLS3タンパクを産生可能な細胞）または被験物質を接触させない細胞画分（PLS3タンパクを産生可能な細胞から調製）のPLS3タンパクの産生量（対照産生量）よりも小さい被験物質を選択する工程。 Specifically, this method can be carried out by performing the following steps (1 ′) to (3 ′).
(1 ′) contacting a test substance with a cell capable of producing PLS3 protein or a cell fraction prepared from the cell,
(2 ′) a step of measuring the production amount (test production amount) of PLS3 protein in the cell or cell fraction contacted with the test substance, and (3 ′) control cells in which the test production amount is not in contact with the test substance. A step of selecting a test substance smaller than the production amount (control production amount) of PLS3 protein in a cell fraction (prepared from a cell capable of producing PLS3 protein) or a cell fraction (prepared from cells capable of producing PLS3 protein) which is not in contact with the test substance.
　かかるスクリーニングに用いられる細胞（対照細胞を含む）としては、内在性または外来性の別を問わず、PLS3遺伝子が発現してPLS3タンパクを産生し得る細胞であればよい。なおPLS3遺伝子の由来も特に制限されず、ヒト由来であっても、またヒト以外のマウスなどの哺乳類やその他の生物種に由来するものであってもよい。好ましくはヒト由来のPLS3タンパクである。かかる細胞として、具体的には、造血細胞以外のPLS3遺伝子を発現しえる細胞、すなわち固形組織細胞、および単離調製された当該細胞の初代培養細胞を挙げることができる。また、定法に従って、PLS3遺伝子のcDNAを有する発現ベクターを導入してPLS3タンパクを産生可能な状態に調製された形質転換細胞を使用することもできる。なお、スクリーニングに用いられる細胞の範疇には、細胞の集合体である組織も含まれる。 The cells used for the screening (including control cells) may be any cells that can express PLS3 gene and produce PLS3 protein regardless of whether they are endogenous or exogenous. The origin of the PLS3 gene is not particularly limited, and may be derived from humans, or derived from mammals such as mice other than humans or other biological species. PLS3 protein derived from human is preferable. Specific examples of such cells include cells capable of expressing a PLS3 gene other than hematopoietic cells, that is, solid tissue cells, and primary cultured cells of the cells isolated and prepared. In addition, transformed cells prepared in a state capable of producing PLS3 protein by introducing an expression vector having PLS3 gene cDNA according to a conventional method can also be used. The category of cells used for screening includes tissues that are aggregates of cells.
　本発明のスクリーニング方法（3-2）の工程（1’）において、被験物質とPLS3タンパク産生可能細胞とを接触させる条件は、特に制限されないが、当該細胞が死滅せず、且つPLS3遺伝子が発現し且つPLS3が産生し得る培養条件（温度、ｐＨ、培地組成など）を選択することが好ましい。 In the step (1 ′) of the screening method (3-2) of the present invention, the condition for bringing the test substance into contact with the cell capable of producing PLS3 protein is not particularly limited, but the cell does not die and the PLS3 gene is expressed. In addition, it is preferable to select culture conditions (temperature, pH, medium composition, etc.) that PLS3 can produce.
　候補物質の選別は、例えば上記条件で被験物質とPLS3タンパク産生可能細胞またはその細胞画分とを接触させることで、PLS3タンパクの産生量を低下させる物質を探索することによって行うことができる。具体的には、被験物質存在下でPLS3タンパク産生可能細胞またはその細胞画分を培養した場合のPLS3タンパク産生量が、被験物質非存在下で上記に対応するPLS3タンパク産生可能細胞またはその細胞画分を培養した場合に得られるPLS3タンパクの産生量（対照産生量）よりも小さいことを指標として、細胞または細胞画分と接触させた当該被験物質を候補物質として選別することができる。 Selection of candidate substances can be performed, for example, by searching for substances that reduce the production amount of PLS3 protein by contacting the test substance with cells capable of producing PLS3 protein or a cell fraction thereof under the above conditions. Specifically, when a PLS3 protein-producing cell or a cell fraction thereof is cultured in the presence of a test substance, the PLS3 protein production amount corresponding to the above in the absence of the test substance or a cell fraction thereof The test substance brought into contact with a cell or a cell fraction can be selected as a candidate substance by using as an index the production amount (control production amount) of PLS3 protein obtained when the fraction is cultured.
　PLS3タンパク産生量の測定（検出、定量）は、PLS3タンパク産生可能細胞またはその細胞画分から得られるPLS3タンパクの量を、当該PLS3タンパクに対する抗体（抗PLS3抗体）（前述する本発明の検出試薬）を利用してウエスタンブロット法や免疫沈降法、ＥＬＩＳＡ等の公知の方法を行うことによって実施することができる。具体的には、ウエスタンブロット法は、一次抗体として本発明の検出試薬を用いた後、二次抗体として125Iなどの放射性同位元素、蛍光物質、ホースラディッシュペルオキシターゼ（HRP）などの酵素等で標識した一次抗体に結合する抗体を用いて標識し、これら標識物質由来のシグナルを放射線測定器(BAS-1800II：富士フィルム社製など)、蛍光検出器などで測定することによって実施できる。また、一次抗体として本発明の検出試薬を用いた後、ECL Plus Western Blotting Detction System (アマシャム ファルマシアバイオテク社製)を利用して、該プロトコールに従って検出し、マルチバイオイメージャーSTORM860(アマシャム ファルマシアバイオテク社製)で測定することもできる。 Measurement (detection and quantification) of PLS3 protein production is performed by measuring the amount of PLS3 protein obtained from cells capable of producing PLS3 protein or its cell fraction, and an antibody against the PLS3 protein (anti-PLS3 antibody) (the detection reagent of the present invention described above) Can be carried out by performing a known method such as Western blotting, immunoprecipitation or ELISA. Specifically, in Western blotting, the detection reagent of the present invention is used as a primary antibody, followed by labeling with a radioisotope such as 125 I as a secondary antibody, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), or the like. The labeling can be performed using an antibody that binds to the primary antibody, and signals derived from these labeling substances are measured with a radiation measuring instrument (BAS-1800II, manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like. Further, after using the detection reagent of the present invention as a primary antibody, ECL Plus Western Blotting Detction System (Amersham Pharmacia Biotech) was used for detection according to the protocol, and multi-bioimager STORM860 (Amersham Pharmacia Biotech) Can also be measured.
　かかる本発明のスクリーニング方法により選別される物質は、PLS3タンパクの産生抑制剤として位置づけることができる。これらの物質は、PLS3タンパクの産生を抑制することによってEM誘導を阻止して、浸潤や転移などの癌進展を抑制する薬物、また術後の再発予防する薬物、予後を向上する薬物の候補物質となる。 A substance selected by the screening method of the present invention can be positioned as a PLS3 protein production inhibitor. These substances are drugs that inhibit EM induction by inhibiting the production of PLS3 protein and suppress cancer progression such as invasion and metastasis, drugs that prevent recurrence after surgery, and drug candidates that improve prognosis It becomes.
　(iii) PLS3の機能を低下させる作用を指標とした物質のスクリーニング方法
　当該スクリーニングは、被験物質の中から、PLS3の機能を低下させる作用を有する物質を、PLS3作用の低下を指標として探索し、本発明薬剤の有効成分として取得する方法である。 (iii) Screening method of substances using the action of reducing the function of PLS3 as an index The screening searches for substances having an action of reducing the function of PLS3 from test substances using the decrease in the action of PLS3 as an index, This is a method for obtaining the active ingredient of the drug of the present invention.
　当該方法は、具体的には下記の工程（1”）～（3”）を行うことによって実施することができる。
（1”）被験物質とPLS3に反応する細胞またはこの細胞から調製した細胞画分を接触させる工程、
（2”）PLS3に反応する細胞とPLS3とを接触させる工程、
（3”）被験物質およびPLS3を接触させた細胞または細胞画分のPLS3の機能または活性を検出する工程、及び
（4”）上記の検出した機能が、被験物質を接触させない対照細胞または細胞画分のPLS3の機能または活性よりも低い被験物質を選択する工程。 Specifically, the method can be carried out by performing the following steps (1 ″) to (3 ″).
(1 ") a step of contacting a test substance with a cell that reacts with PLS3 or a cell fraction prepared from the cell,
(2 ") a step of contacting PLS3 with a cell that reacts with PLS3,
(3 ″) a step of detecting the function or activity of PLS3 in a cell or cell fraction in contact with the test substance and PLS3; and (4 ″) a control cell or cell fraction in which the detected function does not contact the test substance. Selecting a test substance having a lower PLS3 function or activity per minute.
　かかるスクリーニングに用いられるPLS3に反応する細胞としては、PLS3と接触させる事によって何らかの生物活性を示す細胞であればよい。かかる細胞として、具体的には、大腸癌または乳癌由来の細胞株や単離調製された初代培養細胞を挙げることができる。なお、スクリーニングに用いられる細胞の範疇には、細胞の集合体である組織も含まれる。 The cells that react with PLS3 used for such screening may be cells that exhibit some biological activity when brought into contact with PLS3. Specific examples of such cells include colon cancer or breast cancer-derived cell lines and isolated primary cultured cells. The category of cells used for screening includes tissues that are aggregates of cells.
　候補物質の選別は、上記条件で被験物質とPLS3と反応する細胞またはその細胞画分とを接触させて、PLS3の作用を低下させる物質を探索することによって行うことができる。具体的には、被験物質存在下でPLS3に反応する細胞を培養した場合に生じるPLS3の機能が、被験物質非存在下で上記に対応するPLS3に反応する細胞を培養した場合に得られるPLS3の機能（対照機能）よりも低いことを指標として、当該被験物質を候補物質として選別することができる。 Selection of candidate substances can be performed by contacting a test substance with cells that react with PLS3 or a cell fraction thereof under the above conditions to search for substances that reduce the action of PLS3. Specifically, the function of PLS3 that occurs when cells that respond to PLS3 in the presence of a test substance are cultured is that of PLS3 obtained when cells that respond to PLS3 corresponding to the above are cultured in the absence of the test substance. The test substance can be selected as a candidate substance using an index lower than the function (control function) as an index.
　ここで、PLS3の機能としては、前述するようにアクチン繊維を束ねる機能が知られており、その機能測定方法も公知である。 Here, as the function of PLS3, as described above, the function of bundling actin fibers is known, and the function measurement method is also known.
　上記(i)～(iii)のスクリーニング方法で選別された物質は、固形がん、とくに大腸がんまたは乳がん組織細胞においてPLS3遺伝子の発現増加（亢進）を抑制してEMS誘導を抑制する作用を有するものであり、よって大腸がんや乳がんの癌進展（浸潤や転移）を抑制する薬剤の有効成分として使用することが可能である。さらに上記(1)～(3)のスクリーニング方法で選別された物質は、固形がんの患者の術後の再発を予防する薬剤、ならびに予後を向上する薬剤の有効成分として使用することができる。 Substances selected by the above screening methods (i) to (iii) have the effect of suppressing EMS induction by suppressing the increase (enhancement) of PLS3 gene expression in solid cancers, especially colon cancer or breast cancer tissue cells. Therefore, it can be used as an active ingredient of a drug that suppresses cancer progression (invasion or metastasis) of colon cancer or breast cancer. Furthermore, the substance selected by the screening methods (1) to (3) can be used as an active ingredient of a drug for preventing postoperative recurrence of a solid cancer patient and a drug for improving the prognosis.
　上記のスクリーニング方法によって選別された候補物質は、さらに固形がんを有する病態非ヒト動物を用いてスクリーニングにかけることもできる。かくして選別される候補物質は、さらに非ヒト動物を用いた安全性試験、さらに固形がん患者への臨床試験に供してもよく、これらの試験を実施することによって、より実用的な本発明薬剤の有効成分を選別取得することができる。 The candidate substance selected by the above screening method can be further screened using a non-human animal having a solid cancer. Candidate substances thus selected may be further subjected to safety tests using non-human animals, and further to clinical trials for patients with solid cancer. By carrying out these tests, the drug of the present invention is more practical. It is possible to select and acquire the active ingredients.
　このようにして選別された物質は、必要に応じて構造解析を行った後、その物質の種類に応じて、化学的合成、生物学的合成（発酵を含む）または遺伝子学的操作によって、工業的に製造することができ、本発明薬剤の調製に使用することができる。 The material selected in this way is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic engineering, And can be used for the preparation of the medicament of the present invention.
　本発明を、下記実施例等により説明するが、本発明は、かかる実施例に限定されるものではない。また、以下の実験例において、遺伝子操作、細胞培養等には、特に断りのない限り、Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press),Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience)等に記載された方法を用いた。 The present invention will be described with reference to the following examples, but the present invention is not limited to such examples. In the following experimental examples, unless otherwise specified, genetic manipulation, cell culture, etc. unless otherwise noted, Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology Publishing Associates Wiley-Interscience) and the like.
　Ｉ．材料およびその前調製
　後述する実験例で使用する各種の被験試料は、下記の方法に従って調製した。なお、被験試料は、　九州大学病院、帝京大学病院およびその関連病院にて1991-2002年の間に採取されたものについて、書面によるinformed consentを得た後に使用した。具体的には、大腸がん原発巣の腫瘍組織（T）と対応する非癌部組織(N)は、110症例から外科手術時に採取し、腫瘍環流静脈血（VDT）は手術時、末梢血（PB）は手術前に各々177症例から入手した。また対照群として使用する末梢血（PB）（対照PB）は25名の健常者ボランティアより採取し、また腫瘍環流静脈血（VDT）の対照として使用する門脈血（対照VDT）は24名の良性疾患の患者より採取した。 I. Materials and Pre-preparation Various test samples used in the experimental examples described later were prepared according to the following methods. The test samples collected at Kyushu University Hospital, Teikyo University Hospital and related hospitals during 1991-2002 were used after obtaining written informed consent. Specifically, non-cancerous tissue (N) corresponding to tumor tissue (T) of the primary colorectal cancer was collected from 110 cases during surgery, and tumor perfusion venous blood (VDT) was collected during surgery, peripheral blood (PB) were obtained from 177 cases each before surgery. Peripheral blood (PB) used as a control group (control PB) was collected from 25 healthy volunteers, and portal vein blood (control VDT) used as a control for tumor perfusion venous blood (VDT) was collected from 24 people. Collected from patients with benign diseases.
　大腸がん原発巣からの腫瘍組織（T）と非腫瘍組織（N）は、RNA抽出まで-80℃でTissue Tek OCT medium (Sakura, Tokyo, Japan)に包埋しておき、その後RNA抽出してcDNAを調製した。VDTとPBからのRNA抽出は、PAXgene blood RNA kit (Qiagen K.K. GmbH, Germany)を用いて行った。 Tumor tissue (T) and non-tumor tissue (N) from the colorectal cancer primary lesion are embedded in Tissue Tek OCT medium (Sakura, Tokyo, Japan) at -80 ℃ until RNA extraction, and then RNA is extracted. CDNA was prepared. RNA extraction from VDT and PB was performed using PAXgene blood RNA kit (Qiagen K.K. GmbH, Germany).
　ヒト大腸癌細胞株として、結腸癌細胞株LoVoと直腸癌細胞株CaR-1は、Cell Resource Center of Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan)より入手した。それぞれ10% FBSと100 units/mL ペニシリンと 100 units/mL ストレプトマイシンを含んだF12 mediumおよびMEM mediumで培養した(5% CO2, 37 ℃)。なお、以下の実験例ではこれらの細胞株を「大腸癌細胞株」と総称する。 As human colon cancer cell lines, colon cancer cell line LoVo and rectal cancer cell line CaR-1 were obtained from Cell Resource Center of Biomedical Research, Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). The cells were cultured in F12 medium and MEM medium containing 10% FBS, 100 units / mL penicillin and 100 units / mL streptomycin, respectively (5% CO 2 , 37   ° C). In the following experimental examples, these cell lines are collectively referred to as “colon cancer cell lines”.
　II．各種実験手法
（1）リアルタイム定量的RT-PCR
　リアルタイム定量RT-PCRは、表１に記載する各遺伝子に特異的なプライマーを用いて、LightCycler-FastStart DNA Master SYBR Green I キットを用いたLightCycler system (Roche Applied Science, IN, USA)を使用して行った（Ogawa, K., et al. Clin Cancer Res 11, 2889-2893 (2005)参照）。 II. Various experimental methods
(1) Real-time quantitative RT-PCR
Real-time quantitative RT-PCR is carried out using the LightCycler system (Roche Applied Science, IN, USA) using the LightCycler-FastStart DNA Master SYBR Green I kit with primers specific to each gene listed in Table 1. (See Ogawa, K., et al. Clin Cancer Res 11, 2889-2893 (2005)).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　PLS3のmRNAの増幅は、具体的には、最初の変性を95℃で10分、その後の変性を95℃で10秒からなる反応を40サイクル、アニーリングを62℃で10秒、伸長反応を67℃で10秒間行った。なお、PLS3以外の遺伝子のmRNAの増幅は、アニーリングに使用する温度を62℃に代えて60℃、また伸長反応に使用する温度を67℃に代えて65℃を使用する他、上記と同様の条件で行った。なお、mRNA量は内部コントロールであるGAPDH mRNAを用いて標準化した。 Specifically, PLS3 mRNA amplification was performed by first denaturing at 95 ° C for 10 minutes, followed by denaturation at 95 ° C for 10 seconds for 40 cycles, annealing for 10 seconds at 62 ° C, and extension reaction for 67 seconds. C. for 10 seconds. The amplification of mRNA of genes other than PLS3 is the same as described above except that the temperature used for annealing is changed to 60 ° C instead of 62 ° C, and the temperature used for the extension reaction is changed to 67 ° C instead of 67 ° C. Performed under conditions. The amount of mRNA was standardized using GAPDH mRNA as an internal control.
　（2）安定PLS3導入大腸癌細胞（LoVo-PLS3細胞）の樹立
　ヒトPLS3 cDNA をクローニングして、これをpcDNA3.1(+) expression vector (Invitrogen Corp., Carlsbad, CA, USA) に説明書に従って挿入した。配列をシークエンスして、上記ベクターにヒトPLS3 cDNAが正確に挿入されたことを確認した。得られたベクターを、大腸癌細胞株LoVoに、Lipofectamine 2000 method (Invitrogen Corp., Carlsbad, CA, USA)を用いてトランスフェクションした（LoVo-PLS3細胞）。PLS3が導入された「LoVo-PLS3細胞」を、ネオマイシン(600 mg/mL) (Invitrogen Crop., Carlsbad, CA, USA)を用いて選別した。また、コントロール細胞として、上記ベクターに代えてmockベクターを用いて、同様に大腸癌細胞株LoVoをトランスフェクションして、「LoVo-mock細胞」を作成した。 (2) was cloned established human PLS3 cDNA stable PLS3 introduced colon cancer cells (LoVo-PLS3 cells), which pcDNA3.1 (+) expression vector (Invitrogen Corp., Carlsbad, CA, USA) according to the instructions in Inserted. The sequence was sequenced to confirm that the human PLS3 cDNA was correctly inserted into the vector. The resulting vector was transfected into the colon cancer cell line LoVo using the Lipofectamine 2000 method (Invitrogen Corp., Carlsbad, CA, USA) (LoVo-PLS3 cells). “LoVo-PLS3 cells” into which PLS3 had been introduced were selected using neomycin (600 mg / mL) (Invitrogen Crop., Carlsbad, Calif., USA). Further, as a control cell, a mock vector was used instead of the above vector, and the colon cancer cell line LoVo was similarly transfected to prepare “LoVo-mock cells”.
　（3）免疫細胞染色
　LoVo-PLS3細胞とLoVo-mock細胞を、ガラスカバースリップのうえに1.0×105cells/mLの割合で乗せて、48時間インキュベーションした。その後PBSで洗浄し、-20℃の90％メタノールで5分間処理して固定化した。次いで、抗PLS3抗体および抗vimentin抗体 (いずれもSanta Cruz Biotechnology Inc., CA, USA)を用いて、4℃で終夜インキュベーションした。非特異的タンパク質を3 %のFBS（in PBS）で30分間ブロックし、洗浄後、PLS3発現細胞およびVimentin発現細胞を、avidin-biotin-peroxidase method (LSAB kit; DAKO, Kyoto, Japan)を用いて検出した（Masuda, T.A., et al., Cancer Res 62, 3819-3825 (2002)参照）。切片はすべてヘマトキシリンで核染した。 (3) the immune cell staining LoVo-PLS3 cells and LoVo-mock cells, on top of the glass coverslip placed in a ratio of 1.0 × 10 5 cells / mL, and incubated for 48 hours. Thereafter, it was washed with PBS, and fixed with 90% methanol at -20 ° C. for 5 minutes. Subsequently, overnight incubation was performed at 4 ° C. using an anti-PLS3 antibody and an anti-vimentin antibody (both Santa Cruz Biotechnology Inc., CA, USA). Nonspecific proteins were blocked with 3% FBS (in PBS) for 30 minutes, washed, and PLS3-expressing cells and vimentin-expressing cells were removed using the avidin-biotin-peroxidase method (LSAB kit; DAKO, Kyoto, Japan) It was detected (see Masuda, TA, et al., Cancer Res 62, 3819-3825 (2002)). All sections were nuclear stained with hematoxylin.
  （4）蛍光免疫細胞染色
　LoVo-PLS3細胞 と LoVo-mock細胞を、ガラスカバースリップの上に1.0×105cells/mLの割合で乗せて、48時間インキュベーションした。その後PBSで洗浄し、3.7% ホルムアルデヒドで10分間、-20oCの90% メタノールで５分間処理して固定化した。次いでPBSで洗浄した後、細胞を抗F-actin抗体 (Molecular Probes, Eugene, OR, USA) と 抗-E-cadherin抗体 (BD Biosciences Pharmingen, San Diego, CA, USA)を用いて室温で1時間反応させた。すべての被験細胞はDAPIを用いて核染した。細胞はconfocal LSM510 microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY)で励起した後、撮影した（×63magnification）。 (4) Fluorescent immune cell staining LoVo-PLS3 cells and LoVo-mock cells were placed on a glass cover slip at a rate of 1.0 × 10 5 cells / mL and incubated for 48 hours. Thereafter, it was washed with PBS, and fixed by treatment with 3.7% formaldehyde for 10 minutes and -20 ° C. with 90% methanol for 5 minutes. After washing with PBS, cells were treated with anti-F-actin antibody (Molecular Probes, Eugene, OR, USA) and anti-E-cadherin antibody (BD Biosciences Pharmingen, San Diego, CA, USA) for 1 hour at room temperature. Reacted. All test cells were nuclear stained with DAPI. The cells were photographed after excitation with a confocal LSM510 microscope (Carl Zeiss Microimaging, Inc., Thornwood, NY) (× 63magnification).
　（5）浸潤能のIn vitroアッセイ
  細胞の浸潤能を評価するために、既に報告している方法（Masuda, T.A., et al., Cancer Res 62, 3819-3825 (2002)）に準じて、BD BioCoatTMTumor Invasion System (Becton Dickinson, San Jose, CA, USA)を用いてIn vitroアッセイを行った。具体的には、細胞（5×104細胞/well）をチャンバーの上段に置き、チャンバーの下段を化学誘引物質として750μL  F12（10% FBS）で満たした。37℃で48時間インキュベーションした後、細胞膜をCalcein-AM溶液でラベルした。細胞膜を通じて下面に移動した浸潤性細胞の蛍光強度を、Multilabel Plate Counter VICTOR3(PerkinElmer, Inc., USA)を使用して、蛍光プレートリーダー（励起波長485nm／発光波長530nm）で測定した。 (5) In vitro assay of invasion capacity In order to evaluate the invasion capacity of cells, in accordance with the method already reported (Masuda, TA, et al., Cancer Res 62, 3819-3825 (2002)), BD In vitro assays were performed using the BioCoat ™ Tumor Invasion System (Becton Dickinson, San Jose, CA, USA). Specifically, the cells (5 × 10 4 cells / well) placed in the upper chamber was filled with 750μL F12 (10% FBS) and lower chamber as a chemoattractant. After incubation at 37 ° C. for 48 hours, the cell membrane was labeled with Calcein-AM solution. The fluorescence intensity of the invasive cells that migrated to the lower surface through the cell membrane was measured with a fluorescence plate reader (excitation wavelength 485 nm / emission wavelength 530 nm) using Multilabel Plate Counter VICTOR3 (PerkinElmer, Inc., USA).
　（6）遊走能アッセイ
　細胞の遊走能を評価するために、Cultrex 24-Well Cell Migration Assay (TREVIGEN)を用いて、そのマニュアルに従ってIn vitroアッセイを行った。 (6) Migration ability assay In order to evaluate the migration ability of cells, an in vitro assay was performed according to the manual using the Cultrex 24-Well Cell Migration Assay (TREVIGEN).
　（7）PLS3 RNA干渉
　下記に示すPLS3-specific siRNA (Santa Cruz Biotechnology Inc., CA, USA) （配列番号５～１０）を用いてPLS3 抑制実験を行った。
sc-43215 A：CCACGGAUAGAUAUUAACAtt（配列番号５）
            UGUUAAUAUCUAUCCGUGGtt（配列番号６）
sc-43215 B：GAACUCUCUUGGUGUCAAUtt（配列番号７）
            AUUGACACCAAGAGAGUUCtt（配列番号８）
sc-43215 C：GAAGAGUGGCAAUCUAACAtt（配列番号９）
            UGUUAGAUUGCCACUCUUCtt（配列番号１０）。 (7) PLS3 RNA interference are shown below PLS3-specific siRNA (Santa Cruz Biotechnology Inc., CA, USA) and PLS3 inhibition experiments (SEQ ID NO: 5-10) was performed.
sc-43215 A: CCACGGAUAGAUAUUAACAtt (SEQ ID NO: 5)
UGUUAAUAUCUAUCCGUGGtt (SEQ ID NO: 6)
sc-43215 B: GAACUCUCUUGGUGUCAAUtt (SEQ ID NO: 7)
AUUGACACCAAGAGAGUUCtt (SEQ ID NO: 8)
sc-43215 C: GAAGAGUGGCAAUCUAACAtt (SEQ ID NO: 9)
UGUUAGAUUGCCACUCUUCtt (SEQ ID NO: 10).
　また、ネガティブコントロール用のsiRNAとして、SilencerTM Negative Control #1 siRNA (Ambion, USA)を使用した。 Silencer ™ Negative Control # 1 siRNA (Ambion, USA) was used as siRNA for negative control.
　具体的には、トランスフェクション試薬であるLipofectamine RNAi MAX(Invitrogen Crop., Carlsbad, CA, USA)とPLS3-specific siRNA（またはネガティブコントロール用のSilencerTMNegative Control #1 siRNA）を、底が平坦なマイクロタイタープレートの2mL容量の６ウエルの中に添加しインキュベーションした後、ヒト大腸癌細胞株CaR-1（1.5×105cells/well）を添加し、加湿環境（37℃、5% CO2）でインキュベートした。 Specifically, the transfection reagent Lipofectamine RNAi MAX (Invitrogen Crop., Carlsbad, CA, USA) and PLS3-specific siRNA (or Silencer TM Negative Control # 1 siRNA for negative control) were mixed with After adding and incubating in 6 mL wells of 2 mL capacity of the titer plate, human colon cancer cell line CaR-1 (1.5 × 10 5 cells / well) is added and humidified (37 ° C, 5% CO 2 ). Incubated.
　（8）PLS3蛋白質のウエスタンブロット分析
　細胞から抽出した総タンパク質の一部(35μg)を、7.5%のREADY GELS J (Bio-Rad Laboratories, Japan)に供して、電気泳動をかけた。泳動後、抗PLS3抗体(Santa Cruz Biotechnology Inc., CA, USA)（1/100に希釈して使用）と抗原-抗体反応を行い、ECL Detection Reagents (Amersham Biosciences, Piscataway, NJ)を用いて、抗原-抗体反応物を検出した。なお、これらのタンパク質は1/1000に希釈したβ-アクチンのタンパク質(Cytoskelton, Denver, CO)で標準化した。 (8) Western blot analysis of PLS3 protein A portion (35 μg) of the total protein extracted from the cells was subjected to electrophoresis using 7.5% READY GELS J (Bio-Rad Laboratories, Japan). After electrophoresis, perform an antigen-antibody reaction with anti-PLS3 antibody (Santa Cruz Biotechnology Inc., CA, USA) (diluted to 1/100) and use ECL Detection Reagents (Amersham Biosciences, Piscataway, NJ) Antigen-antibody reaction was detected. These proteins were standardized with β-actin protein (Cytoskelton, Denver, CO) diluted to 1/1000.
　（9）フローサイトメトリー
　結腸がん患者2名（１名は肝転移者、もう１名はリンパ節転移者）から、ヘパリンで前処理して末梢血10mlを得た。得られた末梢血を、1500×gで30分間、フィコール密度勾配遠心分離することにより、単核球層を取得した。単核球をフィコエリトリン結合－抗CD45抗体(cat.555483、Becton Dickinson)で染色した。死細胞は7-aminoactinomycin Dで除去した。単核球を、2%FBSを含むPBSに懸濁した。標識された単核球を、BD FACS Vantage SE Cell Sorter Systemを用いて分析および選別し、そのデータをFACSDiva software (Becton Dickinson)で解析した。ゲーティングは、negative-control staining profileに基づいて行った。 (9) Flow cytometry From two colon cancer patients (one with liver metastasis and one with lymph node metastasis), 10 ml of peripheral blood was obtained by pretreatment with heparin. The obtained peripheral blood was subjected to ficoll density gradient centrifugation at 1500 × g for 30 minutes to obtain a mononuclear cell layer. Mononuclear cells were stained with phycoerythrin-conjugated anti-CD45 antibody (cat.555483, Becton Dickinson). Dead cells were removed with 7-aminoactinomycin D. Mononuclear cells were suspended in PBS containing 2% FBS. Labeled mononuclear cells were analyzed and sorted using the BD FACS Vantage SE Cell Sorter System, and the data were analyzed with FACSDiva software (Becton Dickinson). Gating was performed based on a negative-control staining profile.
　（10）循環腫瘍細胞の蛍光免疫細胞染色
　結腸がん患者の末梢血をBD FACSVantage SE Cell Sorter Systemでソートして、CD45(-)循環細胞を取得した。取得したCD45(-)循環細胞を、ガラスカバーグラスの上に播播して37℃で12時間インキュベートした。PBSで洗浄して、癌細胞以外の非接着循環細胞を除去した後、室温で１時間、抗cytokeratin抗体(clone MNF-116、DAKO)および抗PLS3抗体(Santa Cruz Biotechnology)とともにインキュベーションし、次いで-20℃の90％メタノールで固定化した。抗cytokeratin抗体と抗PLS3抗体を検出するために、抗マウスおよび抗ヤギの蛍光標識ウサギ抗体 (A-11060およびA-11078、)を使用して、室温で1時間インキュベーションした。切片はすべてDAPIで染色した。免疫染色された細胞は、共焦点LSM510顕微鏡(Carl Zeiss Microimaging)（倍率×63）で観察した。 (10) peripheral blood fluorescence immunocytochemistry colon cancer patients circulating tumor cells sorted by BD FACSVantage SE Cell Sorter System, CD45 (-) were obtained circulating cells. The obtained CD45 (−) circulating cells were seeded on a glass cover glass and incubated at 37 ° C. for 12 hours. After washing with PBS to remove non-adherent circulating cells other than cancer cells, incubation with anti-cytokeratin antibody (clone MNF-116, DAKO) and anti-PLS3 antibody (Santa Cruz Biotechnology) for 1 hour at room temperature, then- Immobilization with 90% methanol at 20 ° C. To detect anti-cytokeratin and anti-PLS3 antibodies, anti-mouse and anti-goat fluorescently labeled rabbit antibodies (A-11060 and A-11078,) were used for 1 hour incubation at room temperature. All sections were stained with DAPI. The immunostained cells were observed with a confocal LSM510 microscope (Carl Zeiss Microimaging) (magnification × 63).
　（11）免疫組織化学
　ホルマリン固定、パラフィン包埋された大腸がん臨床検体を用いて、PLS3免疫染色を行った。脱パラフィン処理とブロッキング処理後に、抗-PLS3抗体(Santa Cruz Biotechnology Inc., CA, USA)（100倍希釈）を4℃で終夜反応させた。抗原-抗体反応のシグナルは、avidin-biotin-peroxidase method (LSAB kit; DAKO, Kyoto, Japan)を用いて検出した（Masuda, T.A., et al., Cancer Res 62, 3819-3825 (2002)参照）。切片はすべてヘマトキシリンで核染した。 (11) Immunohistochemistry PLS3 immunostaining was performed using formalin-fixed, paraffin-embedded colorectal cancer clinical specimens. After deparaffinization and blocking, anti-PLS3 antibody (Santa Cruz Biotechnology Inc., CA, USA) (100-fold dilution) was reacted overnight at 4 ° C. Signal of antigen-antibody reaction was detected using avidin-biotin-peroxidase method (LSAB kit; DAKO, Kyoto, Japan) (see Masuda, TA, et al., Cancer Res 62, 3819-3825 (2002)) . All sections were nuclear stained with hematoxylin.
　（12）統計分析
　連続変数について、データは平均値±標準偏差(s.d.)として示した。PLS3の発現と臨床病理的な要因との関係、およびin vitro分析データの関係は、Studentのt検定、カイ二乗検定およびANOVAを使用して分析した。全体の生存率と無病生存率のカーブは、手術の日から測定したKaplan-Meier法に従ってプロットし、比較のためにlog-rankテストを使用した。4グループの浸潤能の試験データはANOVAおよびTukeyの多重比較テストで分析した。相違はすべて、p<0.05のレベルで統計的に有意とし、P<0.1のレベルは傾向を示した。潜在的な予後の変数について相対的な多変量の重要性を検討した。PLS3の予後に対する独立した寄与度をCox比例ハザード回帰を用いて試験した。統計分析は、JMP software package(SASInstitute Inc., Cary, CN,USA)を用いて行った。 (12) Statistical analysis For continuous variables, data are shown as mean ± standard deviation (sd). The relationship between PLS3 expression and clinicopathological factors, and the relationship between in vitro analysis data, was analyzed using Student's t-test, chi-square test and ANOVA. Overall and disease-free survival curves were plotted according to the Kaplan-Meier method measured from the day of surgery and the log-rank test was used for comparison. The test data of invasive ability of 4 groups were analyzed by ANOVA and Tukey multiple comparison test. All differences were statistically significant at the level of p <0.05, and the level of P <0.1 showed a trend. We examined the importance of relative multivariate for potential prognostic variables. Independent contribution to prognosis of PLS3 was tested using Cox proportional hazard regression. Statistical analysis was performed using the JMP software package (SAS Institute Inc., Cary, CN, USA).
　実験例１　大腸癌細胞株Lovoにおける、PLS3のEMT誘導
（1）大腸癌細胞におけるPLS3の機能を調べるために、実験手法（2）に記載する方法に従って２つのLoVo細胞株を樹立した。1つは、PLS3を発現する細胞「LoVo-PLS3細胞」であり、もう一つは、コントロール細胞としての「LoVo-mock細胞」である。図１aに、LoVo-PLS3細胞（右側）とLoVo-mock細胞（左側）の画像を示す。PLS3遺伝子が導入され発現することでLoVo細胞は、LoVo-mock細胞が有する敷石状の形態から、線維芽細胞様の形態に変化することが確認された。 Experimental Example 1 EMT induction of PLS3 in colorectal cancer cell line Lovo (1) In order to examine the function of PLS3 in colorectal cancer cells, two LoVo cell lines were established according to the method described in Experimental Method (2). One is a cell “LoVo-PLS3 cell” that expresses PLS3, and the other is a “LoVo-mock cell” as a control cell. FIG. 1 a shows images of LoVo-PLS3 cells (right side) and LoVo-mock cells (left side). By introducing and expressing the PLS3 gene, LoVo cells were confirmed to change from a cobblestone-like form possessed by LoVo-mock cells to a fibroblast-like form.
　（2）LoVo-PLS3細胞に形態変化が認められたことから、LoVo-PLS3細胞および対照のLoVo-mock細胞について、PLS3、上皮間葉移行（EMT:Epithelial-Mesenchymal Transition）に関連するタンパク質（VimentinとE-cadherin）、およびF-actinの発現レベルを、免疫細胞染色および蛍光免疫細胞染色により測定した。 (2) Because LoVo-PLS3 cells have undergone morphological changes, proteins related to PLS3 and epithelial-mesenchymal transition (EMT) are found in LoVo-PLS3 cells and control LoVo-mock cells. And E-cadherin) and F-actin expression levels were measured by immunocytostaining and fluorescent immunostaining.
　具体的には、PLS3と間葉マーカーであるVimentinの発現レベルは実験手法（3）に記載する方法に従って免疫細胞染色により、また上皮マーカーであるE-cadherinとF-actinの発現レベルは実験手法（4）に記載する方法に従って蛍光免疫細胞染色により調べた。免疫細胞染色および蛍光免疫細胞染色の結果を、それぞれ図１bおよび図１cに示す。その結果、LoVo-mock細胞と比較してLoVo-PLS3細胞において、PLS3とVimentinの発現レベルは増強されていたが、E-cadherinの発現レベルは抑制されていることが判明した（(図1b、c)。またLoVo-PLS3細胞において、F-actinの発現レベルが著しく上昇していることから（図１c）、PLS3発現によって細胞内外でF-アクチン脱重合が抑制され、アクチン重合が促進されていることが確認された。 Specifically, the expression levels of PLS3 and mesenchymal marker vimentin were determined by immunocytostaining according to the method described in experimental method (3), and the expression levels of epithelial markers E-cadherin and F-actin were determined by experimental method. According to the method described in (4), it was examined by fluorescent immunocell staining. The results of immune cell staining and fluorescent immune cell staining are shown in FIGS. 1b and 1c, respectively. As a result, the expression levels of PLS3 and Vimentin were enhanced in LoVo-PLS3 cells compared to LoVo-mock cells, but the expression levels of E-cadherin were suppressed ((FIG. 1b, c) In addition, since the expression level of F-actin is markedly increased in LoVo-PLS3 cells (Fig. 1c), F-actin depolymerization is suppressed inside and outside the cell by PLS3 expression, and actin polymerization is promoted. It was confirmed that
　以上のことから、PLS3を発現する細胞（LoVo-PLS3細胞）は、上皮細胞表現型から間葉細胞表現型に切り替わり、上皮間葉移行（EMT）が誘導されていること、また細胞内にF-actinが蓄積されていることが確認された。 Based on the above, PLS3-expressing cells (LoVo-PLS3 cells) are switched from the epithelial cell phenotype to the mesenchymal cell phenotype, and epithelial-mesenchymal transition (EMT) is induced. It was confirmed that -actin was accumulated.
　（3）また作成した２つのLoVo細胞（LoVo-PLS3細胞、LoVo-mock細胞）について、BD BioCoatTM Tumor Invasion System (Becton Dickinson, San Jose, CA, USA)と Cultrex 24 Well Cell Migration Assay (TREVIGEN, Gaithersburg, MD, USA) を使用して、それらのプロトコルに従って、各細胞の浸潤能と遊走能を測定した。各細胞の浸潤能および遊走能の測定に使用したIn vitroアッセイ法の概要を、それぞれ実験手法(5)および(6)に記載する。浸潤能に関する結果を図２左に、および遊走能に関する結果を図２右にそれぞれ示す。図２からわかるように、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）は、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、浸潤能および遊走能のいずれもが約1.5倍増強していた。 (3) The two created LoVo cells (LoVo-PLS3 cells and LoVo-mock cells) are also BD BioCoatTM Tumor Invasion System (Becton Dickinson, San San Jose, CA, USA) and Cultrex 24 Well Cell Cell Migration, GREthergen , MD, USA) was used to measure the invasion ability and migration ability of each cell according to their protocol. The outline of the Invitro assay method used for measuring the invasion ability and migration ability of each cell is described in the experimental methods (5) and (6), respectively. The results relating to invasive ability are shown on the left in FIG. 2, and the results relating to migration ability are shown on the right in FIG. As can be seen from FIG. 2, PLS3SmRNA expressing cells (LoVo-PLS3 cells) are both about 1.5 times more potent in invasion and migration than cells that do not express PLS3 mRNA (LoVo-mock cells). Was.
　（4）さらに各LoVo細胞株（LoVo-PLS3細胞、LoVo-mock細胞）について、細胞増殖性を調べるために、3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Roche Diagnostics Corp., GmbH) assayを行った。結果を図３に示す。図３に示すように、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）は、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、細胞増殖能が有意に低いことが判明した。 (4) Furthermore, in order to investigate cell proliferation of each LoVo cell line (LoVo-PLS3 cell, LoVo-mock cell), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) (Roche Diagnostics Corp., GmbH) assay was performed. The results are shown in FIG. As shown in FIG. 3, it was found that cells that express PLS3 mRNA (LoVo-PLS3 cells) have significantly lower cell proliferation ability than cells that do not express PLS3 mRNA (LoVo-mock cells).
　（5）また各LoVo細胞株（LoVo-PLS3細胞、LoVo-mock細胞）について、抗がん剤（5-FU）への耐性を調べた。LoVo-mock細胞、およびLoVo-PLS3細胞をそれぞれ96 well plateに播いて、24時間後に5-FUを図４に示す濃度（0、1、5および10μg/ml）で処置した。72時間後に、(4)に記載するMTTアッセイを実施し、細胞増殖率から5-FUに対する感受性（耐性）を評価した。結果を図４に示す。図４に示すように、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）のほうが、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、高い5-FU濃度にも関わらず細胞が増殖しており、このことからEMT誘導されたLoVo-PLS3細胞は5-FUに対して耐性であると考えられた。 (5) Each LoVo cell line (LoVo-PLS3 cell, LoVo-mock cell) was examined for resistance to an anticancer agent (5-FU). LoVo-mock cells and LoVo-PLS3 cells were seeded in 96-well plates, and 24 hours later, 5-FU was treated with the concentrations shown in FIG. 4 (0, 1, 5, and 10 μg / ml). 72 hours later, the MTT assay described in (4) was performed, and the sensitivity (resistance) to 5-FU was evaluated from the cell proliferation rate. The results are shown in FIG. As shown in FIG. 4, cells that proliferate PLS3 mRNA (LoVo-PLS3 cells) grow more in spite of higher 5-FU concentrations than cells that do not express PLS3 mRNA (LoVo-mock cells). This suggests that EMT-induced LoVo-PLS3 cells are resistant to 5-FU.
　以上の実験結果から、アクチンの重合と脱重合を調整しながら細胞の形態と遊走能をコントロールすることが知られているPLS3 mRNAの発現によって、大腸癌細胞においてEMTが誘導されること、浸潤能および遊走能が増強すること、細胞増殖能が低下すること、および抗がん剤に対する感受性が弱まる（耐性化する）ことが確認された。 Based on the above experimental results, EMT is induced in colon cancer cells by the expression of PLS3 mRNA, which is known to control cell morphology and migration ability while regulating actin polymerization and depolymerization, invasion ability Further, it was confirmed that the migration ability was enhanced, the cell proliferation ability was lowered, and the sensitivity to the anticancer agent was weakened (tolerated).
　実験例２　LoVo-PLS3細胞およびCaR-1 PLS3 siRNA細胞におけるEMT関連遺伝子の発現
（1）LoVo-mock細胞およびLoVo-PLS3細胞について、実験手法(1)に記載するリアルタイム定量RT-PCR法に従って、10つのEMT関連遺伝子（E-cadherin、TWIST、SNAIL、SMAD4、SLUG、ZEB1、vimentin、FN1、N-cadherin、FOXC2）の発現レベルを調べた。結果を図５aおよび図５bに示す。その結果、図５aに示すように、実験例１(1)で示したように、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）は、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、上皮マーカーであるE-cadherinの発現レベルが低下していることが確認された。また、E-cadherin抑制遺伝子であるTWIST、SNAIL、SMAD4、SLUGおよびZEB1の発現レベルは、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）において、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、有意に高くなっていることが確認された(p<0.05)。また図５bに示すように、間葉マーカーであるvimentin、FN1、N-cadherinおよびFOXC2の発現レベルは、PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）において、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比べて、有意に高くなっていることが確認された(p<0.05)。 Experimental Example 2 Expression of EMT-related genes in LoVo-PLS3 cells and CaR-1 PLS3 siRNA cells (1) For LoVo-mock cells and LoVo-PLS3 cells, according to the real-time quantitative RT-PCR method described in Experimental method (1), The expression levels of 10 EMT-related genes (E-cadherin, TWIST, SNAIL, SMAD4, SLUG, ZEB1, vimentin, FN1, N-cadherin, FOXC2) were examined. The results are shown in FIGS. 5a and 5b. As a result, as shown in FIG. 5a, as shown in Experimental Example 1 (1), cells expressing PLS3 mRNA (LoVo-PLS3 cells) were compared with cells not expressing PLS3 mRNA (LoVo-mock cells). Thus, it was confirmed that the expression level of E-cadherin, an epithelial marker, was reduced. In addition, the expression levels of TWIST, SNAIL, SMAD4, SLUG, and ZEB1 that are E-cadherin suppressor genes are the same as those of cells that express PLS3 mRNA (LoVo-PLS3 cells) and cells that do not express PLS3 mRNA (LoVo-mock cells). It was confirmed that it was significantly higher than that (p <0.05). Further, as shown in FIG. 5b, the expression levels of the mesenchymal markers vimentin, FN1, N-cadherin, and FOXC2 are expressed in cells that express PLS3 mRNA (LoVo-PLS3 cells) but do not express PLS3 mRNA (LoVo- mock cells) were confirmed to be significantly higher (p <0.05).
　この結果は、実験例１で示した「PLS3の外因性発現によって大腸癌細胞においてEMTが誘導されること」を裏付けるものである。 This result confirms that “EMT is induced in colon cancer cells by exogenous expression of PLS3” shown in Experimental Example 1.
　（2）PLS3 mRNAの発現抑制実験を、TGFβによりEMT誘導させて内因性のPLS3 mRNAを多量に発現させた直腸癌細胞株CaR-1を使用して、PLS3 siRNAを用いて実施した。具体的には、実験手法(7)に記載する方法に従って、上記直腸癌細胞株CaR-1を、ネガティブコントロール用siRNA（SilencerTM Negative Control #1 siRNA (Ambion, USA)）またはPLS3 siRNA（PLS3-specific siRNA(Santa Cruz Biotechnology Inc., CA, USA）で処理し、次いでPLS3 mRNAの発現を、実験手法(1)および(8)に記載する方法に従って、リアルタイム定量RT-PCRおよびウェスタンブロット分析によって確認した。結果を図６に示す。 (2) An experiment to suppress the expression of PLS3 mRNA was performed using PLS3 siRNA using a rectal cancer cell line CaR-1 in which a large amount of endogenous PLS3 mRNA was expressed by EMT induction with TGFβ. Specifically, according to the method described in the experimental method (7), the above rectal cancer cell line CaR-1 is transformed into siRNA for negative control (Silencer ™ Negative Control # 1 siRNA (Ambion, USA)) or PLS3 siRNA (PLS3- Treated with specific siRNA (Santa Cruz Biotechnology Inc., CA, USA) and then confirmed PLS3 mRNA expression by real-time quantitative RT-PCR and Western blot analysis according to the methods described in experimental procedures (1) and (8) The results are shown in FIG.
　図６aおよび図６bの各左図に示すように、LoVo-PLS3細胞中のPLS3のmRNA発現レベルおよび蛋白質産生レベルは、LoVo-mock細胞におけるそれらよりも有意に高かった。一方、図６aおよび図６bの各右図に示すように、CaR-1 PLS3 siRNA細胞中のPLS3のmRNA発現レベルおよび蛋白質産生レベルはCaR-1 control siRNA細胞におけるそれらよりも有意に低かった。特にPLS3のmRNA発現レベルは、PLS3 siRNAで処理した細胞（PLS3 siRNA細胞）中で実質的に70％低下しており、PLS3 siRNAによってPLS3のmRNA発現産生が抑制されることが確認された。 As shown in the left diagrams of FIGS. 6a and 6b, the mRNA expression level and protein production level of PLS3 in LoVo-PLS3 cells were significantly higher than those in LoVo-mock cells. On the other hand, as shown in the right diagrams of FIGS. 6a and 6b, the mRNA expression level and protein production level of PLS3 in CaR-1 PLS3 siRNA cells were significantly lower than those in CaR-1 control siRNA cells. In particular, the PLS3 mRNA expression level was substantially reduced by 70% in cells treated with PLS3 siRNA (PLS3 siRNA cells), and it was confirmed that PLS3 siRNA suppresses PLS3 mRNA expression production.
　（3）またPLS3 siRNAで処理したCaR-1細胞（PLS3 siRNA細胞）とネガティブコントロール用siRNAで処理したCaR-1細胞（コントロールsiRNA細胞）について、EMT関連遺伝子（E-cadherin、TWIST、SNAIL、vimentin）の発現レベルを調べた。結果を図５cに示す。その結果、図５cに示すように、PLS3 siRNA細胞におけるvimentin（間葉マーカー）およびTWIST（E-cadherin抑制遺伝子）の発現レベルは、コントロールsiRNA細胞におけるそれらの発現レベルよりも有意に低下していた(p<0.05)。一方、PLS3 siRNA細胞でのE-cadherin（上皮マーカー）の発現レベルは、コントロールsiRNA細胞でのその発現レベルよりも有意に上昇していた(p<0.05)。 (3) EMT-related genes (E-cadherin, TWIST, SNAIL, vimentin) for CaR-1 cells treated with PLS3 siRNA (PLS3 siRNA cells) and CaR-1 cells treated with negative control siRNA (control siRNA cells) ) Expression level was examined. The results are shown in FIG. As a result, as shown in FIG. 5c, the expression levels of vimentin (mesenchymal marker) and TWIST (E-cadherin suppressor gene) in PLS3 siRNA cells were significantly lower than those in control siRNA cells. (p <0.05). On the other hand, the expression level of E-cadherin (epithelial marker) in PLS3 siRNA cells was significantly higher than that in control siRNA cells (p <0.05).
　以上の実験で示すように、外因性PLS3 mRNAを発現する細胞（LoVo-PLS3細胞）中の、vimentin、TWIST、SNAIL、SMAD4、SLUG、ZEB1、FN1、FOXC2およびN-cadherinの発現レベルは、PLS3 mRNAを発現しない細胞（LoVo-mock細胞）と比較して、有意に上昇制御されていた。また、PLS3 siRNAで処理されたCaR-1細胞（内因性PLS3 mRNAを発現する細胞）は、コントロールsiRNAで処理されたCaR-1細胞と比較して、間葉マーカーであるvimentinおよびTWISTの発現が抑制され、上皮マーカーであるE-cadherinの発現が促進されていた。すなわち、PLS3 mRNAの発現をsiRNAで抑制することで、上皮間葉移行（EMT）が、間葉上皮移行（MET）に切り替わることが確認された。これらの知見は、上皮間葉移行（EMT）誘導に関する従来の研究と一致していた（Mani, S.A., et al., Cell 133, 704-715 (2008);Moustakas, A.et al.,Cancer Sci 98, 1512-1520 (2007); Shirakihara, T., et al., Mol Biol Cell 18, 3533-3544 (2007);Mani, S.A., et al., Proc Natl Acad Sci U S A 104, 10069-10074 (2007);Peinado, H., et al., Nat Rev Cancer 7, 415-428 (2007)）。 As shown in the above experiment, the expression levels of vimentin, TWIST, SNAIL, SMAD4, SLUG, ZEB1, FN1, FOXC2 and N-cadherin in cells expressing exogenous PLS3 mRNA (LoVo-PLS3 cells) are PLS3 As compared with cells not expressing mRNA (LoVo-mock cells), the increase was significantly controlled. In addition, CaR-1 cells treated with PLS3 siRNA (cells expressing endogenous PLS3 mRNA) have higher expression of vimentin and TWIST, which are mesenchymal markers, than CaR-1 cells treated with control siRNA. The expression of E-cadherin, an epithelial marker, was suppressed. That is, it was confirmed that epithelial-mesenchymal transition (EMT) switches to mesenchymal epithelial transition (MET) by suppressing the expression of PLS3PL mRNA with siRNA. These findings were consistent with previous studies on epithelial-mesenchymal transition (EMT) induction (Mani, SA, et al., Cell 133, 704-715 (2008); Moustakas, A.et al., Cancer Sci 98, 1512-1520 (2007); Shirakihara, T., et al., Mol Biol Cell 18, 3533-3544 (2007); Mani, SA, et al., Proc Natl Acad Sci U S A 104, 10069- 10074 (2007); Peinado, H., et al., Nat Rev Cancer 7, 415-428 (2007)).
　このことから、PLS3遺伝子は、癌細胞の浸潤や遊走に関係するEMTを誘導する遺伝子（EMT誘導遺伝子）であると考えられる。 Therefore, it is considered that the PLS3 gene is a gene that induces EMT related to cancer cell invasion and migration (EMT-inducible gene).
　実験例３　大腸癌患者における、原発腫瘍中のPLS3 mRNAの発現と予後との関係
（1）大腸がん患者（n=110）における癌組織(T)と正常組織(N)におけるPLS3 mRNAの発現レベルを調べるために、実験手法(1)に記載する方法に従って、リアルタイム定量RT-PCRを行った。 In Experimental Example 3 colon cancer patients, the relationship between the PLS3 mRNA expression and prognosis in primary tumor (1) colorectal cancer patients (n = 110) in cancer tissues (T) and PLS3 mRNA expression in normal tissues (N) To examine the level, real-time quantitative RT-PCR was performed according to the method described in Experimental Procedure (1).
　結果を図７aに示す。図７aに示すように、癌組織(T)におけるPLS3 mRNA発現レベルは1.14±0.88（平均値±s.d.)であり、正常組織(N)の0.94±0.67（平均値±s.d.）よりも著しく高かった（p<0.0001)。 The result is shown in FIG. As shown in FIG. 7a, the expression level of PLS3 mRNA in cancer tissue (T) was 1.14 ± 0.88 (mean ± sd), which was significantly higher than 0.94 ± 0.67 (mean ± sd) in normal tissue (N). (P <0.0001).
　下記表に、当該結腸直腸がん患者（n=110）について、PLS3 mRNA発現（PLS3 mRNA/GAPDH）および臨床病理的な要因との関係を纏めた結果を示す。なお、ここでは、癌組織（T）におけるPLS3 mRNAの発現がGAPDH mRNAの発現に対して２以上である26症例を「高PLS3発現群」(PLS3(T)/GAPDH(T)≧2.0)とし、２未満である84症例を「低PLS3発現群」(PLS3(T)/GAPDH(T)<2.0)とした。 The following table summarizes the results of the relationship between PLS3-mRNA expression (PLS3-mRNA / GAPDH) and clinicopathological factors for the colorectal cancer patients (n = 110). Here, 26 cases in which the expression of PLS3 mRNA in cancer tissue (T) is 2 or more relative to the expression of GAPDH mRNA are defined as “high PLS3 expression group” (PLS3 (T) / GAPDH (T) ≧ 2.0). 84 cases less than 2 were designated as “low PLS3 expression group” (PLS3 (T) / GAPDH (T) <2.0).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　これからわかるように、高PLS3発現群に属する26症例において、低PLS3発現群に属する84症例によりも、リンパ節転移(p=0.06)、リンパ液の浸潤(p=0.025)、静脈浸潤(p=0.013)および肝転移(p=0.068)がいずれも高いことが確認された。すなわち、PLS3発現と、リンパ節転移、リンパ液の浸潤および静脈浸潤のそれぞれとの間には、正の相関があることが判明した。 As can be seen, in 26 cases belonging to the high PLS3 expression group, lymph node metastasis (p = 0.06), lymphatic infiltration (p = 0.025), venous invasion (p = 0.013), compared to 84 cases belonging to the low PLS3 expression group. ) And liver metastases (p = 0.068) were both high. That is, it was found that there was a positive correlation between PLS3 expression and lymph node metastasis, lymph infiltration and venous invasion.
　図７bに、上記高PLS3発現群（26症例）と低PLS3発現群（84症例）について、手術後５年間の生存率を示す（Kaplan-Meier overall survival）。図７bに示すように、高PLS3発現群に属する患者（実線）は、低PLS3発現群に属する患者（破線）より、予後（５年生存率）が有意に不良であった(p=0.0078)。 Fig. 7b shows the survival rate for 5 years after the surgery for the high PLS3 expression group (26 cases) and the low PLS3 expression group (84 cases) (Kaplan-Meier survival). As shown in FIG. 7b, patients belonging to the high PLS3 expression group (solid line) had a significantly worse prognosis (5-year survival rate) than patients belonging to the low PLS3 expression group (dashed line) (p = 0.0078). .
　癌組織(T)におけるPLS3発現の高低、腫瘍サイズ、リンパ節転移、および肝転移の要素について、予後に与える影響をCox比例ハザード回帰を用いて多変量解析した結果を下表に示す。 The following table shows the results of multivariate analysis of the effects of prognosis on the factors of PLS3 expression in tumor tissue (T), tumor size, lymph node metastasis, and liver metastasis using Cox proportional hazard regression.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　これからわかるように、癌組織(T)中のPLS3の発現レベルが、独立した予後因子(p=0.0364)であることが明らかになった。また、その相対危険度（RR）は、リンパ節転移による危険度の次にランクされた。 As can be seen, the expression level of PLS3 in cancer tissue (T) was found to be an independent prognostic factor (p = 0.0364). The relative risk (RR) was ranked next to the risk due to lymph node metastasis.
　（2）大腸がん患者（n=110）をDukes分類に従って４症例（Dukes A：31名、Dukes B：30名、Dukes C：38名、Dukes D：11名）にわけ、各症例の原発巣組織（癌組織）におけるPLS3 mRNAの発現レベルを、実験手法(1)に記載するリアルタイム定量RT-PCRにより、測定した。 (2) Colorectal cancer patients (n = 110) are divided into 4 cases according to the Dukes classification (Dukes A: 31 people, Dukes B: 30 people, DukesDC: 38 people, Dukes D: 11 people). The expression level of PLS3 mRNA in the nest tissue (cancer tissue) was measured by real-time quantitative RT-PCR described in Experimental Procedure (1).
　結果を図８に示す。図８に示すように、各症例の原発巣組織におけるPLS3 mRNA発現レベル（平均値±s.d.)は、Dukes A症例：1.22±0.93、Dukes B症例：1.29±0.9、Dukes C症例：1.36±1.07、Dukes D症例：2.38±1.58であり、原発巣組織でのPLS3 mRNA発現がDukes D（遠隔転移あり）症例で有意に高いことが確認された。この結果から、原発巣組織でのPLS3 mRNA発現が高い症例はEMT誘導により遠隔転移を形成していることがうかがわれる。 The results are shown in FIG. As shown in FIG. 8, the PLS3 mRNA expression level (mean ± sd) in the primary lesion tissue of each case was Dukes A case: 1.22 ± 0.93, Dukes B case: 1.29 ± 0.9, Dukes C case: 1.36 ± 1.07, Dukes D cases: 2.38 ± 1.58, confirming that PLS3 mRNA expression in the primary tissue was significantly higher in Dukes D (with distant metastasis) cases. This result indicates that cases with high PLS3 mRNA expression in the primary lesion tissue formed distant metastasis by EMT induction.
　（3）図９ａに、大腸がん患者の癌(T)組織および非癌(N)組織を、抗PLS3抗体を用いて実験手法(10)に記載する方法に従って免疫組織化学染色した結果を示す。図中、「Ｎ」側は非癌細胞領域であり、「Ｔ」側は癌細胞領域である。矢印は、PLS3を過剰発現した癌細胞を示す。図９bは、a図の一部を拡大した図である。図からわかるように、PLS3遺伝子は、同一組織内でも、正常細胞(Ｎ)よりも癌細胞(Ｔ)でより強く発現していた。また、PLS3遺伝子は、主腫瘍細胞から離れ、非癌細胞側に浸潤している「budding tumor cells」と呼ばれる小癌細胞群において特に強く発現していることが確認された。 (3) FIG. 9a shows the results of immunohistochemical staining of cancer (T) tissue and non-cancer (N) tissue of colorectal cancer patients using an anti-PLS3 antibody according to the method described in experimental method (10). . In the figure, the “N” side is a non-cancer cell region, and the “T” side is a cancer cell region. Arrows indicate cancer cells that overexpressed PLS3. FIG. 9b is an enlarged view of a part of FIG. As can be seen from the figure, the PLS3 gene was expressed more strongly in cancer cells (T) than in normal cells (N) even in the same tissue. In addition, it was confirmed that the PLS3 gene was particularly strongly expressed in a group of small cancer cells called “budding tumor cells” that was distant from the main tumor cells and invaded the non-cancer cell side.
　以上のことから、大腸癌組織中でのPLS3 mRNAの高い発現が、浸潤能、遊走能および予後不良と正の相関関係を示し、大腸がんの原発腫瘍中のPLS3mRNAの多量発現が、予後の独立した予測因子になることが示唆された。 Based on the above, high expression of PLS3 mRNA in colorectal cancer tissues was positively correlated with invasion ability, migration ability and poor prognosis, and high expression of PLS3 mRNA in primary tumors of colorectal cancer It was suggested to be an independent predictor.
　実験例４　患者の還流静脈血(VDT)および末梢血(PB)中のPLS3 mRNAの発現レベルと大腸がん患者における予後との関係
（1）実験手法(1)に記載するリアルタイム定量RT-PCRを使用して、大腸がん患者(n=177)の腫瘍還流静脈血（VDT）および末梢血（PB）中のPLS3 mRNAの発現レベルを測定した。VDTとPB中のPLS3発現レベルについて正常なカットオフ値を決めるために、良性疾病患者(n=24)の門脈血におけるPLS3 mRNA発現レベルおよび健康ボランティア(n=25)のPB中のPLS3 mRNA発現レベルを調べた。 その結果、VDT／門脈血のカットオフ値を0.0569（平均+2 s.d.）、PBのカットオフ値を0.0546（平均+2 s.d.)に決定した。これらのカットオフ値を用いて、大腸がん患者(n=177)を、腫瘍還流静脈血（VDT）および末梢血（PB）毎に、高PLS3発現群(PLS3/GAPDH≧VDT又はPBのカットオフ値)と低PLS3発現群(PLS3/GAPDH＜VDT又はPBのカットオフ値)に分類した（VDT：高PLS3発現群=21名、低PLS3発現群=156名、PB：高PLS3発現群=10名、低PLS3発現群=167名）。 Experimental Example 4 Relationship between expression level of PLS3 mRNA in patients with reflux venous blood (VDT) and peripheral blood (PB) and prognosis in patients with colorectal cancer (1) Real-time quantitative RT-PCR described in experimental method (1) Was used to measure the expression level of PLS3 mRNA in tumor reflux venous blood (VDT) and peripheral blood (PB) of colon cancer patients (n = 177). PLS3 mRNA expression level in portal blood of benign disease patients (n = 24) and PLS3 mRNA in PB of healthy volunteers (n = 25) to determine normal cutoff values for PLS3 expression levels in VDT and PB The expression level was examined. As a result, the cutoff value of VDT / portal blood was determined to be 0.0569 (average +2 sd), and the cutoff value of PB was determined to be 0.0546 (average +2 sd). Using these cut-off values, colon cancer patients (n = 177) were divided into high PLS3 expression groups (PLS3 / GAPDH ≧ VDT or PB) for each tumor return venous blood (VDT) and peripheral blood (PB). Off value) and low PLS3 expression group (PLS3 / GAPDH <VDT or PB cut-off value) (VDT: high PLS3 expression group = 21 people, low PLS3 expression group = 156 people, PB: high PLS3 expression group = 10 people, low PLS3 expression group = 167 people).
　これらの大腸がん患者について、腫瘍還流静脈血（VDT）および末梢血（PB）におけるPLS3 mRNA発現状況と生存率との関係を調べた。生存率は、手術から５年間の未病生存率（病気を発症しないで生存している割合）（DFS）と全生存率（病気発症の有無に関わらす生存している割合）（OS）に分けて調べた。 In these colon cancer patients, the relationship between the PLS3 mRNA expression status and survival rate in tumor reflux venous blood (VDT) and peripheral blood (PB) was examined. Survival rate is 5 years after surgery without disease (proportion of survival without developing disease) (DFS) and overall survival (proportion of survival with or without disease) (OS) I examined it separately.
　腫瘍還流静脈血（VDT）におけるPLS3発現状況と生存率（未病生存率（DFS）、全生存率（OS））との関係を図１０aに、末梢血（PB）におけるPLS3発現状況と生存率（未病生存率（DFS）、全生存率（OS））との関係を図１０ｂに示す。なお、未病生存率（DFS）の評価は、腫瘍以外の要因で亡くなった患者を除いて行った（VDT：高PLS3発現群=15名、低PLS3発現群=136名、PB：高PLS3発現群=7名、低PLS3発現群=144名）。 Fig. 10a shows the relationship between PLS3 expression status in tumor perfusion venous blood (VDT) and survival rate (non-disease survival rate (DFS), overall survival rate (OS)). PLS3 expression status and survival rate in peripheral blood (PB) The relationship between (disease survival rate (DFS), overall survival rate (OS)) is shown in FIG. 10b. In addition, the disease-free survival rate (DFS) was evaluated excluding patients who died due to factors other than tumor (VDT: high PLS3 expression group = 15, low PLS3 expression group = 136, PB: high PLS3 expression) Group = 7, low PLS3 expression group = 144).
　図からわかるように、腫瘍還流静脈血（VDT）（門脈血）および末梢血（PB）のいずれも、高PLS3発現群は、未病生存率(DFS)および全生存率(OS)がともに、低PLS3発現群よりも有意に低いこと、すなわちVDT（門脈血）またはPBにおいて、PLS3mRNAを高発現している大腸がん患者は、再発の危険性が高く、また５年生存率が低く、予後が不良であることが判明した（図１０a、b）。 As can be seen from the figure, both the tumor return venous blood (VDT) (portal blood) and the peripheral blood (PB) had both high disease-free survival (DFS) and overall survival (OS) in the high PLS3 expression group. Patients with colorectal cancer that are highly expressed in PDT3 mRNA in VDT (portal blood) or PB have a higher risk of recurrence and a lower 5-year survival rate. The prognosis was found to be poor (FIGS. 10a and b).
　（2）下記表４および５に、Cox比例ハザード回帰を用いて、当該大腸がん患者（n=151）について、腫瘍還流静脈血（VDT）（門脈血）中のPLS3 mRNA発現（PLS3 mRNA/GAPDH）と臨床病理的な要因との関係を未病生存率（DFS）および全生存率（OS）のそれぞれについて纏めた結果を、また表６および７に、当該大腸がん患者（n=151）について、末梢血（PB）中のPLS3 mRNA発現（PLS3 mRNA/GAPDH）と臨床病理的な要因との関係を未病生存率（DFS）および全生存率（OS）のそれぞれについて纏めた結果を示す。 (2) In Tables 4 and 5 below, using Cox proportional hazard regression, PLS3 mRNA expression (PLS3 mRNA) in tumor perfusion venous blood (VDT) (portal blood) for the colorectal cancer patient (n = 151) / GAPDH) and the relationship between clinicopathological factors, the results of the summary of the disease-free survival rate (DFS) and the overall survival rate (OS) are shown in Tables 6 and 7, respectively. 151) The results of summarizing the relationship between PLS3 mRNA expression (PLS3 mRNA / GAPDH) in the peripheral blood (PB) and clinicopathological factors for both disease-free survival (DFS) and overall survival (OS) Indicates.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　これらの臨床所見は、PLS3 mRNAを過剰発現する癌幹細胞（CRC）が、原発腫瘍および循環細胞の両方に高い悪性度を示すことを示唆している。 These clinical findings suggest that cancer stem cells (CRC) overexpressing PLS3 mRNA show high malignancy in both primary tumors and circulating cells.
　以上のことから、腫瘍還流静脈血（VDT）（門脈血）と末梢血（PB）におけるPLS3 mRNAの高発現が、５年生存率（全存率(p=0.008、p=0.011)(表５、７）)に関しても、また再発（未病生存率(p=0.029、p=0.038)（表４、６)）に関しても、独立した予測因子になることが判明した。 Based on the above, high expression of PLS3 mRNA in tumor perfusion venous blood (VDT) (portal blood) and peripheral blood (PB) is a 5-year survival rate (overall survival rate (p = 0.008, p = 0.011) (Table 5, 7)) and recurrence (non-disease survival (p = 0.029, p = 0.038) (Tables 4, 6)) were found to be independent predictors.
　表８および９に、還流静脈血（VDT）（門脈血）および末梢血（PB）について、高PLS3発現群と臨床病理的要因との関係を示す。 Tables 8 and 9 show the relationship between the high PLS3 expression group and clinicopathological factors for reflux venous blood (VDT) (portal blood) and peripheral blood (PB).
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　この結果からわかるように、VDT（門脈血）における高PLS3発現群（n=21）は、低PLS3発現群(n=156)と比べて、浸潤深度(p=0.04)、リンパ節転移(p=0.007)および肝転移(p=0.01)が大きいことが判明した。VDT（門脈血）と同様、末梢血における高PLS3発現群(n=10)は、低PLS3発現群(n=166)と比較して、浸潤深度(p=0.034)、リンパ節転移(p=0.043)および肝転移(p=0.032)が大きく、より高い悪性度を示した。 As can be seen from this result, in the high PLS3 expression group (n = 21) in VDT (portal blood), compared to the low PLS3 expression group (n = 156), the infiltration depth (p = 0.04), lymph node metastasis ( p = 0.007) and liver metastases (p = 0.01) were found to be large. Similar to VDT (portal vein blood), high PLS3 expression group (n = 10) in peripheral blood compared to low PLS3 expression group (n = 166), depth of invasion (p = 0.034), lymph node metastasis (p = 0.043) and liver metastases (p = 0.032) were large, indicating higher grade.
　図１１に正常組織（図中、左から副腎、骨髄、大脳、小脳、結腸、胎児脳、胎児肝臓、心臓、腎臓、肝臓、肺、肝、胎盤、末梢血、前立腺、唾液腺、骨格筋、小腸、脊髄、脾臓、胃、精巣、甲状腺、気管、子宮）でのPLS3 mRNAの発現分布を示す。これからもわかるようにPLS3遺伝子は固形組織で発現される遺伝子であり、造血細胞では発現されない（Delanote, V., et al., Acta Pharmacol Sin 26, 769-779 (2005)、Delanote, V., et al. Traffic 6, 335-345 (2005).）。すなわち、骨髄、還流静脈血（門脈血）および末梢血において、本来、PLS3のmRNA発現は観察されない。従って、上記の結果から、還流静脈血（門脈血）や末梢血中のPLS3mRNA発現量を測定することで、癌細胞（原発巣細胞）におけるPLS3mRNA発現量を測ることなく、大腸がんの癌進展度（浸潤や転移）および術後の予後を予測できるものと考えられる。 FIG. 11 shows normal tissues (from the left in the figure, adrenal gland, bone marrow, cerebrum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, liver, placenta, peripheral blood, prostate, salivary gland, skeletal muscle, small intestine. , PLS3 mRNA expression distribution in spinal cord, spleen, stomach, testis, thyroid, trachea, uterus). As can be seen, the PLS3 gene is a gene expressed in solid tissues and not expressed in hematopoietic cells (Delanote, V., et al., Acta Pharmacol Sin 26, 769-779 (2005), Delanote, V., et al. Traffic 6, 335-345 (2005).). That is, PLS3 mRNA expression is not originally observed in bone marrow, reflux venous blood (portal vein blood) and peripheral blood. Therefore, from the above results, by measuring the expression level of PLS3 mRNA in reflux venous blood (portal vein blood) and peripheral blood, it is possible to detect cancer of colorectal cancer without measuring the expression level of PLS3 mRNA in cancer cells (primary focus cells). The degree of progression (invasion and metastasis) and postoperative prognosis can be predicted.
  大腸がんの予後因子としてリンパ節転移の有無が非常に重要と考えられている。しかしリンパ節転移陰性大腸がんDukes B症例は既存の臨床病理学的因子での予後予測が困難である。そこで予後予測因子に血中PLS3発現がなりうるかを評価するために、Dukes B症例を151症例まで症例数を追加して末梢血（PB）の PLS3発現を測定した。PBのカットオフ値を用いて、大腸がん患者(n=151)を、高PLS3発現群(PLS3/GAPDH≧PBのカットオフ値)33名と低PLS3発現群(PLS3/GAPDH＜PBのカットオフ値)118名に分類した。これらの大腸がん患者について、末梢血（PB）におけるPLS3 mRNA発現状況と生存率（DFS:手術から５年間の未病生存率、OS：全生存率）との関係を調べた結果を図１２に示す。 この結果から、リンパ節転移陰性であり既存の臨床病理学的因子（腫瘍因子、静脈侵襲、リンパ管侵襲）での予後予測が困難とされるDukes B stageの大腸がんにおいても末梢血中のPLS3発現は予後、再発予測因子となることが確認された。このことから末梢血PLS3発現は、現在使用されている進行度分類をリンパ節転移陰性大腸がんにおいては凌駕していることが明らかとなった。 The presence or absence of lymph node metastasis is considered to be very important as a prognostic factor for colorectal cancer. However, it is difficult to predict the prognosis of Dukes B cases with lymph node-negative colon cancer using existing clinicopathological factors. Therefore, in order to evaluate whether PLS3 expression in blood could be a prognostic predictor, we increased the number of Dukes 症 例 B cases up to 151 cases and measured PLS3 expression in peripheral blood (PB). Using the cut-off value of PB, colon cancer patients (n = 151) were cut into 33 patients with high PLS3 expression (PLS3 / GAPDH ≧ PB cut-off value) and low PLS3 expression group (PLS3 / GAPDH <PB) (Off value) Classified into 118 people. The results of examining the relationship between PLS3 の mRNA expression status in peripheral blood (PB) and survival rate (DFS: disease-free survival rate for 5 years after surgery, OS: overall survival rate) for these colon cancer patients are shown in FIG. Shown in From this result, it was found that the disease in peripheral blood was also observed in Dukes B stage colorectal cancer, which is negative for lymph node metastasis and difficult to predict the prognosis of existing clinicopathological factors (tumor factors, venous invasion, lymphatic invasion). It was confirmed that PLS3 expression is a prognosis and recurrence predictor. From this, it became clear that peripheral blood PLS3 expression surpassed the currently used advanced classification in lymph node-negative colorectal cancer.
　（3）実験手法(9)に記載する方法に従って、大腸がんの患者から採取した末梢血から、定法に従って単核球層（白血球および癌細胞が含まれる）を抽出し、次いで抽出した細胞からCD45を発現していない細胞〔CD45(-)細胞〕を集めた。 (3) Extract the mononuclear cell layer (including leukocytes and cancer cells) from peripheral blood collected from patients with colorectal cancer according to the method described in Experimental Method (9), and then extract from the extracted cells Cells not expressing CD45 [CD45 (−) cells] were collected.
　白血球を始めとする血液細胞はCD45を発現していることから、CD45を発現していないCD45(-)細胞は、血液細胞以外の細胞である。次いで、回収したCD45(-)細胞について、実験手法(10)に記載する方法に従って、抗cytokeratin抗体と抗PLS3抗体を用いて、サイトケラチン染色とPLS3の蛍光2重免染を行った。結果を図１３に示す。図１３中、左図はサイトケラチン染色の結果を、中央図はPLS3染色の結果を、右図は両者を重ねた結果をそれぞれ示す。この図は、末梢液中に癌細胞が存在し、当該癌細胞はPLS3 mRNAを発現していることを示す。血液中を流れる癌細胞は、通常ただちにアポトーシスを受けて死亡する。これに対して、大腸がんの患者の末梢血には、アポトーシス耐性を示す、PLS3 mRNAを発現する遊離癌細胞が存在すると考えられる。 Since blood cells including leukocytes express CD45, CD45 (−) cells not expressing CD45 are cells other than blood cells. Subsequently, the collected CD45 (−) cells were subjected to cytokeratin staining and fluorescent double-immunization of PLS3 using an anticytokeratin antibody and an anti-PLS3 antibody according to the method described in Experimental Procedure (10). The results are shown in FIG. In FIG. 13, the left figure shows the result of cytokeratin staining, the middle figure shows the result of PLS3 staining, and the right figure shows the result of overlapping the two. This figure shows that cancer cells are present in the peripheral fluid, and the cancer cells express PLS3 mRNA. Cancer cells that flow through the blood usually die immediately after undergoing apoptosis. On the other hand, it is considered that free cancer cells expressing PLS3 mRNA, which is resistant to apoptosis, are present in the peripheral blood of colorectal cancer patients.
　実験例５　乳がん患者における、原発巣中のPLS3 mRNAの発現と予後との関係
（1）乳がん患者（n=34）における癌組織(T)と正常組織(N)におけるPLS3 mRNAの発現レベルを調べるために、実験手法(1)に記載する方法に従って、リアルタイム定量RT-PCRを行った。 Experimental Example 5 Relationship between expression of PLS3 mRNA in the primary lesion and prognosis in breast cancer patients (1) Examining the expression level of PLS3 mRNA in cancer tissues (T) and normal tissues (N) in breast cancer patients (n = 34) Therefore, real-time quantitative RT-PCR was performed according to the method described in Experimental Procedure (1).
　結果を図１４aに示す。これに示すように、癌組織(T)と正常組織(N)との間におけるPLS3 mRNA発現レベルに有意差は認められなかった。 The result is shown in FIG. 14a. As shown, there was no significant difference in the expression level of PLS3 mRNA between the cancer tissue (T) and the normal tissue (N).
　下記表に、当該乳がん患者（n=102）について、PLS3 mRNA発現（PLS3 mRNA/GAPDH mRNA）および臨床病理的な要因との関係を纏めた結果を示す。なお、ここでは、癌組織（T）におけるPLS3 mRNAの発現がGAPDH mRNAの発現に対して4.1以上である13症例を「高PLS3発現群」(PLS3(T)/GAPDH(T)≧4.1)とし、4.1未満である89症例を「低PLS3発現群」(PLS3(T)/GAPDH(T)<4.1)とした。 The table below shows the results of the relationship between PLS3 mRNA expression (PLS3 mRNA / GAPDH mRNA) and clinicopathological factors for the breast cancer patients (n = 102). Here, 13 cases in which the expression of PLS3 mRNA in cancer tissue (T) is 4.1 or more relative to the expression of GAPDH mRNA are referred to as “high PLS3 expression group” (PLS3 (T) / GAPDH (T) ≧ 4.1). 89 cases less than 4.1 were designated as “low PLS3 expression group” (PLS3 (T) / GAPDH (T) <4.1).
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　これから、高PLS3発現群に属する13症例において、低PLS3発現群に属する89症例によりも、ホルモン（エストロゲン、プロゲステロン）受容体陰性症例が多く、PLS3発現と、ホルモン受容体陰性との間には、正の相関があると考えられた。 From now on, in 13 cases belonging to the high PLS3 expression group, more cases of hormone (estrogen, progesterone) receptor negative than 89 cases belonging to the low PLS3 expression group, and between PLS3 expression and hormone receptor negative, There was a positive correlation.
　図１４bに、上記高PLS3発現群（17症例）と低PLS3発現群（77症例）について、手術後１０年間の生存率を示す（Kaplan-Meier overall survival）。図１４bに示すように、高PLS3発現群に属する患者は、低PLS3発現群に属する患者より、予後（１０年生存率）が有意に不良であった(p<0.05)。 FIG. 14b shows the survival rate for 10 years after surgery for the high PLS3 expression group (17 cases) and the low PLS3 expression group (77 cases) (Kaplan-Meier all survival). As shown in FIG. 14b, patients belonging to the high PLS3 expression group had a significantly worse prognosis (10-year survival rate) than patients belonging to the low PLS3 expression group (p <0.05).
　癌組織(T)における、PLS3 mRNA発現の高低、リンパ節転移、およびプロゲステロン受容体（陰性／陽性）の要素について、予後（術後の全生存率）に与える影響を、Cox比例ハザード回帰を用いて単変量解析および多変量解析した結果を下表に示す。 Cox proportional hazard regression was used to determine the effects of high and low PLS3 mRNA expression, lymph node metastasis, and progesterone receptor (negative / positive) components on prognosis (survival after surgery) in cancer tissues (T). The table below shows the results of univariate analysis and multivariate analysis.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　これからわかるように、癌組織(T)中のPLS3の発現レベルが、独立した予後因子(p=0.0392)であることが明らかになった。また、その相対危険度（RR）は、リンパ節転移による危険度の次にランクされた。 As can be seen, the expression level of PLS3 in cancer tissue (T) was an independent prognostic factor (p = 0.0392). The relative risk (RR) was ranked next to the risk due to lymph node metastasis.
　実験例６　末梢血(PB)および骨髄中のPLS3 mRNAの発現レベルと乳がん患者における予後との関係
（1）実験手法(1)に記載するリアルタイム定量RT-PCRを使用して、乳がん患者(n=341)の末梢血（PB）中のPLS3 mRNAの発現レベルを測定した。なお、カットオフ値は、非担癌患者のPLS3発現の95％信頼区間上限とした。このカットオフ値（0.0025）を用いて、乳がん患者(n=341)を、末梢血（PB）を検体として、高PLS3発現群(PLS3/GAPDH＞0.0025)と低PLS3発現群(PLS3/GAPDH≦0.0025)に分類した（高PLS3発現群=108名、低PLS3発現群=233名）。 Experimental Example 6 Relationship between expression level of PLS3 mRNA in peripheral blood (PB) and bone marrow and prognosis in breast cancer patients (1) Using real-time quantitative RT-PCR described in experimental method (1), breast cancer patients (n = 341) The expression level of PLS3 mRNA in peripheral blood (PB) was measured. The cut-off value was the upper limit of the 95% confidence interval for PLS3 expression in non-cancer-bearing patients. Using this cut-off value (0.0025), breast cancer patients (n = 341), peripheral blood (PB) as samples, high PLS3 expression group (PLS3 / GAPDH> 0.0025) and low PLS3 expression group (PLS3 / GAPDH ≦ 0.0025) (high PLS3 expression group = 108, low PLS3 expression group = 233).
　これらの乳がん患者について、末梢血（PB）におけるPLS3発現状況と生存率との関係を調べた。生存率は、手術から2500日間の全生存率（病気発症の有無に関わらす生存している割合）（OS）と未病生存率（病気を発症しないで生存している割合）（DFS）とに分けて調べた。末梢血（PB）におけるPLS3発現状況と生存率（全生存率（OS）、未病生存率（DFS））との関係を図１５に示す。なお、未病生存率（DFS）の評価は、腫瘍以外の要因で亡くなった患者を除いて行った（高PLS3発現群=108名、低PLS3発現群=232名、合計561名）。 For these breast cancer patients, the relationship between the PLS3 expression status in peripheral blood (PB) and the survival rate was examined. Survival rate is the total survival rate (survival rate with or without disease) (OS) and non-disease survival rate (rate of survival without disease) (DFS) It was divided and examined. FIG. 15 shows the relationship between the PLS3 expression status in peripheral blood (PB) and the survival rate (overall survival rate (OS), non-disease survival rate (DFS)). The disease-free survival rate (DFS) was evaluated by excluding patients who died due to factors other than tumors (high PLS3 expression group = 108, low PLS3 expression group = 232, total 561).
　図１５からわかるように、末梢血（PB）の高PLS3発現群において、全生存率(OS)および未病生存率(DFS)がともに、低PLS3発現群よりも有意に低いこと（p<0.0001）、すなわちPBにおいて、PLS3mRNAを高発現している乳がん患者は、再発の危険性が高く、また５年生存率が低く、予後が不良であることが判明した。 As can be seen from FIG. 15, in the peripheral blood (PB) high PLS3 expression group, both the overall survival rate (OS) and non-disease survival rate (DFS) are significantly lower than the low PLS3 expression group (p <0.0001). ) In other words, it was found that breast cancer patients that highly express PLS3 mRNA in PB have a high risk of recurrence, a low 5-year survival rate, and a poor prognosis.
　（2）上記(1)と同様にして、乳がん患者(n=577)の骨髄中のPLS3 mRNAの発現レベルを測定した。なお、カットオフ値は、正常者のPLS3発現の95％信頼区間上限とした。このカットオフ値（0.105）を用いて、乳がん患者(n=577)を、骨髄を検体として、高PLS3発現群(PLS3/GAPDH＞0.105)と低PLS3発現群(PLS3/GAPDH≦0.105)に分類した（高PLS3発現群=121名、低PLS3発現群=456名）。 (2) In the same manner as in (1) above, the expression level of PLS3 (mRNA in the bone marrow of breast cancer patients (n = 577) was measured. The cut-off value was the upper limit of the 95% confidence interval for PLS3 expression in normal subjects. Using this cut-off value (0.105), breast cancer patients (n = 577) are classified into high PLS3 expression group (PLS3 / GAPDH> 0.105) and low PLS3 expression group (PLS3 / GAPDH ≦ 0.105) using bone marrow as a specimen. (High PLS3 expression group = 121, low PLS3 expression group = 456).
　これらの乳がん患者について、骨髄におけるPLS3発現状況と生存率との関係を調べた。生存率は、手術から2500日間の全生存率（OS）および未病生存率(DFS)について調べた。骨髄におけるPLS3発現状況と全生存率（OS）および未病生存率(DFS)との関係を図１６に示す。 For these breast cancer patients, the relationship between the PLS3 expression status in the bone marrow and the survival rate was examined. Survival was examined for overall survival (OS) and disease free survival (DFS) at 2500 days after surgery. FIG. 16 shows the relationship between the PLS3 expression status in the bone marrow and the overall survival rate (OS) and the disease free survival rate (DFS).
　図１６からわかるように、骨髄の高PLS3発現群において、全生存率(OS) および未病生存率(DFS)が、低PLS3発現群よりも有意に低いこと（OS:p=0.0038、DFS:p=0.0235）、すなわち骨髄において、PLS3mRNAを高発現している乳がん患者は、再発の危険性が高く、また５年生存率が低く、予後が不良であることが判明した。 As can be seen from FIG. 16, in the bone marrow high PLS3 expression group, the overall survival rate (OS) and the disease-free survival rate (DFS) are significantly lower than those in the low PLS3 expression group (OS: p = 0.0038, DFS: p = 0.0235), that is, breast cancer patients that highly express PLS3 mRNA in the bone marrow were found to have a high risk of recurrence, a low 5-year survival rate, and a poor prognosis.
　（3）下記表に、Cox比例ハザード回帰を用いて、当該乳がん患者（n=343）について、末梢血（PBT）中のPLS3 mRNA発現（PLS3 mRNA/GAPDH mRNA）と臨床病理的な要因との関係を纏めた結果を示す。 (3) In the table below, using Cox proportional hazards regression, PLS3 が ん mRNA expression (PLS3 mRNA / GAPDH mRNA) in peripheral blood (PBT) and clinicopathological factors for the breast cancer patient (n = 343) The result of summarizing the relationship is shown.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　これから、高PLS3発現群に属する110症例において、低PLS3発現群に属する233症例によりも、腫瘍サイズ、リンパ節転移、リンパ節浸潤、遠隔転移、ホルモン（プロゲステロン）受容体陰性症例が有意に多く、PLS3発現と、これらの臨床病理学的因子との間には、正の相関があると考えられた。 From 110 cases belonging to the high PLS3 expression group, tumor size, lymph node metastasis, lymph node invasion, distant metastasis, hormone (progesterone) receptor negative cases are significantly more than the 233 cases belonging to the low PLS3 expression group, There was a positive correlation between PLS3 expression and these clinicopathological factors.
　（4）末梢血における、PLS3 mRNA発現の高低、リンパ節転移、およびプロゲステロン受容体（陰性／陽性）の要素について、予後（術後の全生存率）に与える影響を、Cox比例ハザード回帰を用いて単変量解析および多変量解析した結果を下表に示す。 (4) Cox proportional hazard regression was used to determine the effects of high and low PLS3 mRNA expression, lymph node metastasis, and progesterone receptor (negative / positive) components on the prognosis (postoperative overall survival) in peripheral blood. The table below shows the results of univariate analysis and multivariate analysis.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　これからわかるように、原発巣中と同様に、末梢血中のPLS3のmRNA発現レベルが、独立した予後因子(p=0.031)であることが明らかになった。 As can be seen, it was revealed that the PLS3 mRNA expression level in peripheral blood was an independent prognostic factor (p = 0.031), as in the primary lesion.
　以上のことから、すなわち、乳がんについても、大腸がんと同様に、癌細胞におけるPLS3mRNA発現量を測ることなく、末梢血や骨髄中のPLS3mRNA発現量を測定することで、術後の予後や再発の可能性を予測できると考えられる。 From the above, in other words, for breast cancer, as with colon cancer, measuring PLS3 mRNA expression level in peripheral blood and bone marrow without measuring PLS3 mRNA expression level in cancer cells enables postoperative prognosis and recurrence. It is thought that this possibility can be predicted.
　また、本発明の実験から、下記のことがいえる：
1）PLS3遺伝子は、上皮間葉転移（EMT）を誘導する遺伝子である、
2）原発腫瘍、骨髄、血液（門脈血、末梢血）でのPLS3 mRNA発現は、固形がん、特に大腸がんや乳がんの患者の再発および予後不良に関係し、予後不良の危険度の高い患者を識別するための高精度なマーカーになりえる。またPLS3によるEMT制御は、固形がん、特に大腸がんの癌進展（浸潤、転移）を制御するうえで臨床的に有用である。
3）末梢血中のPLS3発現は、Dukes B stageの大腸がんにおいても予後、再発予測因子となり得る。特に、リンパ節転移陰性大腸がんにおいては、当該末梢血中のPLS3発現は、現在使用されている進行度分類を凌駕していることから臨床的に有用である。 Also, from the experiments of the present invention, the following can be said:
1) PLS3 gene is a gene that induces epithelial-mesenchymal transition (EMT),
2) PLS3 mRNA expression in the primary tumor, bone marrow, and blood (portal vein blood, peripheral blood) is related to recurrence and poor prognosis in patients with solid cancer, especially colorectal cancer and breast cancer, and the risk of poor prognosis It can be a highly accurate marker for identifying high patients. Moreover, EMT control by PLS3 is clinically useful in controlling cancer progression (invasion, metastasis) of solid cancer, particularly colorectal cancer.
3) PLS3 expression in peripheral blood can be a prognosis and recurrence predictor even in Dukes B stage colorectal cancer. In particular, in lymph node metastasis-negative colorectal cancer, the expression of PLS3 in the peripheral blood is clinically useful because it exceeds the currently used progression classification.
配列番号３～４および１１～３４は、表１に記載する各遺伝子に特異的なプライマーの塩基配列を示す。配列番号５～１０は、PLS3遺伝子のsiRNAの塩基配列を示す。 SEQ ID NOs: 3 to 4 and 11 to 34 show the base sequences of primers specific to each gene described in Table 1. SEQ ID NOs: 5 to 10 show the base sequences of siRNAs of the PLS3 gene.

Claims (20)

  1. PLS3遺伝子の塩基配列（配列番号１）若しくはそれに相補的な塩基配列にストリンジェントな条件でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチド、またはPLS3タンパクを認識する抗体を含む、固形がん患者の再発予後因子PLS3を測定するための検査薬。 A solid cancer patient comprising a polynucleotide having at least 15 bases that hybridizes under stringent conditions to a base sequence of the PLS3 gene (SEQ ID NO: 1) or a base sequence complementary thereto, or an antibody that recognizes a PLS3 protein A test drug for measuring the prognosis factor PLS3.
  2. 固形がんが、大腸がんまたは乳がんである請求項１に記載する検査薬。 The test agent according to claim 1, wherein the solid cancer is colorectal cancer or breast cancer.
  3. 少なくとも請求項１の検査薬を含有する、固形がん患者の再発予後因子PLS3を測定するための検査薬キット。 A test kit for measuring PLS3, a prognostic factor for recurrence of solid cancer patients, comprising at least the test drug of claim 1.
  4. 上皮間葉移行細胞を検出し測定するために使用される、請求項１に記載する検査薬または請求項３に記載する検査薬キット。 The test drug according to claim 1 or the test drug kit according to claim 3, which is used for detecting and measuring epithelial-mesenchymal transition cells.
  5. 固形がん患者から採取した試料中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程を有する、当該患者について固形がんの再発または予後を予測する方法。 A method for predicting recurrence or prognosis of solid cancer for a patient, the method comprising measuring a PLS3 gene expression level or PLS3 protein production level in a sample collected from the solid cancer patient.
  6. 上記被験試料が、固形がん組織、骨髄、または血液である請求項５に記載する方法。 The method according to claim 5, wherein the test sample is a solid cancer tissue, bone marrow, or blood.
  7. 上記血液が、末梢血である請求項６に記載する方法。 The method according to claim 6, wherein the blood is peripheral blood.
  8. （１）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程、
    （２）上記で測定したPLS3遺伝子発現量（被験発現量）またはPLS3タンパク産生量（被験産生量）を、対照者から採取された上記被験試料に対応する試料（対照試料）のPLS3遺伝子発現量（対照発現量）またはPLS3タンパク産生量（対照産生量）と対比する工程、
    を有し、
    （３）対照発現量または対照産生量に比して被験発現量または被験産生量が高い場合に、固形がん患者について再発の可能性が高いか、または予後不良であると決定する、請求項５に記載する方法。     (1) measuring the PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient;
    (2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production)
    Have
    (3) When the test expression level or the test production level is higher than the control expression level or the control production level, it is determined that the possibility of recurrence is high or the prognosis is poor for a solid cancer patient. 5. The method according to 5.
  9. 被験者から採取された試料（被験試料）におけるPLS3遺伝子発現量（被験発現量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3遺伝子発現量（対照発現量）、または被験者から採取された試料（被験試料）におけるPLS3タンパク産生量（被験産生量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3タンパク産生量（対照産生量）を、請求項１に記載する検査薬を用いて測定する、請求項５に記載する方法。 PLS3 gene expression level (test expression level) in the sample collected from the subject (test sample) and PLS3 gene expression level (control expression level) in the sample (control sample) corresponding to the test sample collected from the control, or the subject The amount of PLS3 protein production (test production amount) in the sample collected from the test sample (test sample) and the amount of PLS3 protein production (control production amount) in the sample (control sample) corresponding to the test sample collected from the control The method according to claim 5, wherein the measurement is performed using the test agent described in 1.
  10. 固形がん患者から採取した試料中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程を有する、上皮間葉移行した癌細胞を検出する方法。 A method for detecting cancer cells that have migrated to epithelial-mesenchymal cells, comprising measuring a PLS3 gene expression level or a PLS3 protein production level in a sample collected from a solid cancer patient.
  11. 上記被験試料が、固形がん組織、骨髄、または血液である請求項１０に記載する方法。 The method according to claim 10, wherein the test sample is solid cancer tissue, bone marrow, or blood.
  12. 上記血液が、末梢血である請求項１１に記載する方法。 The method according to claim 11, wherein the blood is peripheral blood.
  13. （１）固形がん患者から採取した試料（被験試料）中のPLS3遺伝子発現量またはPLS3タンパク産生量を測定する工程、
    （２）上記で測定したPLS3遺伝子発現量（被験発現量）またはPLS3タンパク産生量（被験産生量）を、対照者から採取された上記被験試料に対応する試料（対照試料）のPLS3遺伝子発現量（対照発現量）またはPLS3タンパク産生量（対照産生量）と対比する工程、
    を有し、
    （３）対照発現量または対照産生量に比して被験発現量または被験産生量が高い場合に、固形がん患者の癌細胞または遊離癌細胞が上皮間葉移行していると決定する、請求項１０に記載する方法。     (1) measuring the PLS3 gene expression level or PLS3 protein production level in a sample (test sample) collected from a solid cancer patient;
    (2) The PLS3 gene expression level (test expression level) or PLS3 protein production level (test production level) measured above is the PLS3 gene expression level of the sample (control sample) corresponding to the test sample collected from the control person (Control expression level) or PLS3 protein production (control production)
    Have
    (3) When the test expression level or the test production level is higher than the control expression level or the control production level, it is determined that the cancer cells or free cancer cells of the solid cancer patient have transitioned to epithelial mesenchyme Item 11. The method according to Item 10.
  14. 被験者から採取された試料（被験試料）におけるPLS3遺伝子発現量（被験発現量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3遺伝子発現量（対照発現量）、または被験者から採取された試料（被験試料）におけるPLS3タンパク産生量（被験産生量）および対照者から採取された被験試料に対応する試料（対照試料）におけるPLS3タンパク産生量（対照産生量）を、請求項４に記載する検査薬を用いて測定する、請求項１０に記載する方法。 PLS3 gene expression level (test expression level) in the sample collected from the subject (test sample) and PLS3 gene expression level (control expression level) in the sample (control sample) corresponding to the test sample collected from the control, or the subject The amount of PLS3 protein production (test production amount) in the sample collected from the test sample (test sample) and the amount of PLS3 protein production (control production amount) in the sample (control sample) corresponding to the test sample collected from the control The method according to claim 10, wherein measurement is performed using the test agent described in 4.
  15. PLS3遺伝子の発現を抑制するsiRNAまたはPLS3に対する中和抗体を有効成分として含む、医薬組成物。 A pharmaceutical composition comprising, as an active ingredient, a siRNA that suppresses expression of the PLS3 gene or a neutralizing antibody against PLS3.
  16. 固形がんの再発抑制または予後向上のために用いられるか、または固形がんの癌進展を抑制するために用いられる請求項１５に記載する医薬組成物。 The pharmaceutical composition according to claim 15, which is used for suppressing recurrence of solid cancer or improving prognosis, or for suppressing cancer progression of solid cancer.
  17. 固形がんが大腸がんまたは乳がんである請求項１５に記載する医薬組成物。 The pharmaceutical composition according to claim 15, wherein the solid cancer is colon cancer or breast cancer.
  18. 下記の工程を有する、PLS遺伝子の発現を抑制する物質のスクリーニング方法：
    （1）被験物質と、PLS3遺伝子を発現可能な細胞とを接触させる工程、
    （2）被験物質を接触させた細胞のPLS3遺伝子の発現量（被験発現量）を測定する工程、及び
    （3）上記の被験発現量が、被験物質を接触させない対照細胞のPLS3遺伝子の発現量（対照発現量）よりも小さい場合の被験物質を選択する工程。     A screening method for a substance that suppresses the expression of a PLS gene, comprising the following steps:
    (1) contacting the test substance with a cell capable of expressing the PLS3 gene;
    (2) a step of measuring the expression level (test expression level) of the PLS3 gene in the cell contacted with the test substance, and (3) the expression level of the PLS3 gene in the control cell where the test expression level is not in contact with the test substance. A step of selecting a test substance when it is smaller than (control expression level).
  19. 下記の工程を有する、PLS3の機能を抑制する物質のスクリーニング方法：
    （1’）被験物質とPLS3タンパクを産生可能な細胞またはこの細胞から調製した細胞画分とを接触させる工程、
    （2’）被験物質を接触させた細胞またはその細胞画分のPLS3タンパク産生量（被験産生量）を測定する工程、及び
    （3’）上記の被験産生量が、被験物質を接触させない対照細胞もしくはその細胞画分のPLS3タンパク産生量（対照産生量）よりも小さい場合の被験物質を選択する工程。     A screening method for a substance that suppresses the function of PLS3, comprising the following steps:
    (1 ′) contacting a test substance with a cell capable of producing PLS3 protein or a cell fraction prepared from the cell,
    (2 ′) a step of measuring the PLS3 protein production amount (test production amount) of the cell contacted with the test substance or its cell fraction, and (3 ′) a control cell in which the above test production quantity does not contact the test substance. Alternatively, a step of selecting a test substance when the PLS3 protein production amount (control production amount) of the cell fraction is smaller.
  20. 固形がんの患者に対して再発予防または予後向上に有効な成分、または固形がんの癌進展抑制に有効な成分を探索するための方法である、請求項１８または１９に記載するスクリーニング方法。 The screening method according to claim 18 or 19, which is a method for searching for a component effective for preventing recurrence or improving prognosis for a solid cancer patient, or a component effective for suppressing cancer progression of solid cancer.
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CN107446949A (en) * 2017-07-25 2017-12-08 国家纳米科学中心 PLS3 recombinant proteins eukaryon expression plasmid and its construction method and application

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