JP2013198496A - AIDING METHOD OF PROGNOSIS DETERMINATION OF OVARIAN CANCER WHICH USES COPY NUMBER OF α-ACTININ-4 GENE AS MARKER, AND KIT FOR PROGNOSIS DETERMINATION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new diagnostic method of cancer.SOLUTION: A method of aiding prognosis determination of ovarian cancer in a mammal includes a step of counting copy numbers of α-actinin-4 gene in cells originated from mammalian localization suspected to be ovarian cancer. The method indicates the adverse prognosis of ovarian cancer when the copy numbers of the gene is 4 or more.

Description

  The present invention relates to a method for assisting prognosis determination of ovarian cancer and a kit for prognosis determination.

  “Cancer” is a term often used to describe various malignant neoplasms. Most cancers invade surrounding tissues, metastasize to several places, and are likely to recur even when removed, leading to death if treatment is inadequate. Therefore, for cancer treatment, it is important to detect cancer as early as possible and to give sufficient treatment to the patient.

  For example, pancreatic cancer is cancer that originates from the pancreas, and 90% or more of them are known to be pancreatic duct cancer that originates from cells related to exocrine secretion, particularly cells of the pancreatic duct carrying pancreatic juice. Because the pancreas is surrounded by many organs such as the stomach, duodenum, spleen, small intestine, large intestine, liver, and gallbladder, it is very difficult to find pancreatic cancer. On the other hand, pancreatic cancer has the property that it is easy to spread to other organs from the initial stage and to easily metastasize. Therefore, early detection is essential for the treatment of pancreatic cancer.

  Ovarian cancer is a cancer that originates in the ovary and is known to show almost no symptoms in the early stage. Therefore, in the case of metastatic ovarian cancer, metastasis is often already seen at the time of initial diagnosis. Therefore, early detection is essential for the treatment of ovarian cancer.

  In general, tumor markers that enable diagnosis by blood tests are useful for early detection of cancer. For example, CA19-9, CEA, Dupan-2 and the like are known as tumor markers for pancreatic cancer. Further, βHCG, CA125, STN, SLX and the like are known as tumor markers for ovarian cancer. However, it is often difficult to detect cancer early using these tumor markers. Therefore, development of a new marker for cancer is desired.

  Furthermore, various anticancer agents are used for cancer chemotherapy depending on the primary tumor origin, tissue type, and the like. For example, anticancer agents such as gemcitabine and fluorouracil are used for chemotherapy of pancreatic cancer. However, even if these anticancer agents are used, a therapeutic effect is often not obtained sufficiently. Ovarian cancer is relatively easy to succeed as an anticancer drug, and multi-drug combination therapy mainly consisting of cisplatin is often used for its chemotherapy, but there are many cases where the therapeutic effect is not sufficiently obtained. Therefore, development of another molecular target therapeutic agent having a different mechanism of action from these drugs is expected.

  α-actinin-4 is a molecule isolated as an actin-binding protein involved in cancer cell invasion / metastasis (Non-patent Document 1: Honda et al., J Cell Biol. 1998 Mar 23; 140 (6) : 1383-93). It has also been reported that the expression of α-actinin-4 protein is involved in cell motility and invasiveness (Non-patent Document 2: Honda et al., Gastroenterology. 2005 Jan; 128 (1): 51-62) .

Honda et al., J Cell Biol. 1998 Mar 23; 140 (6): 1383-93 Honda et al., Gastroenterology. 2005 Jan; 128 (1): 51-62

  The inventors of the present invention have a statistically significant difference in the prognosis of cases in which the expression of α-actinin-4 (ACTN4) protein in pancreatic duct cancer tissue is enhanced, and further, the prognosis is poor. It was found that the number of copies of the α-actinin-4 (ACTN4) gene was significantly increased in cases where protein expression was enhanced. In addition, the present inventors have found that an increase in the copy number of the ACTN4 gene is also observed in ovarian cancer, and thus is a phenomenon commonly seen in cancer. Furthermore, the present inventors have found that the pancreatic cancer cell line in which the expression of ACTN4 gene is suppressed has a lower cell growth rate than the control cell line, and further, the pancreatic cancer cell line in which the expression of ACTN4 gene is suppressed It was found that the ability of tumor formation when the cells were orthotopically transplanted into the pancreas of SCID mice was attenuated with a statistically significant difference compared to the cell line. The present invention is based on these findings.

  Accordingly, an object of the present invention is to provide a method for assisting in determining the prognosis of ovarian cancer and a kit for determining the prognosis.

  The method for assisting in the determination of the prognosis of ovarian cancer according to the present invention is a method for assisting in the determination of the prognosis of ovarian cancer in a mammal, and is obtained from a lesion part suspected to be ovarian cancer in the mammal. Measuring the copy number of the α-actinin-4 gene in the isolated cells, and determining that the prognosis of the ovarian cancer is poor when the copy number of the gene is 4 or more, is there.

  The kit for determining the prognosis of ovarian cancer according to the present invention is a kit for determining the prognosis of ovarian cancer in a mammal, and is obtained from a lesion part suspected to be ovarian cancer in the mammal. A reagent capable of measuring the copy number of the α-actinin-4 gene in the obtained cells, and the prognosis of the ovarian cancer is judged to be poor when the copy number of the gene is 4 or more It is a kit.

  According to the present invention, it is possible to predict the prognosis of ovarian cancer and select an appropriate treatment method. This is a particularly important advantage given that cancer is generally difficult to cure.

FIG. 1 is a photomicrograph showing specific examples of a case determined to be strongly positive (left figure) and a case determined to be weakly positive / negative (right figure) in the evaluation of the expression of α-actinin-4 in pancreatic cancer cells. It is. FIG. 2 is a graph showing survival curves prepared by the Kaplan-Meier method for two groups of strong positive cases and weak positive / negative cases of α-actinin-4 gene expression. FIG. 3 is a bar graph showing the copy number of the ACTN4 gene in cancer cells from patients with invasive pancreatic duct cancer. FIG. 4 is a graph comparing the cell growth of pancreatic cancer cell lines (Cont-1 and Cont-2) with the cell growth of the same cell lines (KD-1 and KD-2) knocked down in the expression of the ACNT4 gene. is there. FIG. 5 shows the size of a tumor obtained by injecting a pancreatic cancer cell line (Cont-1 and Cont-2) into the pancreas of a mouse and the same cell line (KD-1 and It is a graph which compares with the size of the tumor obtained by inject | pouring KD-2). FIG. 6 is a microscope showing specific examples of a case determined to be weakly positive / negative (left diagram) and a case determined to be strongly positive (right diagram) in the evaluation of the expression of α-actinin-4 in ovarian cancer cells. It is a photograph. FIG. 7 shows two groups of strongly positive cases and weakly positive / negative cases of α-actinin-4 protein expression, and 2 cases of ACTN4 gene copy number increased cases (copy number ≧ 4) and non-increased cases (copy number <4). It is a graph which shows the survival curve of the ovarian cancer patient created by the Kaplan-Meier method about the group.

  The cancer subject to the present invention is ovarian cancer. In the present invention, it is preferable to target cancers that are difficult to obtain therapeutic effects by anticancer agents, that is, cancers that have low sensitivity to chemotherapy. Furthermore, in the present invention, it is preferable to target ovarian cancer in which the ACTN4 gene is amplified in the cancer cells.

  The ovarian cancer that is the subject of the present invention is a cancer that forms in the ovary. The tissue type of ovarian cancer is not particularly limited, but is preferably clear cell adenocarcinoma. Moreover, as ovarian cancer, ovarian cancer in which the therapeutic effect by an anticancer agent is difficult to be obtained, that is, ovarian cancer with low sensitivity to chemotherapy is particularly preferable.

  As used herein, the term “poor prognosis” means, for example, a survival rate of less than 20%, preferably less than 15%, more preferably less than 10%, most preferably less than 5% after 5 years of cancer resection. It means that there is.

  In the present invention, “α-actinin-4” or “ACTN4” used as a diagnostic index and a treatment target in the present invention is α-actinin-4 (ACTN4) derived from a mammal to be a subject of diagnosis or treatment. Can do. The sequence of the ACTN4 gene and the amino acid sequence encoded thereby are well known in the art, and examples thereof include the human-derived ACTN4 sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 (Ensembl ID: ENSG00000100402 and ENST000000252699). The sequence of human-derived ACTN4 is also registered in NCBI access numbers: NM_004924 and NP_004915. The locus of the ACTN4 gene is also well known in the art. For example, the human ACTN4 gene is located at residues 43, 830, 167 to 43, 913, 010 of the 19th chromosome. For mammals other than humans, similar genes and proteins corresponding to human-derived ACTN4 can be used.

  Herein, it has been demonstrated that enhanced expression of α-actinin-4 (ACTN4) is a poor prognostic factor for pancreatic cancer, particularly invasive pancreatic ductal cancer. Furthermore, a correlation between the amplification of the ACTN4 gene in cells and the enhanced expression of the gene has been demonstrated. In the present specification, it is further demonstrated that the ACTN4 gene is amplified not only in pancreatic cancer but also in ovarian cancer. Therefore, it is shown that the amplification of the gene is a phenomenon commonly observed in cancer. Has been. Therefore, by examining the increased expression of ACTN4 in tissues or cells obtained from a lesion part suspected of being pancreatic cancer in a mammal (human or mammal other than human) as a subject, the lesion is pancreatic cancer. It can be diagnosed whether or not there is. Furthermore, by examining the number of copies of the ACTN4 gene in cells obtained from a lesion that is suspected of being cancerous, it is possible to diagnose whether the lesion is cancerous.

  The tissue or cells from the lesion to be diagnosed may be a surgical sample removed by surgery from a patient, or may be a biopsy sample obtained by an endoscope or the like. For example, it is advantageous to use a biopsy sample from a patient as an object of diagnosis when examining a treatment plan including determination whether or not the lesion should be surgically removed.

  In the diagnostic method according to the first aspect of the present invention, by examining the copy number of the ACTN4 gene in a cell obtained from a lesion part suspected of being cancerous, it is determined whether or not the lesion is cancerous. To detect cancer. This method includes the step of measuring the copy number of the ACTN4 gene in cells obtained from the lesion, and if the copy number exceeds 2, it is determined that the subject mammal is cancerous. (Diagnosis) is done.

  According to a preferred embodiment of the present invention, the cancer is pancreatic cancer. That is, according to this embodiment, there is provided a method for detecting pancreatic cancer in a mammal, wherein the copy number of α-actinin-4 gene in a cell obtained from a lesion part suspected of being pancreatic cancer in the mammal is determined. A method comprising the step of measuring is provided. In this method, when the copy number of the gene exceeds 2, it is indicated that pancreatic cancer exists in the mammal.

  According to another preferred embodiment of the invention, said cancer is ovarian cancer. That is, according to this embodiment, there is provided a method for detecting ovarian cancer in a mammal, which is a copy of an α-actinin-4 gene in a cell obtained from a lesion portion suspected of being ovarian cancer in the mammal. A method is provided comprising the step of measuring the number. In the method, when the copy number of the gene exceeds 2, it is indicated that ovarian cancer is present in the mammal.

  Standard methods known in the art can be used for measuring the copy number of the ACTN4 gene. The sequence of the ACTN4 gene and the amino acid sequence encoded thereby are as described above, and those skilled in the art will measure the copy number of the ACTN4 gene using standard methods by referring to these sequences. be able to. Methods for measuring gene copy number include, inter alia, hybridization and amplification based assays.

  As an assay based on hybridization, there is a Southern blotting method. In this method, genomic DNA is fragmented, separated by electrophoresis, transferred to a membrane, and hybridized to a specific probe of ACTN4. By comparing the intensity of the hybridization signal from this specific probe with the signal intensity from a control probe that hybridizes to the same genomic region without an increase in copy number, the relative ACTN4 gene copy number was obtained. It is done.

  According to a preferred embodiment of the present invention, the measurement of the copy number of the ACTN4 gene is carried out by fluorescence in situ hybridization (FISH) method (Angerer, Meth. Enzymol. 152, 649) using a probe specific for the gene on the genome. , 1987). In general, in situ hybridization mainly involves immobilizing a tissue or cell to be analyzed, pre-hybridizing the tissue or cell, and hybridizing a specific probe to a nucleic acid in the tissue or cell. This is carried out by soaking, washing to remove the unbound specific probe, and detecting the hybridized specific probe. Specific probes used for such applications are labeled with a fluorescent reporter. By counting the number of fluorescence in one cell nucleus, the copy number of the ACTN4 gene in that cell can be obtained.

  As another hybridization-based assay, the comparative genomic hybridization (CGH) method can also be used. In the CGH method, DNA is extracted from test cells to be analyzed and normal cells as controls, each DNA is labeled with a fluorescent dye of a different color, and a mixed solution consisting of equal amounts of these labeled DNAs is prepared. Store the mixed solution at 37 ° C. in the dark for 2-3 days. This results in competitive binding between the test DNA and the control DNA for all sites on the chromosome. As a result, if there is an increase in the gene copy number in the test DNA, the fluorescence of the color labeled with the test DNA is strongly observed at the position corresponding to the gene on the slide. If there is a decrease, the fluorescence of the color labeled control DNA is strongly observed at the position corresponding to that gene on the slide.

  Amplification-based assays include quantitative nucleic acid amplification methods. In this method, since the ACTN4 gene in the genome serves as a template for the amplification reaction, an amplification product in an amount corresponding to the copy number can be obtained. Specific nucleic acid amplification methods include quantitative PCR methods such as real-time quantitative PCR methods.

  According to a preferred embodiment of the present invention, when the copy number of the ACTN4 gene in the cell obtained from the lesion is 4 or more, more preferably 6 or more, the subject mammal has cancer. It is determined (diagnostic) that it exists.

  In the diagnostic method according to the second aspect of the present invention, it is determined whether or not the lesion is pancreatic cancer by examining the enhanced expression of ACTN4 in a tissue or cell obtained from a lesion portion suspected of being pancreatic cancer, Thereby, pancreatic cancer is detected. This method includes a step of measuring the expression level of the ACTN4 gene in a tissue or cell obtained from the lesion, and when the expression level is higher than that of the corresponding normal tissue or cell, that is, When expression is enhanced, it is determined (diagnosed) that pancreatic cancer is present in the subject mammal.

  Standard methods known in the art can be used to measure the expression level of the ACTN4 gene. The expression level of a general gene is typically measured using the amount of mRNA or protein as an index. Similarly, in the present invention, the expression level of the ACTN4 gene can be measured using the amount of mRNA or protein of ACTN4 as an index. The sequence of the ACTN4 gene and the amino acid sequence encoded thereby are as described above, and those skilled in the art will measure the expression level of the ACTN4 gene using a standard method by referring to these sequences. be able to.

  According to a preferred embodiment of the present invention, the step of measuring the expression level of the gene comprises measuring the amount of ACTN4 protein in the tissue or cell. The amount of ACTN4 protein can be measured by a method well known in the art. For example, the amino acid sequence of ACTN4 protein can be exemplified by that represented by SEQ ID NO: 2, and those skilled in the art can appropriately measure the amount of the protein by referring to this sequence.

  The amount of ACTN4 protein can be suitably measured, for example, by using an antibody that binds to the protein. Such an antibody is preferably an antibody specific for the ACTN4 protein. The antibody may be a polyclonal antibody or a monoclonal antibody. Quantification of ACTN4 protein is preferably performed by immunohistochemical staining using such antibodies. Or you may use the immunoblot method etc. which quantify this using an antibody after electrophoresis the protein extract obtained from the said tissue or cell.

  According to another preferred embodiment of the present invention, the step of measuring the expression level of the gene comprises quantifying the mRNA of ACTN4 in the tissue or cell. For example, as the mRNA of ACTN4, one containing the nucleotide sequence represented by SEQ ID NO: 1 (where t in the sequence is converted to u) can be mentioned, and those skilled in the art refer to this sequence. Thus, it is possible to appropriately quantify the ACTN4 mRNA. Specific methods for quantification include, for example, a method of performing electrophoresis after amplification by RT-PCR using a specific primer pair.

  The normal tissue or cell as a comparison control may be a normal tissue or cell from the same individual as the subject, or may be a normal tissue or cell from a different individual. Alternatively, the expression level of the ACTN4 gene may be measured for normal tissues or cells from a plurality of different individuals, and the average value may be used as a comparison control. Furthermore, when appropriate values such as mean value + standard deviation are set from the data of expression levels in multiple normal tissues or cells measured in advance, and the expression level of the test tissue or cells is higher than the appropriate value It may be determined that there is enhanced expression of the ACTN4 gene. “Corresponding normal tissue or cell” includes normal pancreatic tissue or cells.

  In order to carry out the detection / diagnosis method according to the present invention as described above, necessary reagents can be put together into a kit. Therefore, according to the present invention, a kit for diagnosis of cancer (preferably pancreatic cancer or ovarian cancer) in a mammal is provided, which kit measures the copy number of the α-actinin-4 gene in a cell. A reagent capable of being produced. Such a reagent is selected according to the specific method to be carried out, and is preferably a probe specific to the gene on the genome, which can be used in the FISH method.

  The kit according to another aspect of the present invention is a kit for diagnosing pancreatic cancer in a mammal, comprising a reagent capable of measuring the expression level of α-actinin-4 gene in a tissue or cell. As such a reagent, a reagent for quantifying the ACTN4 protein in the tissue or cells, for example, an antibody that specifically binds to the ACTN4 protein can be preferably used. Alternatively, such a reagent may be a reagent for quantifying ACTN4 mRNA in the tissue or cells, for example, a specific primer used for RT-PCR using ACTN4 mRNA as a template and other RT-PCR primers. It may be a reagent.

  The kit according to the present invention may further contain other reagents, reaction containers, instructions, etc., depending on the specific method to be carried out.

  In the present specification, it has been demonstrated that by suppressing the expression of the ACTN4 gene, the proliferative ability of the pancreatic cancer cell line is reduced and the tumor-forming ability of the pancreatic cancer cell line in the pancreas is attenuated. In general, when the gene copy number of an oncogene is amplified and the protein expression is remarkably enhanced with the amplification, the function is often enhanced also in cell biology. In pancreatic cancer, an increase in the copy number of ACTN4 gene and an increase in the expression of ACTN4 protein were observed, and the enhancement of cell motility was confirmed by this amplification of ACTN4 gene. Furthermore, cells in which the ACTN4 gene was knocked down were suppressed in the destructive invasion mode in pancreatic cancer. In the present specification, amplification of the ACTN4 gene has been confirmed for ovarian cancer as well as pancreatic cancer. Therefore, it is considered that the decrease in the proliferation ability of the cancer cells and the decrease in the tumor formation ability due to suppression of the expression of the ACTN4 gene are also observed in ovarian cancer cells, and also in other cancers. Therefore, it is considered that cancer can be treated by inhibiting ACTN4.

  Accordingly, the present invention provides a method for treating or preventing cancer comprising administering to a subject a therapeutically effective amount of an ACTN4 inhibitor. Furthermore, according to the present invention, there is provided the use of an ACTN4 inhibitor in the manufacture of a medicament for treating cancer.

  According to a preferred embodiment of the present invention, the cancer is pancreatic cancer. That is, according to this embodiment, in a method for treating or preventing pancreatic cancer, comprising administering a therapeutically effective amount of an ACTN4 inhibitor to a subject, and in the manufacture of a medicament for treating pancreatic cancer The use of an ACTN4 inhibitor is provided.

  According to another preferred embodiment of the invention, said cancer is ovarian cancer. That is, according to this embodiment, a method of treating or preventing ovarian cancer, comprising administering to a subject a therapeutically effective amount of an ACTN4 inhibitor, and a medicament for treating ovarian cancer There is provided the use of an ACTN4 inhibitor in manufacture.

  Treatment of cancer includes, for example, prevention of cancer recurrence or metastasis. Furthermore, the subject to be treated or prevented is preferably a mammal, such as a human or non-human mammal.

  According to a preferred embodiment of the invention, the ACTN4 inhibitor is a small molecule such as a drug. Such a small molecule can be obtained by screening by a method known in the art using the inhibitory activity of ACTN4 as an index.

  According to a preferred embodiment of the present invention, the ACTN4 inhibitor is an antibody that specifically binds to the ACTN4 protein. The antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

  According to a preferred embodiment of the present invention, the ACTN4 inhibitor is an antisense nucleic acid molecule that specifically suppresses the expression of the ACTN4 gene. The antisense method is a well-known technique for suppressing the expression of a specific gene. In one specific example, the antisense nucleic acid molecule is an antisense RNA having a length of about 10 to 40 bases designed based on the sequence of the 5 'coding region of the ACTN4 gene. In another embodiment, the antisense nucleic acid molecule is a DNA oligonucleotide designed to be complementary to the sequence of a region involved in transcription of the ACTN4 gene. Such an antisense nucleic acid molecule can be easily designed based on the sequence of the ACTN4 gene as shown in SEQ ID NO: 1.

  According to a preferred embodiment of the present invention, the ACTN4 inhibitor is an siRNA nucleic acid molecule that specifically suppresses the expression of the ACTN4 gene. As used herein, “siRNA nucleic acid molecule” means not only siRNA itself but also a longer double-stranded RNA molecule capable of introducing siRNA into a target cell. siRNA nucleic acid molecules are well-known tools that can suppress the expression of specific genes by RNA interference (RNAi) (Elbashir, S.M. et al., Nature 411, 494-498, 2001). The siRNA typically comprises a 19-21 base pair nucleotide sequence that is homologous to a sequence specific to the target gene mRNA. The double-stranded RNA molecule described above typically comprises a longer nucleotide sequence that is homologous to a sequence specific for the mRNA of the target gene. Such siRNA nucleic acid molecules can be easily designed based on the sequence of the ACTN4 gene as shown in SEQ ID NO: 1. Further, the siRNA nucleic acid molecule can be expressed by an appropriate vector delivered into the cell. Therefore, the ACTN4 inhibitor may be a vector that expresses an siRNA nucleic acid molecule that specifically suppresses the expression of the ACTN4 gene. Such vectors can be readily constructed by standard procedures well known in the art (Bass, BL, Cell 101, 235-238, 2000; Tavernarakis, N. et al., Nat. Genet 24, 180-183, 2000; Malagon, F. et al., Mol. Gen. Genet. 259, 639-644, 1998; Parrish, S. et al., Mol. Cell 6, 1077-1087, 2000) .

  The ACTN4 inhibitor can be administered by an appropriate route such as topical, intravenous, subcutaneous, intramuscular, oral, rectal, mucosal and the like. The therapeutically effective amount is appropriately determined by a doctor or veterinarian according to the severity of symptoms, the age of the subject, the effectiveness of the specific drug used, the route of administration, the frequency of administration, and the like. Generally, the therapeutically effective amount is about 0.001 to about 1000 mg / kg body weight, preferably about 0.01 to about 10 mg / kg body weight, more preferably about 0.01 to about 1 mg / kg per day. Weight is kg.

  The ACTN4 inhibitor can be administered with a pharmaceutically acceptable carrier. Therefore, according to the present invention, there is provided a pharmaceutical composition for treating cancer, comprising an ACTN4 inhibitor and a pharmaceutically acceptable carrier.

  According to a preferred embodiment of the present invention, the cancer is pancreatic cancer. That is, according to this embodiment, there is provided a pharmaceutical composition for treating pancreatic cancer, comprising an ACTN4 inhibitor and a pharmaceutically acceptable carrier.

  According to another preferred embodiment of the invention, said cancer is ovarian cancer. That is, according to this embodiment, a pharmaceutical composition for treating ovarian cancer comprising an ACTN4 inhibitor and a pharmaceutically acceptable carrier is provided.

  Pharmaceutically acceptable carriers such as vehicles, excipients, diluents and the like are appropriately selected by those skilled in the art depending on the route of administration, the properties of the specific drug used, and the like.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention.

Example 1: Relationship between pancreatic cancer and increased expression of α-actinin-4 gene
As a sample, formalin from 173 patients with no previous history who underwent excision of invasive pancreatic duct cancer at National Cancer Center Central Hospital (Japan) from January 1990 to October 2003 A resected specimen embedded in fixed paraffin was used. For these samples, the expression of α-actinin-4 protein was examined by immunohistochemical analysis. The primary antibody used for the immunohistochemical analysis was an anti-human α-actinin-4 rabbit polyclonal antibody prepared at the National Cancer Center Research Institute (Japan). Immunostaining was performed by the ABC method using an avidin-biotin complex.

  In the evaluation of α-actinin-4 expression in pancreatic cancer cells, the expression of α-actinin-4 that is clearly higher in staining than the vascular endothelium on the same section is a strong positive, and the staining of vascular endothelium is equivalent or lower (Including those that were not stained at all) were classified as weak positive or negative and classified into these two categories. This classification was performed by three researchers who were unaware of other clinical data from the patient from whom the sample was taken.

  Examples of samples classified into each category are shown in FIG. In FIG. 1, the left figure is a case determined as strong positive, and the right figure is a case determined as weak positive / negative. “VE” shown in the figure is vascular endothelium, and “cancer” is pancreatic cancer cells.

  Next, the above classification of anti-α-actinin-4 antibody staining properties and the risk factors of various clinicopathological factors for the survival time of pancreatic cancer patients were analyzed by univariate analysis and multivariate analysis of the Cox proportional hazard model. analyzed. The results are shown in Table 1 below.

  According to the results of univariate analysis shown on the left side of Table 1, UICC stage classification, primary lesion size, presence / absence of lymph node metastasis, presence / absence of distant organ metastasis, lymphatic vessel invasion, and α-actinin-4 protein It has been shown that expression intensity in pancreatic cancer cells can be a risk factor for poor prognosis.

  Furthermore, on the right side of Table 1, the UICC stage classification, primary tumor size, presence or absence of lymph node metastasis, presence or absence of distant organ metastasis, lymphatic invasion, which were shown to be a prognostic factor by univariate analysis Only the expression intensity of α-actinin-4 protein in pancreatic cancer cells is shown by the results of analysis by multivariate analysis. According to this result, the presence or absence of lymph node metastasis, the presence or absence of distant organ metastasis, and the expression intensity of α-actinin-4 protein in pancreatic cancer cells showed statistically significant differences, which are risk factors for poor prognosis. It was shown that there is. That is, it was shown that the expression intensity of α-actinin-4 protein in pancreatic cancer cells is a prognostic factor independent of other clinicopathological factors examined this time.

  Subsequently, survival curves were prepared by Kaplan-Meier method for the two groups of α-actinin-4 gene expression strongly positive and weak positive / negative cases classified as described above, and examined by Cox-Mantel test. . The obtained survival curve is shown in FIG. According to FIG. 2, it can be seen that 109 cases that were strongly positive cases had a statistically significant difference and a poor prognosis compared to 64 cases that were weakly positive / negative cases of α-actinin-4 expression. (P <0.00001).

<< Example 2: Increase in the copy number of the ACTN4 gene in pancreatic cancer >>
In order to examine the copy number of the α-actinin-4 (ACTN4) gene, a specific probe for the ACTN4 gene existing on the long wall of chromosome 19 was prepared, and FISH (Fluorescence in Situ) was applied to the paraffin section of the resected pancreatic cancer specimen. Hybridization) analysis was performed.

  From among 173 resected pancreatic cancer cases described in Example 1, 29 cases in which α-actinin-4 expression was determined to be strongly positive immunohistochemically and 17 cases in which weakly positive / negative were determined to be arbitrarily selected After selection, the copy number of the ACTN4 gene was measured using the FISH method. The FISH method was performed using a PathVysion DNA probe kit (Abbott Molecular, Des Plaines, IL). The specific probe used for hybridization was a denatured bacterial artificial chromosome (BAC) probe containing the ACTN4 (RP11-118P21) locus. A denatured BAC probe containing the AKT2 (CTB-166E20) locus was also used. These probes were labeled using SpectrimOrange (Abbott Molecular, Des Plaines, IL). Hybridization was performed at 37 ° C. for 14-18 hours. Samples were counterstained with 4 ', 6-diamidino-2-phenylindole (DAPI). The analysis is performed by two or more judges, and each judge counts the fluorescent signal present in the nucleus for 10 or more pancreatic cancer cells for one case, and the average value is the copy number for each case. did. FIG. 3 shows the number of copies for each case as a bar graph.

  When the number of signals exceeding 4 was defined as gene amplification, 11 out of 29 cases (37.9%) in which ACTN4 gene expression was strongly positive were determined to be gene amplification, and in weak positive / negative cases One of 17 cases (5.9%) was determined to be gene amplification. The frequency of gene amplification in the strong positive case and the weak positive / negative case was determined using the chi-square test, and the strong positive case was higher with a statistically significant difference (P = 0.017). .

  From this, it was considered that the enhanced expression of α-actinin-4 protein in the invasive pancreatic cancer cells observed in Example 1 was due to amplification of the gene encoding AC-actinin-4 (ACTN4). .

<< Example 3: Treatment effect of pancreatic cancer by suppressing expression of ACTN4 gene (in vitro experiment) >>
Two pancreatic cancer cell lines (KD-1 and KD) in which α-actinin-4 protein was constantly reduced by introducing shRNA (short hairpin RNA) against ACNT4 gene into BxPC-3 cells, which are pancreatic cancer cell lines. -2). Specifically, a double-stranded oligonucleotide (5′-ggatggtcttgccttcaat-3 ′; SEQ ID NO: 3) targeting ACNT4 mRNA was synthesized and was synthesized in the pBAsi-hU6 Neo plasmid (Takara Bio, Otsu, Japan). Cloned into. The resulting plasmid was transfected into BxPC-3 cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). 24 hours later, the transfection medium was replaced with RPMI 1640 medium containing 0.4 mg / ml G418 (Geneticin, Invitrogen), and neomycin resistant clones were selected. Two clones were selected from the clones thus obtained and named KD-1 and KD-2, respectively.

KD-1 cells and KD-2 cells were seeded at 2 × 10 ⁵ cells / well in a 6-well tissue culture cluster, and the number of cells was counted every 24 hours. As a control sample, the same experiment was performed on two control cell lines (Cont-1 and Cont-2) into which the pBAsi-hU6 Neo plasmid itself was introduced. Three samples were prepared for each cell line, and the average value ± standard deviation of the counted number of cells was used as data for each cell line. The result is shown in FIG.

  According to FIG. 4, it becomes clear that the cell line in which the expression of the ACNT4 gene is knocked down is suppressed in cell growth compared to the control line.

<< Example 4: Treatment effect of pancreatic cancer by suppressing expression of ACTN4 gene (in vivo experiment) >>
Each of the cell lines (KD-1 and KD-2) and the control cell lines (Cont-1 and Cont-2) in which the protein expression of α-actinin-4 was constantly suppressed was placed in the pancreatic part of SCID mice at 1 ×. 10 ⁶ were injected. These mice were raised for 35 days and then dissected, and the maximum diameter of the tumor formed in the pancreas was measured under a stereomicroscope. The result is shown in FIG.

  FIG. 5 shows that the tumor-forming ability in the pancreas of the ACTN4 knockdown cell line is lower than that of the control cell line with a statistically significant difference. Histologically, the control cell line showed invasive growth that destroyed the pancreatic parenchyma, but the knockdown cell line showed only tumor growth in the pancreatic ductal epithelium and loosely connected tissue. There was no invasive growth destroying the.

  According to these results, it is clear that the cell line in which the expression of the ACNT4 gene is knocked down has a markedly reduced tumor-forming ability as compared with the control line.

Example 5: Relationship between ovarian cancer and increase in copy number of α-actinin-4 gene
<A. Relationship between ovarian cancer and increased expression of α-actinin-4 gene>
As a sample, a formalin-fixed paraffin-embedded specimen of 136 cases of advanced ovarian cancer that was excised at the Department of Obstetrics and Gynecology, National Defense Medical College Hospital from January 1987 to December 2005 was used. . For these samples, the expression of α-actinin-4 protein was examined by immunohistochemical analysis. The primary antibody used for the immunohistochemical analysis was an anti-human α-actinin-4 rabbit polyclonal antibody prepared at the National Cancer Center Research Institute (Japan). Immunostaining was performed by the ABC method using an avidin-biotin complex.

  In the evaluation of the expression of α-actinin-4 in ovarian cancer cells, those having a clearly higher staining ability than the vascular endothelium on the same section are strongly positive, and the staining ability is the same or lower than that of the vascular endothelium. Things (including those that were not stained at all) were classified as weak positive or negative and classified into these two categories.

  Examples of samples classified into each category are shown in FIG. In FIG. 6, the left figure is a case determined as weak positive / negative, and the right figure is a case determined as strong positive. “VE” shown in the figure is the vascular endothelium.

  In this analysis, among 136 cases of advanced stage ovarian cancer, 82 cases (60.3%) showed increased expression of α-actinin-4 protein.

<B. Increased copy number of ACTN4 gene in ovarian cancer>
In order to examine the copy number of the α-actinin-4 (ACTN4) gene, a specific probe for the ACTN4 gene existing on the long wall of chromosome 19 was prepared, and FISH (Fluorescence in Situ Hybridization method was used.

  For 136 cases of advanced stage ovarian cancer described in A above, the copy number of the ACTN4 gene was measured using the FISH method. The FISH method was performed using a PathVysion DNA probe kit (Abbott Molecular, Des Plaines, IL). The specific probe used for hybridization was a denatured bacterial artificial chromosome (BAC) probe containing the ACTN4 (RP11-118P21) locus. The probe was labeled with SpectrimOrange (Abbott Molecular, Des Plaines, IL). Hybridization was performed at 37 ° C. for 14-18 hours. Samples were counterstained with 4 ', 6-diamidino-2-phenylindole (DAPI). The analysis is performed by two or more judges, and each judge counts the fluorescent signal present in the nucleus for 20 or more ovarian cancer cells for one case, and the average value is the number of copies per case. It was.

  As shown in Table 2 below, when a signal having 4 or more signals is defined as gene amplification (increase in copy number), 27 out of 82 cases (32.9%) in which ACTN4 gene expression is strongly positive Was determined to be gene amplification, and 2 of 54 cases (3.7%) were determined to be gene amplification in weak positive / negative cases. The frequency of gene amplification in the strong positive case and the weak positive / negative case was determined using a chi-square test, and the strong positive case was higher with a statistically significant difference (P = 0.000016). .

<C. Association between increased expression of α-actinin-4 protein and increased copy number of ACTN4 gene and life prognosis of patients with advanced stage ovarian cancer>
Two groups of α-actinin-4 protein expression strongly positive and weakly positive / negative cases classified as described above, ACTN4 gene copy number increased cases (copy number ≧ 4) and non-increased cases (copy number < About 2 groups of 4), the survival curve was created by Kaplan-Meier method. The obtained survival curve is shown in FIG.

  The left figure of FIG. 7 is a survival curve prepared for two groups of a strong positive case and a weak positive / negative case of α-actinin-4 protein expression. According to this survival curve, it can be seen that 82 strong positive cases have a poorer prognosis than 54 weak positive / negative cases. Further, the difference between the weak positive / negative cases and the strong positive cases was shown to be a statistically significant difference by Log-rank test and generalized Wilcoxon test (Log-rank test: P = 0). 0.089; Generalized Wilcoxon test: P = 0.0306).

  The right figure of FIG. 7 is a survival curve prepared for two groups of an increased case of ACTN4 gene copy number (copy number ≧ 4) and a non-increased case (copy number <4). According to this survival curve, it can be seen that 29 cases with increased copy number have a poorer prognosis than 107 cases with no increase in copy number. Further, the difference between this non-increased copy number case and the increased case was shown to be a statistically significant difference by the Log-rank test (P = 0.00107).

  Furthermore, from the comparison of the left and right diagrams of FIG. 7 and the statistical P value, the increase in the copy number of the ACTN4 gene is more strictly compared to the expression of α-actinin-4 protein. It has been shown that the prognosis of patients can be predicted.

<D. Clinicopathological prognostic factor analysis of advanced stage ovarian cancer using Cox proportional hazard model>
The risk rate for each clinicopathological factor for the survival period of patients with advanced stage ovarian cancer, the risk rate for staining of anti-α-actinin-4 antibody, and the risk rate for increased ACTN4 gene copy number Minute data were used for analysis by univariate and multivariate analysis of the Cox proportional hazards model. The results are shown in Table 3 below.

  According to the results of univariate analysis shown on the left side of Table 3, FIGO stage classification, clear-cell adenocarcinoma, residual tumor diameter at the first operation, and increased ACTN4 gene copy number, risk of poor prognosis It has been shown that it can be a factor. On the other hand, it has been shown that the serous adenocarcinoma tissue type can be a good prognostic factor in patients with advanced stage ovarian cancer. Moreover, it was shown that the increase in the copy number of the ACTN4 gene has a stronger influence on the prognosis of patients with advanced stage ovarian cancer than the expression of α-actinin-4 protein.

  In addition, on the right side of Table 3, on the right-hand side of FIG. 3, the FIGO stage classification, specific tissue types (clear cell adenocarcinoma and serous adenocarcinoma), which were shown to be prognostic factors by univariate analysis, the residual tumor diameter at the first surgery The results of multivariate analysis of only the copy number of ACTN4 gene and the extraction of risk factors are shown. According to this result, it was shown that the increase in the copy number of the ACTN4 gene is a prognostic factor independent of the clinical stage and clear cell adenocarcinoma as well as the serous adenocarcinoma and the residual tumor diameter at the first operation.

  That is, the increase in the copy number of the ACTN4 gene is a poor prognostic factor independent of other clinicopathological factors examined this time, and more strictly compared with the expression of α-actinin-4 protein. It was shown that the prognosis of cancer-bearing patients can be predicted.

<E. Association between increased copy number of ACTN4 gene and various clinicopathological factors>
The association between an increased copy number of the ACTN4 gene and various clinicopathological factors in ovarian cancer was determined using a chi-square test. The results are shown in Table 4 below.

  According to Table 4, the increase in the copy number of the ACTN4 gene is closely related to clear-cell adenocarcinoma, tumors with high histological / cytological atypia, and tumors showing resistance to chemotherapy. It was shown that. According to this result, an increase in the copy number of the ACTN4 gene is frequently seen in clear cell adenocarcinoma, especially in ovarian cancer, which is less sensitive to chemotherapy, and is associated with low responsiveness to postoperative chemotherapy. It became clear that

  SEQ ID NO: 3: RNAi target sequence

Claims (7)

A method for assisting in determining the prognosis of ovarian cancer in a mammal, comprising:
Measuring the copy number of the α-actinin-4 gene in cells obtained from a lesion suspected of being ovarian cancer in the mammal;
And the method determines that the prognosis of the ovarian cancer is poor when the copy number of the gene is 4 or more.   The method according to claim 1, wherein the copy number of the gene is measured by a FISH method using a probe specific to the gene on the genome.   The method of claim 1 or 2, wherein the cell is a surgical sample or biopsy sample from the mammal.   The method according to any one of claims 1 to 3, wherein the ovarian cancer is ovarian cancer with low sensitivity to chemotherapy. A kit for determining the prognosis of ovarian cancer in a mammal,
A reagent capable of measuring the copy number of the α-actinin-4 gene in cells obtained from a lesion that is suspected of being ovarian cancer in the mammal;
And the kit determines that the prognosis of the ovarian cancer is poor when the copy number of the gene is 4 or more.   The kit according to claim 5, comprising a probe specific for the gene on the genome.   The kit according to claim 5 or 6, wherein the ovarian cancer is ovarian cancer having low sensitivity to chemotherapy.