JP6683986B2 - Cancer stem cell molecular marker - Google Patents

Description

  The present invention relates to a molecular marker for detecting cancer stem cells from a subject and a technique related thereto.

  Cancer stem cells are considered to be a major cause of cancer recurrence and metastasis, and the importance of targeting cancer stem cells in cancer treatment has been pointed out. However, the ratio of the number of cancer stem cells to the whole tumor tissue is small (Non-Patent Document 4), and it is extremely difficult to specifically recognize and treat cancer stem cells. The method for detecting cancer stem cells, which has been pointed out in many papers, uses a normal stem cell detection system, so it has little difference from normal stem cells, and it is common with indicators that reflect tumor growth or invasion. Therefore, in most cases, a trait marker of cancer cells having high proliferative activity is used as an index of cancer stem cells.

Currently, as cancer stem cell markers, as shown in Table 1, cell surface markers such as CD133, CD24, and CD44 are known (Non-Patent Documents 1 to 7), but they are diverted markers of normal stem cells. Since it is a molecule that is also expressed in somatic stem cells (tissue stem cells), its specificity as a cancer stem cell marker is not necessarily high, and it can cause side effects when it is targeted as a therapeutic target. For these reasons, therapies targeting cancer stem cells have not been established, but the issues of “metastasis / invasion”, “recurrence after aging”, and “anticancer drug resistance” in cancer treatment cannot be overcome. . Identification of new molecular markers is needed to detect cancer stem cells in more types of cancer.
The development of cancer stem cell detection technology and new therapeutic methods targeting cancer stem cells will be extremely important issues for future cancer medicine.
It is known that the expression of the gene encoding PRDM14 is specifically increased in breast cancer and ovarian cancer as compared with normal tissues (Patent Document 1), but PRDM14 gene product is expressed in cancer stem cells. It is not known to be used as a marker.

International Publication No. 2008/038832

Shu Zhang et al., Cancer Res. 68: (11) 4311-4320, 2008 Shih-Hwa Chiou et al., Clin. Cancer Res. 14 (13) 4085-4095, 2008 Gang-Ming Zou, J. Cell. Physiol. 217: 598-604, 2008 Cheong J. Lee et al., J Clin Oncol 26: 2806-2812, 2008 Madhuri Kakarala et al., J Clin Oncol 26: 2813-2820, 2008 Craig D. Peacock et al., J Clin Oncol 26: 2883-2889, 2008 Xing Fan et al., J Clin Oncol 26: 2821-2827, 2008

  An object of the present invention is to provide a molecular marker useful for detecting cancer stem cells.

  In order to solve the above-mentioned problems, the present inventors, while continuing diligent research, use a plurality of cancer cell lines derived from solid cancers of breast cancer and pancreatic cancer, and express PRDM14 to induce cancer. The inventor of the present invention found that the stem cell trait of Escherichia coli can be induced both in vitro and in vivo to construct an in vivo therapeutic model using PRDM14 chimeric RNAi, and that they have tumor-reducing / metastasis-suppressing effects. Has been completed.

That is, the present invention relates to a molecular marker for detecting cancer stem cells from a subject by expressing the PRDM14 gene.
The present invention relates to a molecular marker containing a PRDM14 gene product, preferably a PRDM14 gene product which is an mRNA and / or a polypeptide, or a fragment thereof.

In the present invention, the subject is a group consisting of breast, lung, esophagus, stomach, large intestine, liver, pancreas, cervix, uterine body, ovary, kidney, prostate, bladder, testis, thyroid, adrenal gland, lymph node and blood. The present invention relates to a molecular marker, which is a cell population derived from one or more cells or tissues selected from
The present invention also relates to a method of determining the presence or absence of cancer stem cells or determining the degree of cancer stem cell induction in a subject using the molecular marker as an index.

  The present invention includes the step of detecting the gene product by mRNA when the molecular marker comprises the PRDM14 gene product or a fragment thereof; and detecting the gene product by an RT-PCR method; The PRDM14 gene product is a polypeptide, and the step of detecting the gene product with a reagent that specifically reacts with the gene product is included; when the humoral factor is included, the humoral factor is specific with the humoral factor. Method, which comprises a step of detecting with a chemically reactive reagent, preferably an antibody.

The present invention also relates to a kit for determining the presence or absence of cancer stem cells, the kit including a reagent for detecting the molecular marker.
In the present invention, the reagent for detecting is a probe and / or primer having a base sequence complementary to the PRDM14 gene for detecting mRNA which is the product of PRDM14 gene; detecting the polypeptide which is the product of PRDM14 gene The present invention relates to a kit, which is an antibody for detecting a polypeptide that is a humoral factor.

The present invention also relates to a method for determining the degree of cancer or cancer stem cell induction using the kit.
Furthermore, the present invention is a nucleic acid used for suppressing the expression of PRDM14 gene in cancer stem cells, wherein the nucleic acid is one or more selected from the group consisting of antisense, siRNA and shRNA. Which is a nucleic acid.

Furthermore, the present invention relates to a pharmaceutical composition containing the nucleic acid, preferably further containing an anticancer agent.
The present invention also relates to a pharmaceutical composition for treating or preventing cancer.
The present invention also relates to a method of inhibiting the function of cancer stem cells using the nucleic acid.

  According to the present invention, it was revealed that the PRDM14 gene had low expression in normal tissues and increased expression in tumor tissues and was extremely deeply involved in stem cell traits of cancer cells. Histological diagnosis), and a humoral factor that is elevated by overexpression of the gene has been identified, and by serodiagnosis using it, whether the patient's tumor has a cancer stem cell trait, that is, "metastasis / invasion" , It will be a reference marker to judge "recurrence after years".

  Since the PRDM14 gene has low expression in normal tissues and increased expression in tumor tissues, it is most suitable as a therapeutic target because side effects are unlikely to occur. It also has an effect on cells in which cell proliferation is vigorous, but rather has a very specific effect on a group of cells that become stem cells and produce cancer cells. When used in combination with existing anti-cancer agents, cell groups with a fast cell cycle are existing anti-cancer agents, and cancer cells related to "metastasis / invasion" and "recurrence after aging" are PRDM14 inhibitors (such as siRNA). It is possible to induce cell death, which leads to the cure of cancer.

FIG. 1-A is a graph showing the expression level of PRDM14 mRNA in various cancer tissues. FIG. 1-B is a graph showing the expression level of PRDM14 mRNA in 177 cases of breast cancer tissue and 5 cases of normal breast tissue. From the left side of the graph, 5 cases show normal tissues, and the other cases show values in breast cancer tissues. FIG. 1-C is an image in which the expression level of PRDM14 protein in each of breast cancer (a) and pancreatic cancer (b) was analyzed by an immunohistochemistry method. FIG. 1-D shows the expression level of PRDM14 mRNA in various tumor cell lines derived from breast cancer and pancreatic cancer. FIG. 1-E is an image (strong magnification) obtained by analyzing the expression level of PRDM14 protein in breast cancer tissue by an immunohistochemistry method. FIG. 1-F shows the expression levels of PRDM14 in a breast cancer cell line, a PRDM14 forcibly expressing strain of the breast cancer cell line, and an expression-suppressed strain by shRNA. After immunoprecipitation with the FLAG tag carried in the expression vector, Western blot was performed with the PRDM14 antibody to detect only PRDM14 expressed by gene transfer, not endogenous PRDM14.

FIG. 2-A is a graph showing the results of MTT assay using the PRDM14-introduced strain (solid line) and the control vector-introduced strain (dotted line). FIG. 2-B is a graph showing the results of MTT assay for the purpose of evaluating the viability of breast cancer cells by treatment of PRDM14-specific siRNA alone or in combination with various anticancer agents. FIG. 2C is a graph showing the results of MTT assay for the purpose of evaluating the viability of pancreatic cancer cells treated with a specific siRNA against PRDM14 alone or in combination with an anticancer agent (GEM: gemcitabine). is there. FIG. 2-D shows the regimen of the MTT assay when the upper part uses the specific siRNA alone and the lower part uses the siRNA in combination with, for example, ADR (adriamycin) as an anticancer agent. FIG. 2-E is a flow cytometry analysis result showing that the SP fraction disappeared by treating a breast cancer cell line with a specific siRNA against PRDM14. FIG. 2-F is a flow cytometry analysis result showing that the SP fraction disappeared by treating the pancreatic cancer cell line with a specific siRNA against PRDM14. FIG. 2-G is the result of flow cytometry analysis showing that cell death was induced in a breast cancer cell line by knocking down (KD) PRDM14 with a low concentration of siRNA. The area of the right half divided into four is a cell group in which cell death was induced. Specifically, the lower right 1/4 indicates early cell death and the upper right 1/4 indicates late cell death. FIG. 2-H is an analysis result of flow cytometry showing that cell death was induced in a pancreatic cancer cell line by knocking down (KD) PRDM14 with a low concentration of siRNA. Similarly, the right half region divided into four is a cell group in which cell death was induced. Specifically, the lower right 1/4 indicates early cell death and the upper right 1/4 indicates late cell death.

FIG. 2-I shows the results of the Tumor sphere assay of PRDM14-introduced strains. The lower row shows the results of staining the formed Tumor spheres with antibodies of stem cell marker molecules (Nanog, Oct3 / 4, SSEA1, SSEA4). FIG. 2-J shows the result of the proliferation assay performed on a culture dish having general adhesiveness using the PRDM14-introduced strain. FIG. 2-K is a graph showing the expression level (mRNA) of PRDM14 after siRNA treatment of breast cancer cell lines. FIG. 2-L is the result of flow cytometry analysis showing an increase in the SP fraction in a PRDM14 stable expression strain. When analyzing SP cells, the reserpine-added analysis group serves as a negative control. In the figure, the area surrounded by the solid line is the Hoechst 33342 weakly positive or negative fraction. Since this fraction almost disappeared in the negative control containing reserpine, it was confirmed that this fraction was the SP cell fraction. FIG. 2-M is a flow cytometric analysis showing that the SP fraction disappeared in a breast cancer cell line in which PRDM14 was constantly knocked down (KD) with respect to breast cancer cells expressing PRDM14 using shRNA. The result. FIG. 2-N shows the analysis result of the flow cytometry of the breast cancer cell line which combined-treated with siRNA with respect to PRDM14 and ADR (adriamycin). FIG. 2O shows the results of flow cytometry analysis of pancreatic cancer cell lines in which siRNA against PRDM14 was treated in combination with GEM (gemcitabine).

FIG. 3-A shows the results of q-ChIP-PCR (chromatin immunoprecipitation-real-time PCR), in which the transcription factor PRDM14 is a specific transcription factor (Nanog) that is important for the maintenance of cancer stem cells. , Oct3 / 4). PRDM14 suggests the possibility of directly controlling the expression of those transcription factors. FIG. 3-B is a chart showing that PRDM14 can influence “metastasis / invasion” and “recurrence after aging” of cancer. "EMT" means epithelial-mesenchymal transition. FIG. 3-C is a graph showing the results of ELISA of RANTES / CCL5, CXCR1, CXCR2, and CXCR3 measured using the culture supernatant of a PRDM14-introduced breast cancer cell line as a sample. FIG. 3-D is a graph showing the results of ELISA of RANTES / CCL5, CXCR1, CXCR2, and CXCR3 measured using the culture supernatant of another breast cancer cell line into which PRDM14 was introduced as a sample.

FIG. 4-A is a graph showing the change over time in tumor size of a breast cancer cell line in which the PRDM14 gene was forcibly expressed. FIG. 4-B is an image of a nude mouse in which a breast cancer cell line in which the PRDM14 gene was forcibly expressed was orthotopically transplanted (mammary gland). FIG. 4-C shows pathological findings of lungs and lymph nodes obtained from nude mice orthotopically transplanted with a breast cancer cell line in which PRDM14 gene was forcibly expressed. FIG. 4-D is a graph showing the time-dependent change in tumor size of a breast cancer cell line in which shRNA for the PRDM14 gene was introduced and the PRDM14 gene was constitutively KD. FIG. 4-E shows images at the endpoint of a lung metastasis formation model (nude mouse) obtained by tail vein injection of a breast cancer cell line in which the PRDM14 gene was forcibly expressed. FIG. 4-F shows the pathological findings of the lung of a lung metastasis formation model (nude mouse) obtained by tail vein injection of a breast cancer cell line in which the PRDM14 gene was forcibly expressed. FIG. 4-G is a graph showing the change over time in tumor size when another breast cancer cell line in which PRDM14 gene was forcibly expressed was orthotopically transplanted. FIG. 4-H is an image of a nude mouse orthotopically transplanted with another breast cancer cell line in which the PRDM14 gene was forcibly expressed. FIG. 4-I is a pathological finding of a transplanted tumor piece of a nude mouse in which a breast cancer cell line in which PRDM14 gene was forcibly expressed was orthotopically transplanted, and green (fluorescence by an antibody against fragmented caspase 3) caused apoptosis. Cells (red) (fluorescence from anti-CD31 antibody) indicate blood vessels.

FIG. 5-A shows a therapeutic model in which PRDM14 gene-specific siRNA was administered alone (tumor local) with DDS agent (PEI) or combined with ADR (intraperitoneal administration) to nude mice orthotopically transplanted with breast cancer cell line. 3 is a graph showing changes in tumor volume with time in FIG. FIG. 5-B is a therapeutic model in which siRNA specific to PRDM14 gene was administered alone (tumor local) with DDS agent (PEI) or combined with ADR (intraperitoneal administration) to nude mice transplanted orthotopically with breast cancer cell lines. Is an image of. FIG. 5-C is a nude mouse treated with another breast cancer cell line orthotopically and then treated with a PRDM14 gene-specific siRNA alone (tumor local) with a DDS agent (PEI) or in combination with ADR (intraperitoneal administration). 3 is a graph showing the change over time in the tumor volume of the. FIG. 5-D is an image of nude mice treated with another breast cancer cell line orthotopically and then treated with PRDM14 gene-specific siRNA alone or in combination with an anticancer agent. FIG. 5-E is an image of nude mice treated with siRNA specific for PRDM14 gene in a lung metastasis model produced by tail vein administration of a breast cancer cell line, and pathological findings of the removed lung. FIG. 5F is a graph showing changes over time in the lung weight of nude mice treated with PRDM14 gene-specific siRNA in a lung metastasis model prepared by tail vein administration of a breast cancer cell line. Figure 5-G shows that in nude mice transplanted orthotopically with a breast cancer cell line, PRDM14 gene-specific siRNA was administered alone (intravenous injection) together with a DDS agent (calcium phosphate micelle) for intravenous administration, or docetaxel (DOCE). The administration regimen when a combination (intraperitoneal administration) model was prepared, a graph showing the change over time in the tumor volume and tumor weight obtained in the model, the weight of the obtained tumor tissue piece, and an image of the nude mouse Show. This is because the intravenous administration of siRNA is indispensable for treating metastatic lesions, and siRNA is used together with a DDS agent (calcium phosphate micelle).

FIG. 5-H shows a regimen for single administration of PRDM14 gene-specific siRNA together with a DDS agent (PEI) (tumor local injection), or concurrent administration of ADR. FIG. 5I shows a regimen for single administration (intravenous administration) of siRNA together with PEI which is a DDS agent. FIG. 5-J shows a regimen for intraperitoneal administration of siRNA together with DEI PEI as a DDS agent, or intraperitoneal administration in combination with DOC. FIG. 5-K shows the time-dependent change in tumor volume of nude mice co-treated with local administration of siRNA alone or intraperitoneal administration of docetaxel (DOC) after orthotopic implantation of a breast cancer cell line. FIG. 5-L is an image of a nude mouse that was co-transplanted with a breast cancer cell line and then treated with local administration of siRNA alone or intraperitoneal administration of docetaxel (DOC). FIG. 5-M shows the change over time in tumor weight and body weight of nude mice co-treated with siRNA-only local tumor administration or intraperitoneal administration of docetaxel (DOC) after orthotopic implantation of a breast cancer cell line. FIG. 5N is an image of a tumor graft of a nude mouse that was co-transplanted with a breast cancer cell line and then treated with local administration of siRNA alone or docetaxel (DOC) by intraperitoneal administration. FIG. 5O shows the pathological findings of tumor grafts in nude mice that were treated with local administration of siRNA alone or intraperitoneal administration of docetaxel (DOC) after orthotopic transplantation of breast cancer cell lines. FIG. 6 is a diagram in which survival rates of PRDM14-positive breast cancer cases and PRDM14-negative breast cancer cases are plotted against the elapsed time from the onset of breast cancer. FIG. 7-A shows a change over time in the size (tumor diameter) of a tumor formed by subcutaneously transplanting a pancreatic cancer cell line in which a PRDM14 gene was constitutively knocked down by introducing shRNA against the PRDM14 gene into nude mice. It is a graph showing. When shRNA # 3 is introduced, the tumor diameter finally approaches the control (cntl shRNA), but since the pancreatic cancer cell line has the differentiation ability to produce mucus, the mucus accumulates inside the tumor, Tumor size increased. Constantly suppressing the expression of PRDM14 suppresses tumor growth in an in vivo system even in a pancreatic cancer cell line. FIG. 7-B shows a mouse subcutaneous tumor image excised at the endpoint following the course in vivo in the tumorigenicity experiment (Example 11) using a pancreatic cancer cell line. FIG. 7-C is a graph showing the weight of the excised tumor shown in FIG. 7-B. With respect to the tumor weight of shRNA # 3, the tumor weight after aspirating and removing the mucus (containing no cell components) inside the tumor is shown. FIG. 7-D shows a histopathological image (HE staining) of the excised tumor shown in FIG. 7-B. Tumors with PRDM14 knockdown had significantly smaller tumor size compared to tumors formed from cells into which control shRNA (cntl shRNA) was introduced. In addition, since metaplasia and glandular secretion were observed in the tumor when PRDM14 was knocked down, it was found that the tumor has a tendency to differentiate. FIG. 8-A shows the results of flow cytometric analysis of expression of CD44 and CD24 on the cell surface of PRDM14-expressing breast cancer cell lines in which PRDM14 was knocked down by PRDM14 gene-specific shRNA. . FIG. 8-B is an index of differentiation of each cell with respect to a tumor formed by subcutaneously transplanting a breast cancer cell line and a pancreatic cancer cell line in which PRDM14 expression was reduced by shRNA, into a mouse. The result of immunostaining using a molecule-specific antibody is shown. The upper row is from a breast cancer cell line and the lower row is from a pancreatic cancer cell line. FIG. 9 is a result of flow cytometric analysis of SP fractions when PRDM14 gene expression was suppressed by PRDM14 gene-specific siRNA in a cell line of clear cell cancer (kidney cancer) expressing PRDM14. Indicates. When treated with control siRNA (left panel), about 3% SP fraction was observed, but when treated with PRDM14 gene-specific siRNA (right panel), SP fraction decreased to 0.83%. It is possible that stemness is suppressed.

  In the figure, "cntl" represents a control, "Negative cntl" represents a negative control, and "mock" represents a mock.

Hereinafter, the present invention will be described in detail.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, the terms and terms used in connection with cell and tissue culture, molecular biology, immunology, microbiology, gene and protein and nucleic acid chemistry described herein, and their techniques are well known in the art. , Shall be used normally.
Also, unless stated otherwise, the methods and techniques of the invention are described in accordance with conventional methods well known in the art to the various general and more specialized areas cited and discussed herein. As described in the other references. Such documents include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring. Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (supplement of 1992 and 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology- 4th Ed., Wiley & Sons (1999); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1990); and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1999), etc. Is mentioned.

  The terms used in analytical chemistry, synthetic organic chemistry and medicinal chemistry and medicinal chemistry as described herein, as well as their experimental procedures and techniques, are those well known and commonly used in the art. Standard techniques should be used for chemical syntheses, chemical analyses, drug manufacture, formulation and delivery, and treatment of subjects.

  In the present specification, unless otherwise specified, the name of a protein is represented by capital letters or katakana, and the gene encoding the protein is referred to as "gene" in the above capital letters or katakana. Alternatively, the above capital letters or katakana shall be underlined. Therefore, unless otherwise specified, for example, when simply described as “PRDM14”, it means a protein that is itself PRDM14 (PR domain-containing protein 14), and when described as “PRDM14 gene”, PRDM14 And the expression "PRDM14" also means the gene encoding PRDM14.

  PRDM14 used in the present invention is a protein having a PR domain and a zinc finger domain, which is called PR domain-containing protein 14 (also known as PFM11 or MGC59730), and is, for example, the same as the amino acid sequence represented by SEQ ID NO: 1 or Proteins containing substantially identical amino acid sequences are included. Here, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, more preferably about 90% with the amino acid sequence represented by SEQ ID NO: 1. Examples thereof include amino acid sequences having a homology of not less than%, more preferably not less than about 95%, and most preferably not less than about 98%.

  Further, the PRDM14 gene used in the present invention is, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a hybrid specifically with the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions. A DNA encoding a protein containing a soybean base sequence and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is included. Here, as the DNA containing the nucleotide sequence specifically hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions, for example, about 60% or more of the nucleotide sequence represented by SEQ ID NO: 2, DNA containing a nucleotide sequence having a homology of preferably about 70% or more, more preferably about 80% or more, further preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more. Can be used. Here, “stringent conditions” are usually conditions of 42 ° C., 2 × SSC and 0.1% SDS, preferably 65 ° C., 0.1 × SSC and 0.1% SDS. Is.

In the present specification, the term “cancer” is also used as a “malignant tumor”, which is not limited to “carcinoma”.
Although there may be differences in the nucleotide sequences of the PRDM14 gene due to polymorphisms, isoforms, etc., not only between different animals but also between the same animals, even if the nucleotide sequences differ, it is possible to encode PRDM14. As long as it is included in the PRDM14 gene.

  The present invention relates to a molecular marker for detecting cancer stem cells from a subject by expressing the PRDM14 gene. Since the molecular marker of the present invention can be commonly used for a plurality of cancer stem cells such as lung cancer cells, breast cancer cells, colon cancer cells, and pancreatic cancer cells, it can be used as a versatile cancer stem cell molecular marker. It is extremely effective. In addition, it is also used as a marker for determining the malignancy of the cancer tissue state by the expression of the PRDM14 gene, whether it is a state in which metastasis or recurrence is likely to be induced, or a prognostic marker for predicting the prognosis by anticancer drug treatment. be able to. Alternatively, in the anticancer drug treatment, it may be used as a marker for judging the timing of switching from one anticancer drug to another anticancer drug, using an increase in the expression of PRDM14 gene as an index.

  In the present invention, “expression of a gene” refers to a series of biological reactions that start from the transcription of the gene and include translation. For quantifying the expression level of mRNA encoding PRDM14, known techniques such as hybridization techniques (Northern hybridization method, dot hybridization method, RNase protection assay, cDNA microarray, etc.), gene amplification technology (reverse transcription polymerase chain reaction) are used. A reaction (RT-PCR) (including competitive RT-PCR, real-time PCR, etc.) and the like can be used. When a hybridization technique is used, an oligonucleotide or a polynucleotide capable of hybridizing to a polynucleotide encoding PRDM14 or a part thereof can be used as a probe, and when a gene amplification technique is used, the oligonucleotide can be used. Alternatively, the polynucleotide can be used as a primer. The “polynucleotide encoding PRDM14 or a part thereof” includes both DNA and RNA, and includes, for example, mRNA, cDNA, cRNA and the like. Therefore, the nucleotides constituting the oligonucleotide or polynucleotide may be either deoxyribonucleotides or ribonucleotides. In quantifying the expression amount of mRNA encoding PRDM14 in the present invention, the base length of the oligonucleotide used is not particularly limited, but is usually 15 to 100 bases, preferably 17 to 35 bases. The base length of the nucleotide is not particularly limited, but is usually 50 to 1000 bases, preferably 150 to 500 bases.

  The “gene expression” according to the present invention can also include a humoral factor whose expression is induced by the expression of PRDM14. The expression of PRDM14 can also be measured via these humoral factors. The humoral factors that can indirectly detect and measure the expression of PRDM14 include CCL5, GRO (Growth Related Oncogene, or CXCL1, 2, 3), solubilized CD40L, and the like. These humoral factors can also be used as "serum markers" for detecting the expression of PRDM14, that is, for detecting the state in which cancer stem cells are induced.

  In quantifying the expression level of PRDM14, known techniques and known protein analysis techniques, for example, Western blotting method based on antigen-antibody reaction using anti-PRDM14 antibody or a fragment thereof, dot blotting method, immunoprecipitation method, ELISA, immunization A histochemical staining method (IHC) or the like can be used. “Significantly high expression of a gene” means that the expression level of mRNA (expression level) and / or the expression level of a polypeptide including a protein (expression level) is statistically significant as compared with a control. High (high), or preferably at least 1.5 times, more preferably at least 2 times, further preferably at least 3 times, most preferably at least 5 times higher (high). The expression level of the PRDM14 gene is based on the expression level of this internal standard gene with a gene (for example, a housekeeping gene such as β-actin gene or GAPDH gene) whose expression level does not vary significantly between tissues or individuals as an internal standard gene. It is preferable to correct it.

  "Cancer stem cell" refers to a cell having the properties of a stem cell among cancer cells. Stem cells are cells that maintain their differentiation potential even after undergoing cell division. Cancer stem cells were stained with Hoechst fluorescent dye (Hoechst33342), and detected by using a UV laser (wavelength of about 350 nm) as excitation light by using flow cytometry. Side Population (SP) fraction Is concentrated to. The SP fraction is a fraction that is not stained by excreting the dye to the outside of the cell via the ABC transporter or the like with respect to the main population (MP) fraction that is stained by the Hoechst fluorescent dye. It means that. Stem cells can also be identified by marker molecules such as Oct3 / 4, Nanog, SSEA1 and SSEA4. On the other hand, the “normal cell” refers to a cell having a normal function in the activity of a living body or a tissue. Normal cells may include somatic stem cells, but are preferably mature cells.

  The molecular marker of the present invention preferably contains a PRDM14 gene product or a fragment thereof, more preferably the PRDM14 gene product is mRNA and / or polypeptide. The “gene product” refers to a molecule produced by a series of biological reactions associated with gene expression, such as mRNA and endogenous polypeptide (further, protein).

A "subject" is preferably a breast, lung, esophagus, stomach, large intestine, liver, pancreas, cervix, from any living individual, preferably a vertebrate, more preferably a mammal, even more preferably a human individual. A cell population derived from one or more cells or tissues selected from the group consisting of endometrium, ovary, kidney, prostate, bladder, testis, thyroid, adrenal gland, lymph node, blood and lymph. The "living individual" may be healthy or may be suffering from any disease, but when treatment for various cancers is intended, the living individual suffering from cancer is Or organisms affected experimentally, for example, rodents such as mice, rats, gerbils, guinea pigs, cats such as cats, pumas, tigers, deer, deer animals such as deer, rabbits, dogs, etc. , Mink, sheep, goat, cow, horse, monkey, human and the like are preferable.
As "cancer", among the cancers derived from these "subjects", preferably solid cancer, more preferably solid cancer consisting of breast cancer, pancreatic cancer and renal cancer, or refractory cancer. (Breast cancer, pancreatic cancer, biliary tract cancer) is preferable.

  In the present specification, "expressed" means that the gene product can be confirmed by a method known to those skilled in the art, such as RT-PCR, in situ hybridization, immunoassay, chromatography and the like. . In addition, “not detected” means that the gene product cannot be confirmed by the above method for confirming the gene product.

  The present invention also relates to a method of determining the presence or absence of cancer stem cells or determining the degree of cancer stem cell induction in cancer in the subject using the molecular marker as an index. The “determination” is determined to include cancer stem cells when the molecular marker of the present invention is detected, and the subject to be determined may include normal cells and / or cancer cells that are not cancer stem cells. Further, in the “determination of the degree of induction of cancer stem cells in cancer”, for example, as shown in FIG. 1-C, the expression level of PRDM14 protein is increased along the stage classification (clinical progression stage classification) of cancer. Therefore, the change over time in a subject derived from the same tissue or organ of the same individual corresponds to the degree of cancer stem cell induction. However, in breast cancer, the expression level of the PRDM14 protein may not be increased along the stage classification of cancer, which is due to the nature of breast cancer in the background of cancer, that is, estrogen receptor, progesterone receptor that is a triple marker, This is because they are subdivided on the basis of combinations of HER2 molecules and the like and cannot be discussed in a uniform manner (populations with different biological backgrounds). In addition, since PRDM14 has no strong correlation with these molecules, it is presumed that improvement in survival rate can be expected in both cases of triple negative breast cancer for which no existing treatment method has been established up to now, or in combination with other categories. On the other hand, in pancreatic cancer, it correlates with cancer staging.

  The determination may be an in vivo or in vitro determination. As used herein, the term “in vitro determination” refers to determination of a tissue or cell collected from a living body after it has grown in an in vitro environment such as a culture solution. On the other hand, the “in vivo determination” means a determination directly in a living body or a determination immediately after or after immobilization of a tissue or a cell from a living body. Tissues or cells are collected by, for example, but not limited to, incision, cell aspiration, blood collection, and urine collection. When making the determination in vivo, in vivo detection methods known to those skilled in the art may be used. The in vivo determination may be performed using a known detection method such as blood test, in situ hybridization, in situ PCR, immunohistostaining and the like. When the determination is performed in vitro, it may be performed using an in vitro detection method known to those skilled in the art, such as, but not limited to, immunohistological staining and RT-PCR. When performing RT-PCR, the number of cycles is preferably 30 to 35 cycles. In vitro determination also includes determination of tumor tissue after tissue culture.

  When the molecular marker contains a PRDM14 gene product or a fragment thereof, the determination method of the present invention preferably includes a step of detecting the gene product by mRNA and detecting the gene product by an RT-PCR method. In this case, a reagent such as a probe or a primer that specifically binds to mRNA is used.

  Further, when the molecular marker contains the PRDM14 gene product or a fragment thereof, it is preferable that the PRDM14 gene product is a polypeptide, and the step of detecting the gene product with a reagent that specifically reacts with the gene product is included. . On the other hand, when the molecular marker contains a humoral factor, it preferably includes a step of detecting the humoral factor with a reagent that specifically reacts with the humoral factor. For their detection, a reagent that specifically binds to a peptide such as an antibody or a ligand is used. For example, the antibody may be a polyclonal antibody and / or a monoclonal antibody.

  The present invention relates to a kit for determining the presence or absence of cancer stem cells, the kit including at least a reagent for detecting the molecular marker. As a "reagent for detecting", for example, a probe and / or primer having a base sequence complementary to the PRDM14 gene for detecting mRNA which is a product of the PRDM14 gene is a polypeptide which is a product of the PRDM14 gene. The antibody may be an antibody or a ligand for detecting the protein, or an antibody for detecting a polypeptide that is a humoral factor. The "probe and / or primer having a base sequence complementary to a gene" is a DNA or RNA having a complementary sequence so as to specifically bind to a partial sequence of the gene sequence, for example, fluorescent light. It may be optionally labeled with a label, a radiation label, or the like. In addition, the kit may include ancillary reagents suitable for the mode of use, such as a reaction buffer and a reaction accelerator.

  By detecting the detection of cancer stem cells or the induction of cancer stem cells using the kit, whether the target is cancer, or if it is cancer, its prognosis / year-old recurrence risk and cancer in cancer A method for determining the degree of stem cell induction is also included in the present invention.

  The present invention also relates to a nucleic acid used for suppressing the expression of PRDM14 gene in cancer stem cells, which nucleic acid exerts an inhibitory action in the process of transcription of PRDM14 gene into mRNA or translation of mRNA into protein. Examples include nucleic acids that inhibit the expression of a polynucleotide that encodes a protein or a peptide that is a part thereof that contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. it can. Here, the "polynucleotide encoding a protein or a peptide which is a part thereof" containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 may be DNA or RNA. May be.

  Such nucleic acid is preferably one or more selected from the group consisting of antisense, siRNA and shRNA. siRNA is a contiguous 18 sequence of a polynucleotide RNA (for example, mRNA of PRDM14) that encodes a protein having the same or substantially the same amino acid sequence as SEQ ID NO: 1 or a peptide that is a part thereof. It is preferable to include a sense strand sequence of .about.28 nucleotides and an antisense strand sequence which is a complementary sequence thereof, and further comprising a sense strand sequence of 19 to 24 nucleotides continuous from the polynucleotide RNA and a complementary sequence thereof. It is particularly preferable that it contains a certain antisense strand sequence. Known knowledge can be used to select suitable siRNA sequences (eg, Dykxhoorn DM et al., Nature Rev. Mol. Cell Biol. 4; 457-67 (2003); Khvorova A et al., Cell, 115: 209-16 (2003)).

Examples of preferred siRNA sequences in the present invention are shown in Table 2 below.

  The present invention further contemplates that the selected siRNA sequences include deletions, substitutions or additions of 1-3, preferably 1-2, nucleotides in the sequence so long as they allow cleavage of the target mRNA. It may be mutated. The mutation is preferably located on the 5'side, since the mutation on the 3'side from the center tends to cause inactivation. It should be noted that the selected siRNA is one that does not show a so-called off-target effect (the effect of suppressing the expression of a gene other than the target gene partially homologous to the used siRNA) during clinical use. preferable. Therefore, in order to avoid the off-target effect, it is desirable to confirm in advance that there is no cross-reactivity for the candidate siRNA by using a gene chip or the like.

  The shRNA that can be used in the present invention contains a single-stranded loop sequence that covalently binds between a sense strand sequence and an antisense strand sequence thereto, and is processed by the intracellular RNase Dicer. siRNA is RNA that is formed. At the 3 ′ end of the hairpin-type DNA encoding siRNA, a poly T sequence consisting of 1 to 6, preferably 1 to 5 Ts, for example, 4 or as a transcription termination signal sequence or due to overhang. TTTTs or TTTTTTs composed of 5 Ts are connected. The shRNA as a siRNA precursor transcribed from the vector DNA desirably has an overhang consisting of 2 to 4 Us at the 3 ′ end of its antisense strand. Due to the presence of the overhang, sense RNA and antisense RNA can have increased stability against degradation by nucleases. In the present invention, a known loop sequence can be appropriately used as the single-stranded loop sequence.

  Another embodiment of siRNA in the present invention is formed from tandem DNA, which comprises a DNA sequence encoding the sense strand and a DNA sequence encoding the antisense strand in the 5 ′ → 3 ′ direction. Sense RNA and antisense RNA, which are consecutively contained, and which consist of a sequence in which a promoter is linked to the 5'end of each chain and a poly-T sequence is linked to the 3'end of each chain, and which are produced simultaneously after transcription in cells. And hybridize together to form siRNA. It is preferable that the poly T sequence is composed of 1 to 5, and especially 4 to 5 Ts as a transcription termination signal sequence, as in the above. Further, similarly to the hairpin type, the generated siRNA may have an overhang composed of 2 to 4 U at the 3'end of the sense and / or antisense strand.

  In addition, antisense DNA that specifically inhibits the expression of PRDM14 gene can be integrated into the downstream of an appropriate promoter sequence and administered to a subject as an antisense RNA expression vector. Furthermore, an antisense RNA expression vector, siRNA expression vector, or shRNA expression vector that specifically inhibits the expression of PRDM14 gene can be prepared, and these vectors can be administered to a subject. That is, in the present invention, a DNA encoding an antisense RNA, siRNA or shRNA that specifically inhibits the expression of the PRDM14 gene is incorporated into an expression vector, and the expression of the PRDM14 gene is specifically regulated under the control of an appropriate promoter. Is transcribed into RNA that inhibits. Expression vectors used in the present invention include plasmid vectors and viral vectors.

  A plasmid vector can be prepared using a known method, and a commercially available vector, for example, psiHIV-U6 (backbone of a lentivirus shRNA expression vector for PRDM14 gene; GeneCopoea), pReceiver-Lv102 (PRDM14 gene). Backbone of lentivirus expression vector EX-W1089-Lv102; GeneCopoea), piGENE (registered trademark) U6 vector, piGENE (registered trademark) H1 vector, piGENE (registered trademark) tRNA vector (Takara Bio Inc.), etc. (Brummelkamp TR et al., Science, 296: 550-3, 2002; Lee NSet al., Nature Biotechnology, 20: 500-5, 2002; M. Miyagishi et al., Nature Biotechnology, 20: 497- 500, 2002; Paul CPet al., Nature Biotechnolog y, 20: 505-8, 2002; edited by Yoshinori Tahi et al., RNAi experiment protocol, Yodosha, 2003, etc.).

  In general, a plasmid vector can contain a drug resistance gene, a transcription termination sequence, a restriction enzyme cleavage site, a replication origin, etc., in addition to a DNA sequence and a promoter encoding the antisense RNA, siRNA, shRNA of the present invention.

  Further, as the viral vector, for example, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, etc. can be used. The viral vector is preferably modified so that it lacks the self-replicating ability. Methods for producing viral vectors are known to those skilled in the art. The above viral vector can be introduced into cells by directly injecting the vector into the affected area and infecting the cells.

  The present invention also relates to pharmaceutical compositions containing the nucleic acids. The pharmaceutical composition of the present invention preferably further contains an anticancer drug or a cancer vaccine, and if necessary, for example, a drug having an antitumor effect, an adjuvant, a pharmaceutically acceptable carrier (for example, Agents, binders, disintegrants, lubricants, stabilizers, preservatives, pH adjusters, flavoring agents, diluents, solvents for injections, etc.) and the like. Such a pharmaceutical composition is preferably for treating cancer or preventing cancer. In addition, the pharmaceutical composition of the present invention may contain a label, a nanocapsule, or the like that enables specific delivery to a target tissue.

  As the "anticancer agent", a known anticancer agent effective for treating various cancers, for example, adriamycin (ADR), gemcitabine (GEM), fluorouracil, tamoxifen, anastrozole, aclarubicin, doxorubicin (DOC) , Tegafur, cyclophosphamide, irinotecan, cytarabine, paclitaxel, docetaxel, epirubicin, carboplatin, cisplatin (CDDP), thiotepa, or pharmaceutically acceptable salts thereof.

  Examples of the administration route of the pharmaceutical composition of the present invention include oral administration and parenteral administration (for example, intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, local administration, etc.), Examples of dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections and suspensions. For example, in the case of local administration, the affected part can be exposed by surgery and the therapeutic agent of the present invention can be directly administered to the cancer tissue by means of a syringe or the like, and in the case of non-local administration, an optimal DDS agent Can be performed by a simple route of intravenous administration (by intratumoral administration of tumor feeding). The preferred routes of administration are intravenous and topical.

  Although the dose and the number of administrations vary depending on the intended action and effect, administration method, treatment period, age, weight, sex, etc. of an individual organism, expression of antisense DNA, antisense RNA, siRNA, shRNA against PRDM14 gene or these is expressed. When the vector is used, the dose is appropriately selected from the range of usually 0.01 mg / kg to 1 g / kg, preferably 0.1 mg / kg to 500 mg / kg per day for an adult when the living individual is a human. The number of administrations can be appropriately selected from the range of once to several times a day. However, the dose is not limited to the above range because it may vary depending on various conditions.

  Furthermore, the present invention relates to a method of inhibiting the function of cancer stem cells using the nucleic acid. Nucleic acid that also functions as a molecular marker of the present invention has a sequence complementary to a part or all of mRNA specifically expressed in cancer stem cells, and suppresses the expression, thereby having the function of cancer stem cells. It is thought that this can be suppressed. Therefore, a nucleic acid for suppressing the expression of DNA that functions as the molecular marker of the present invention is also included in the present invention.

  Methods for suppressing the expression include, but are not limited to, for example, expression of RNAi and repressor. The nucleic acid may have any length as long as it has a sufficient number of bases to suppress the expression of DNA.

The nucleic acid may be a nucleic acid analog in addition to DNA and RNA, but is preferably DNA and / or RNA from the viewpoint of versatility and the like.
This suggests that cancer stem cells may be specifically and / or efficiently attacked by suppressing the expression of DNA that functions as the molecular marker of the present invention. That is, it is suggested that the above nucleic acid can be applied to gene transfer therapy.

  When antisense, siRNA or shRNA that specifically inhibits the expression of PRDM14 gene is used, it can be formulated by a known method and administered to an individual organism. For example, antisense RNA or siRNA that specifically inhibits the expression of PRDM14 gene can be directly introduced into a target tissue, and in such a case, they can be introduced into, for example, Lipofectamine (registered trademark), Lipofectin. (R) and Cellfectin (R) (Invitrogen) and other liposomes and ion complexes (JP 2011-010549 JP, JP 2010-233499 JP, WO2009 / 113645, WO2008 / 062909) and the like to form a complex It can also be injected.

  The following experimental examples describe the present invention more specifically, and do not limit the scope of the present invention in any way. Those having ordinary knowledge and skills as those skilled in the art can make various modifications to the embodiments shown in the following experimental examples without departing from the spirit of the present invention. include.

<Example 1> Gene expression analysis (1) PRDM14 gene expression in cancer patient specimens (Fig. 1-A)
To investigate the gene expression profile of PRDM14 in malignant tumor, breast cancer (normal tissue: 4, tumor tissue: 24), lung cancer (normal tissue: 4, tumor tissue: 19), esophageal cancer (normal tissue: 3, tumor tissue: 18), gastric cancer (normal tissue: 5, tumor tissue: 14), colon cancer (normal tissue: 7, tumor tissue: 13), liver cancer (normal tissue: 3, tumor tissue: 17), pancreatic cancer ( Normal tissue: 5, tumor tissue: 17), cervical cancer (normal tissue: 4, tumor tissue: 9), uterine body cancer (normal tissue: 5, tumor tissue: 17), ovarian cancer (normal) Tissue: 3, tumor tissue: 21), kidney cancer (normal tissue: 5, tumor tissue: 18), prostate cancer (normal tissue: 5, tumor tissue: 21), bladder cancer (normal tissue: 2, tumor) Tissue: 22), testicular tumor (normal tissue: 6, tumor tissue: 19), thyroid cancer Normal RNA: 3, tumor tissue: 18), adrenal cancer (normal tissue: 5, tumor tissue: 10), lymphoma (normal tissue: 3, tumor tissue: 34), total RNA was extracted and cDNA was extracted. The PRDM14 gene was synthesized and analyzed for expression of the PRDM14 gene using a ViiA7 real-time PCR analyzer (Lifetechnology) by the comparative ΔCt (delta / delta Ct) method using the cyber green method. The β-actin gene was used as an internal standard.

  PRDM14 expression was increased at the mRNA level in breast cancer, lung cancer, esophageal cancer, pancreatic cancer, cervical cancer, ovarian cancer, renal cancer, urinary system tumor, and testicular tumor. In particular, there was a marked increase in breast cancer, lung cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, and testicular tumor. In cancer tissues where these increases were remarkable, it is easily inferred that the degree of induction of cancer cells into cancer stem cells is high due to the expression of the PRDM14 gene. It is not strange that there is cancer tissue whose expression level is reversed in the normal part and the tumor part because it is contained only.

(2) Expression of PRDM14 gene in breast cancer patient samples (Fig. 1-B)
In addition to (1) above, in order to examine the gene expression profile of PRDM14 in breast cancer, total RNA obtained from 177 cases of invasive breast cancer breast cancer tissue and 5 cases of normal mammary gland tissue was synthesized, cDNA was synthesized, and the cyber green method was used. By the comparative ΔCt method used, the expression of PRDM14 gene was analyzed using a ViiA7 real-time PCR analyzer (Lifetechnology). The β-actin gene was used as an internal standard.
Increased expression was observed in 148/177 cases of breast cancer compared to normal mammary gland.
Further, as shown in FIG. 6, it was found that the PRDM14-positive breast cancer cases have a poorer prognosis than the PRDM14-negative breast cancer cases.

[Regarding qRT-PCR reaction]
The primers for PRDM14 and β-actin were both purchased from Origene, and hPRDM14 was Origene's HK205840, β-actin was Origene's HP204660, and GAPDH was Origene's HP205798.
The PCR reaction was performed at 96 ° C. for 5 seconds at 60 ° C. with a total of 25 μL of a solution containing 5 μl of cDNA, 12.5 μl of 2 × Power SYBR (registered trademark) PCR Master Mix (Life Technologies), 1 μl of primer (10 μM) and 6.5 μl of water. It was carried out for 15 seconds and 40 cycles. The experimental results showed the expression of PRDM14 gene with respect to the ratio of the expression amount of PRDM14 gene to the expression amount of β-actin gene (that is, the expression amount of PRDM14 gene / the expression amount of β-actin gene).

(3) Expression analysis of PRDM14 gene in tumor cell lines (Fig. 1-D (a) (b))
Expression analysis of the PRDM14 gene was performed using cell lines derived from breast cancer and pancreatic cancer, the expression of which is enhanced in clinical cases and the involvement of cancer stem cells is concentrated. Breast cancer cell lines BT474, BT549, MCF7, MDA-MB-231, MDA-MB-436, MDA-MB-468, HCC1937, SKBr-3 and T-47D, pancreatic cancer cell line PK-1, Total RNA was similarly extracted from Panc-1 and BxPC-3, and MCF10A which is a mammary gland cell line (non-neoplastic). Using the total RNAs obtained from these, the gene expression of PRDM14 was analyzed by the real-time PCR method as in the above (1) and (2). Using the GAPDH gene as an internal standard, the expression of the PRDM14 gene in each sample is shown in FIG. 1-D according to the ratio of the expression amount of the PRDM14 gene to the expression amount of the GAPDH gene (that is, the expression amount of the PRDM14 gene / the expression amount of the GAPDH gene). Shown in (a) and (b) (experiments were performed three times for each sample).

<Example 2> Detection of PRDM14 protein by immunostaining / western blotting 161 Japanese breast cancer patients (stage 0: 20 cases, stage I: 42 cases, stage II: 50 cases, stage III: 38 cases) , Stage IV: 11 cases) surgical specimen cancer tissue was fixed with formalin and paraffin sections were prepared by a conventional method, and surgical specimens obtained from pancreatic cancer patients (stage II: 9 cases, stage) III: 147 cases, stage IV: 13 cases), using 4 anti-PRDM14 antibodies (ABGENT, AP1214A, 50 times) as the primary antibody against 169 Tissue Microarray (Biomax US, Accumax) samples. After overnight incubation at ℃, goat anti-rabbit antibody labeled with streptavidin (DAKO) was used as the secondary antibody. Immunostaining was performed using EnVision-Plus (DAKO). The tissue of the normal part of the above case was used as a control. The nuclear staining was performed using hematoxylin.

The results are shown in Fig. 1-C (magnification is 400 times). In the case where the expression of PRDM14 gene is increased, it was confirmed that the expression of PRDM14 was also increased, and the increase in gene expression caused the increase in protein expression.
Moreover, the expression of the PRDM14 gene product of the cancer cell line was confirmed by Western blotting (FIG. 1-D (c)). After SDS-PAGE, blocking was performed with 5% BSA, and after incubation overnight at 4 ° C. with an anti-PRDM14 antibody (Millipore, # AB4350, 1000 times) as a primary antibody, a HRP-labeled secondary antibody (anti-rabbit antibody) was used. : GE, NA9340-1ML) according to a standard method to detect the expression of PRDM14 gene product.

<Example 3> Involvement of PRDM14 in cell proliferation In order to clarify the role of PRDM14 in carcinogenesis, cell proliferation when PRDM14 gene was introduced into a breast cancer cell line was examined.
The lentivirus expression vector EX-W1089-Lv102 (GeneCopoeia) of PRDM14 gene was used. The vector is a breast cancer cell line with extremely low expression of MDA-MB-231 cells, medium expression of HCC1937 cells, high expression of MCF7 cells, and non-expression non-cancerous cells of MCF10A cells. The cells into which the gene was introduced by the method and the construct was introduced were selected with the drug marker Puromycin, and mRNA was detected by the real-time PCR method, and the protein expression was detected by the FLAG tag to confirm the introduction (Fig. 1-F (a )).

Similarly, lectin virus shRNA expression vector (psiHIV-U6) for PRDM14 gene was introduced into HCC1937 cells with moderate expression and MCF7 cells with high expression, and cells into which the construct had been introduced were selected by the drug marker Puromycin. Further, the cells were sorted by EGFP using a cell sorter FACS Aria (BD) multiple times. Finally, mRNA was detected by the real-time PCR method (FIG. 1-F (c) (d)) and protein expression was detected by the western blotting method (anti-PRDM14 antibody (Millipore, # AB4350)) to knock the gene. The down efficiency was confirmed. The cntl sh and shRNA # 1 to 4 used were all manufactured by Genecopoeia, and their sequences are shown in the table below.

Using the transgenic strain, the viability of the cells was measured by the MTT method, and the colony forming ability was analyzed. The cells were seeded on the culture dish with the constant expression strain, and after 3 weeks, the cells were fixed with methanol and stained with Giemsa solution, and the number of colonies was measured. As a control, cells into which only the backbone expression vector was introduced were used to compare the colony forming ability.
The results are shown in FIGS. 2-A and 2-J.

<Example 4> Effect on proliferation of breast cancer cells in therapeutically targeting PRDM14 In order to analyze in detail whether PRDM14 can be applied to a therapeutic target, breast cancer cell lines MDA-MB-231, HCC1937, and MCF7. Was transfected with siRNA against PRDM14 gene to examine whether or not it exhibits an antitumor effect, and further examined the action of siRNA against PRDM14 gene in combination with an anticancer agent.

Seven types of siRNAs against the PRDM14 gene were selected by an in silico program, their gene expression suppressing effects were treated with siRNAs in the above three types of breast cancer cells, and 72 hours later, total RNA was extracted according to a standard method, and the qRT-PCR method was used. As a result of examination, the sequences that exhibited common effects in these breast cancer cells were three sequences of siRNA # 2, siRNA # 3, and siRNA # 5 (Fig. 2-K).
Of these, siRNA # 5 for the 3-UTR sequence was excluded. This is because the phenomenon that the 3-UTR sequence is shortened in many tumors is known, and it has been reported that the tumor is not affected by the endogenous miRNA. It was decided to use siRNA # 2, siRNA # 3 for the coding region of the gene (their sequences are shown in Table 2). The control siRNA used was "Universal Negative" manufactured by RNAi Co., Ltd.).

  When the cancer cells were seeded on the plate, the above siRNA (5 nM) was introduced into the cells by the reverse transfection method using RNAiMAX reagent (Lifetechnologies). After 24 hours, the medium was replaced with a normal medium, and 72 hours later, the medium was subjected to various assays. When used in combination with an anticancer drug, cisplatin (CDDP) (25 μM), adriamycin (1 μM) and docetaxel (0.5 μM) were added to the medium after 48 hours, and 72 hours later, the cells were added by the MTT method. The survival rate was measured. The results are shown in Figure 2-B.

  In the cell line in which the PRDM14 gene expression was transiently reduced by a low concentration of siRNA, the cell survival rate was lower than that of the control cell. Furthermore, when a low-concentration anticancer drug generally used in a cultured cell line was used for a short time, the cell viability was synergistically decreased. Therefore, it is suggested that the expression of PRDM14 is involved in the decrease in anticancer drug sensitivity (FIG. 2-B).

<Example 5> Effect on proliferation of pancreatic cancer cells in therapeutically targeting PRDM14 By the same method as in Example 4, three types of pancreatic cancer cell lines used in Example 1 (3) were used. Similar experiments were conducted. At that time, the concentration of gemcitabine at which approximately half of the cells survived was previously determined using a gemcitabine hydrochloride solution. When siRNA against PRDM14 was used to knockdown the PRDM14 gene in three types of pancreatic cancer cell lines that overexpress PRDM14, and when gemcitabine hydrochloride solution was used in combination, the final concentrations were 10 μM, 5 μM, and 5 μM, respectively. Was added to. As a result, as in the case of the breast cancer cell line, in the cell line in which the PRDM14 gene expression was transiently decreased by the low concentration of siRNA, the cell viability was decreased as compared with the control cell. . Furthermore, when gemcitabine hydrochloride solution was used together for a short time, the viability of cells was synergistically decreased. Therefore, it is suggested that the expression of PRDM14 is involved in the reduction of anticancer drug sensitivity in pancreatic cancer cells (FIG. 2-C).

<Example 6> Analysis of SP fraction of cancer cells:
Cancer stem cells are stained with Hoechst fluorescent dye (Hoechst33342), and when detected by using a UV laser (wavelength of about 350 nm) as excitation light using flow cytometry, they are concentrated in Side Population (SP) fractions. The SP fraction refers to a fraction that is not stained by discharging the dye to the outside of the cell via the ABC transporter or the like with respect to the Main Population (MP) fraction stained with the Hoechst fluorescent dye.

(I) Preparation of Reagent As a medium, a DMEM medium containing 5% anti-fetal calf serum (FCS) was prepared and warmed to 37 ° C. Verapamil was adjusted to 50 mM and diluted to 5 mM with 5% FCS + DMEM. Hoechst 33342 was adjusted to 250 μg / mL with 5% FCS + DMEM.

(Ii) Preparation of cells for flow cytometry (FACS) Cells were suspended in 4 mL of 5% FCS + DMEM and the number of cells was counted. Further, 5% FCS + DMEM was added to adjust the cell concentration to 1 × 10 ⁶ cells / ml, and 1 mL was collected in a Falcon tube for verapamil (+). Verapamil (+) and the remaining cells (verapamil (-)) were incubated in a water bath at 37 ° C for 10 minutes. After the incubation, a verapamil solution was added to verapamil (+) so that the final concentration of verapamil was 50 μM, and then, for verapamil (+) and verapamil (−), the final concentration of Hoechst 33342 was 2.5 μM to 5 μM. Hoechst 33342 solution was added so as to have a concentration of 0.0 μM.

  The plate was incubated with shaking at 37 ° C. for 90 minutes, and immediately cooled on ice. The supernatant was removed by centrifugation at 800 rpm and 4 ° C. for 3 minutes. The cells were suspended in 1 × PBS + 5% FCS solution and transferred to an ice-cooled FACS tube. The supernatant was removed again by centrifugation at 800 rpm and 4 ° C. for 3 minutes, and the cells were suspended in 1 × PBS + 5% FCS solution. The supernatant was removed by centrifugation again in the same manner, and suspended in 500 μL of IX PBS + 5% FCS to which EDTA was added so that the final concentration was 2 mM. It was pipetted and passed through a FACS cell strainer. 0.5 μL of 1 mg / mL propidium iodide (PI) was added and subjected to FACS at a flow rate of 1000 cells / sec.

(Iii) Flow cytometry (FACS)
As the flow cytometer, BD FACS Aria (manufactured by BD) was used. The operation of FACS was performed according to the instruction manual. First, verapamil (-) cells are flown to check whether the SP fraction cells can be detected. If confirmed, the SP fraction is gated and verapamil (+) cells are flown to sprinkle the SP fraction cells. I confirmed that it disappeared. If the cells disappeared, the cells in the fraction were determined to be SP fraction cells, and the number and ratio of cells in the fraction were analyzed by FlowJo software (Tomy Digital Biology).

  Three types of siRNA # 2 and siRNA # 3 used in breast cancer cell line MCF7, HCC1937 cells (FIG. 2-E), and Example 1 (3) for the purpose of analyzing the SP fraction by PRDM14 gene knockdown were used. 72 hours after introduction into the pancreatic cancer cell line (Fig. 2-F) and further into the kidney cancer cell line CaKi-1 cells (Fig. 9), the SP fraction was analyzed by the above method. Separately, the PRDM14 gene constant expression strain prepared by the above method and the breast cancer cells whose gene expression was constantly decreased by the shRNA vector were also analyzed (FIG. 2-M). In Figure 2-L, reserpine was used to quench the SP fraction.

The above siRNA (5 nM) was introduced into the cells by reverse transfection using RNAiMAX reagent (Lifetechnologies) when the cancer cells were seeded on the plate. After 24 hours, the medium was replaced with a normal medium, and 72 hours later, the medium was subjected to various assays (Fig. 2-E, F).
In the combined use with an anticancer agent, adriamycin (1 μM) or gemcitabine hydrochloride solution (5 μM) was added 48 hours later, and analysis was performed 72 hours later.

<Example 7> Analysis of apoptosis of cancer cells Target cancer cells were stained with Annexin V which can detect early apoptosis and PI (propidium iodide) which can detect late apoptosis.
The cells were washed twice with cold PBS and resuspended in 1 × Binding buffer to a cell concentration of ˜1 × 10 ⁶ cells / mL. 100 μL of cell suspension (˜1 × 10 ⁵ cells) was added to a ⁵ mL Falcon tube, and FITC-labeled annexin V reagent (5 μL) and PI (2 μL) were added to each test tube. The test tubes were gently mixed, incubated at room temperature in the dark for 15 minutes, 400 μL of 1 × Binding buffer was added to each test tube, and the measurement was performed with a flow cytometer within 1 hour.

When the cancer cells were seeded on the plate, the siRNA (5 nM) was introduced into the cells by reverse transfection using RNAiMAX reagent (Lifetechnologies). After 24 hours, the medium was replaced with a normal medium, and 72 hours later, the medium was subjected to various assays (Fig. 2-G, H).
When combined with an anticancer agent, adriamycin (1 μM) or gemcitabine hydrochloride solution (5 μM) was added 48 hours later, and analysis was performed 72 hours later (FIG. 2-N, O).

<Example 8> Tumor sphere assay using cancer cells:
Sphere assay was performed using breast cancer cell lines. Using an Ultra-Low Attachment 6 well plate (Corning) that does not attach epithelial cells by specially processing the plate surface, epidermal growth factor (EGF) against 25 mL of serum-free medium (F12 medium) 20 ng / mL and basic fibroblast growth factor (bFGF) were added so as to be 20 ng / mL, and further, 0.5 mL of B27 supplement (Life Technologies) was added, and the mixture was cultured for 2 weeks to be spherical. It was investigated whether or not colonies were formed.

  As a result, the sphere-forming ability of breast cancer cells (MDA-MB-231, HCC1937) into which the PRDM14 gene had been introduced was enhanced, and an increase in stem cell markers was observed. Specifically, fluorescent antibodies (R & D Systems, Human Embryonic Stem Cell, SC008) against Nanog, Oct3 / 4, SOX2, SSEA1, and SSEA4 were immunostained according to the protocol, and observed with a confocal microscope. The enhanced expression of Nanog, Oct3 / 4, SSEA1, and SSEA4 at the protein level was observed (Fig. 2-I).

<Example 9> Change in expression of a gene responsible for stem cell property by PRDM14 gene From the above, by introducing the PRDM14 gene into tumor cells, the SP fraction increased, resistance to apoptosis, and further enhancement of sphere-forming ability were observed. It was It was also found that suppressing the expression reduces the SP fraction, makes it susceptible to apoptosis, and ultimately enhances drug sensitivity.
Therefore, in order to identify a gene regulated by the PRDM14 gene, a quantitative RT-PCR method using tRNA extracted from a breast cancer cell line (MCF7, HCC1937, MDA-MB-231, MCF10A) into which the PRDM14 gene was introduced. was carried out based the ^{RT 2 Profiler PCR Arrays (QIAGEN)} . Specifically, we conducted a panel of apoptosis-related molecules, drug resistance-related molecules, and stem cell-related transcription factors. Some of the results are shown below (Table 4).

  A ChIP assay was also performed to verify whether it was a direct transcriptional regulation of the PRDM14 gene product for some of the target genes. Specifically, for the purpose of expressing a fusion protein of Halo tag and PRDM14 in breast cancer cell lines MDA-MB-231 and HCC1937 cells, lentivirus vector EX-W1089-Lv110 (GeneCopoeia) was introduced into those cells, and puromysin was introduced. After selection with, the positive cells were selected by detecting the Halo tag by Western blotting. Using these cells, a fragment of genomic DNA to which the PRDM14 gene product binds was obtained by immunoprecipitation according to the HaloCHIP ™ System (Promega) protocol. Quantitative PCR was performed using these DNA fragments as templates (q-ChIP-PCR method). The primers used were EpiTect ChIP qPCR Primer Assay For Human NANOG (QIAGEN; NM_024865.2 (-) 05Kb: GPH1002937 (-) 05A), EpiTect ChIP qPCR Primer Assay For Human POU5F1 (QIAGEN; NM_002701.4 (-) 06Kb: GPH1024787 (-) 06A), EpiTect ChIP qPCR Primer Assay For Human POU5F1 (QIAGEN; NM_002701.4 (-) 03Kb: GPH1024787 (-) 03A). As the control, ChIP-qPCR Human IGX1A Negative Control (QIAGEN) was used. The results are shown in Figure 3-A.

<Example 10> Identification of humoral factors that fluctuate depending on PRDM14 gene The PRDM14 gene product can be detected by RT-PCR, Western blotting and immunohistological methods, but biopsy tissues are required. Therefore, it is necessary to identify a humoral factor that can be used as a companion marker for serodiagnosis. At the same time, in the in vitro culture system, the change in cell proliferation due to the introduction of the PRDM14 gene was minimal, whereas when the transgenic tumor cell line was orthotopically transplanted to the mammary gland of nude mice in the in vivo experimental system, the tumor of the PRDM14 gene introduced tumor cell line was detected. It was confirmed that growth was remarkably promoted and lymph node metastasis was caused. Therefore, it was decided to comprehensively measure the cytokines and chemokines secreted in the culture supernatant of the PRDM14 gene-introduced tumor cell line.

  Therefore, Luminex 200 system (Luminex system Luminex system, technology based on MAP (Multiple Analyte Profiling) technology) was used for analysis. Luminex (registered trademark) beads bound to an antibody that specifically binds to a target protein are allowed to react with a target antigen in a liquid phase, and the beads are colored with a fluorescent dye, which makes it possible to distinguish up to 100 kinds of antibodies. Further, another antibody against the antigen protein is reacted with a fluorescent-labeled (biotinylated secondary antibody and streptavidin-PE) secondary antibody to form a bead-antibody-target protein complex. While flowing Luminex (registered trademark) beads one by one by flow cytometry, the diameter and color of the beads are measured with a red laser and an APD sensor, and the fluorescence amount on the bead surface is measured with a green laser and a photomultiplier tube. This makes it possible to simultaneously quantify each of the antigens to be analyzed in the sample. After the measurement, a standard curve is created from the fluorescence intensity of the standard solution and the protein concentration, and the concentrations of a plurality of target proteins are converted from the functional formula. This time, 41 kinds of cytokines and chemokines shown in the table below were comprehensively measured.

  Among these, RANTES / CCL5 (R & D), CXCR1 (R & D), CXCR2 (USCN), CXCR3 (USCN), and CD40L (R & D), which had reproducibility, were found in the culture supernatant of the PRDM14 gene-introduced tumor cell line by the ELISA method. The quantification experiment was carried out by repeating the cytokine, chemokine, secreted by the mouse three times (Fig. 3-B, C).

<Example 11> Tumor forming ability experiment using orthotopic transplant model The tumor forming ability was confirmed by inoculating the nude mice with the PRDM14 gene-expressing tumor cells (MDA-MB-231, HCC1937) prepared in the above Examples.
1 × 10 ⁶ PRDM14 gene-expressing tumor cells were suspended in 100 μL of 1 × PBS on ice and mixed with 100 μL of Matrigel (BD). 100 μL of cell matrigel mixture was orthotopically inoculated into the mammary gland (fat pad) of nu / nu mouse (obtained from CLEA Japan, Inc.), and when tumor formation began, the major and minor diameters were measured and the spheroid was obtained. Was calculated and the volume was compared. The results are shown in Figure 4-A.
Furthermore, tumor cells (derived from breast cancer cell line HCC1937 and derived from pancreatic cancer cell line PK-1) in which the expression of PRDM14 gene was suppressed by the shRNA vector prepared in the above Example were orthotopically inoculated by the same method, and tumor The lengths of the major axis and the minor axis were measured, and the volume was calculated by approximating to a spheroid and compared. The respective results are shown in FIG. 4-D and FIG. 7-A. Furthermore, regarding PK-1 origin, the mouse subcutaneous tumor image excised at the endpoint after further in vivo follow is shown in FIG. 7-B, the tumor weight is shown in FIG. 7-C, and its pathological tissue image ( HE staining) is shown in Figure 7-D.

As a result, (i) almost no tumor was formed when the expression of PRDM14 gene was suppressed by shRNA vector, and (ii) the introduction of PRDM14 gene increased the SP fraction cells, and (iii) conversely Since the SP fraction cells are reduced when suppressed, the PRDM14 gene product is considered to be a major factor in tumor formation of cancer stem cells.
After euthanasia, the mice were necropsied to examine the tumor weight, lymph node metastasis, and lung metastasis, and the tumor strain in which the PRDM14 gene had been introduced had a heavier tumor, with more lymph node metastasis and lung micrometastase than controls. Met. Frozen sections were prepared from the obtained tumor tissues, and immunohistochemistry was performed using fragmented caspase-3 (Cleaved Caspase-3) antibody (Cell Signaling: SA1E, # 9664) and anti-CD31 antibody (BD: # 55027). As a result of a clinical study, it was found that there were almost no cells that fell into apoptosis in the tumor mass formed by the cancer cells into which the PRDM14 gene had been introduced, and the density of tumor blood vessels was high (FIG. 4-I).

<Example 12> Lung metastasis model experiment MDA-MB-231 cells known to form lung metastases from the tail vein of nu / nu mice were used. Specifically, 1 × 10 ⁶ cells of PRDM14 gene-expressing tumor cells (MDA-MB-231) and control tumor cells (tumor cells into which the backbone of PRDM14 expression vector was introduced) were injected into the tail vein of nude mice. Each animal was injected intravenously. One month later, the animals were euthanized and lung metastases were confirmed. As a result, a large metastatic lesion was observed in the PRDM14-introduced strain. On the other hand, in the control, only a few micrometastases were observed (Fig. 4-E, F).

<Example 13> Preparation of local treatment model for orthotopic graft / pulmonary metastasis MDA-MB expressing PRDM14 gene and found to form tumors in nude mice as in Example 11. -231 cells and HCC1937 cells (both wild type and not transgenic) were orthotopically transplanted to nude mice, and used at the stage when the tumor size became constant, using siRNA and an existing anticancer agent in combination. A model was created to start treatment with. The dose of siRNA was 1 mg / mouse body weight (kg), which is an extremely low concentration judged to have a specific therapeutic effect of siRNA, and PEI (in vivo jet), which is a commercially available drug delivery system for in vivo use. It was prepared according to the protocol using PEI: Polyplus Transfection Co.) and directly injected into the tumor 3 times / week. On the other hand, regarding the anticancer drug, adriamycin (1 mg / mouse body weight (kg)) or docetaxel (5 mg / mouse body weight (kg)) was intraperitoneally administered once a week to the anticancer drug administration group. . As a result, the siRNA of PRDM14 gene alone had the minimal effect on the tumor, and the combined use of the above anticancer agents synergistically resulted in the minimal effect on the tumor (FIGS. 5-A to D).

Further, for the purpose of confirming the effect on metastatic lesions, 1 × 10 ⁶ cells of MDA-MB-231, which easily forms lung metastases, were intravenously injected per mouse through the tail vein. One week after the injection, a combination of siRNA for the PRDM14 gene and DDS (PEI) was intravenously administered according to the intravenous administration protocol of the in vivo jet PEI. One month after the start of administration, the mice were euthanized and lung metastases were confirmed. Although siRNA # 2 had a poor effect, siRNA # 3 showed almost no metastatic foci, and the lung weight was also small with a significant difference (FIGS. 5-E and F).

Example 14 Preparation of Intravenous Treatment Model for Orthotopic Grafts PRDM14 gene-expressing tumor-forming HCC1937 cells were orthotopically transplanted to a tumor after exceeding a certain tumor diameter, and then treated. A therapeutic model was prepared by mixing the nucleic acid for use with a DDS agent (calcium phosphate micelle) developed in the Kataoka laboratory of the University of Tokyo and injecting it intravenously through the tail vein of a mouse as a mixture. Specifically, 1 × 10 ⁶ HCC1937 cells were suspended in 100 μL of 1 × PBS on ice and mixed with 100 μL of Matrigel (BD). 100 μL of cell matrigel mixture was orthotopically inoculated into the mammary gland (fat pad) of nu / nu mouse (obtained from CLEA Japan, Inc.), and when tumor formation began, the major and minor diameters were measured and the spheroid was obtained. Was calculated and the volume was compared. Nucleic acid was administered at 1 mg / mouse body weight (kg) as in the above example, prepared by mixing DDS agent (calcium phosphate micelle) developed in the Kataoka laboratory of the University of Tokyo, and administered intratumorally 3 times / week. Injected directly into. In addition, the DOC used for standard treatment of breast cancer was reduced from the normally used amount (5 mg / mouse body weight (kg)) and used together once weekly by intraperitoneal administration. As a result, a prominent tumor suppressing effect was obtained without adverse events (Fig. 5-G).

  The DDS agent was produced as follows based on JP-A-2011-231220. The N-methyl-2-pyrrolidone (NMP) used was from Sigma; β-benzyl-N-carboxy-L-aspartic anhydride (BLA-NCA) was from NOF CORPORATION; diethylenetriamine (bis (2-amino) Ethyl) amine) is Tokyo Kasei Kogyo Co., Ltd .; acetic acid and hydrochloric acid are purchased from Wako Pure Chemical Industries Ltd .; 2-propionic-3-methylmaleic anhydride (CDM anhydride) is described in the literature (A. Naganawa, Y. Ichikawa, M. Isobe, Tetrahedron 1994, 50, 8969-8982; DB Rozema, K. Ekena, DL Lewis, AG Loomis, JA Wolff, Bioconjugate Chem. 2003, 14, 51-57).

Synthesis of poly (ethylene glycol) -poly (2-[(2-aminoethyl) amino] ethylaspartamide): mPEG-pAsp (DET) (2a):

According to the method disclosed in JP-A-8-310970, β-benzyl-N-using α-methoxy-ω-amino-poly (ethylene glycol) (mPEG-NH ₂ , MW: 12,000) as an initiator. Carboxy-L-aspartic anhydride (BLA-NCA) is subjected to ring-opening polymerization to give poly (ethylene glycol) -b-poly (β-benzyl-L-aspartate) (mPEG-) represented by the following formula (1). b-PBLA) was obtained. The degree of polymerization of “PBLA” units in mPEG-b-PBLA was 96 according to ¹ H-NMR measurement.

Then, mPEG-b-PBLA was dissolved in NMP in an ice bath according to the method described in ChemMedChem 1 (2006) 439-444, and diethylenetriamine (DET, relative to the benzyl group of the PBLA segment was diluted with the same volume of NMP. 100 eq.) Was added, and the mixture was reacted at 0 ° C. (ice bath) for 4 hours with stirring. The reaction was stopped by the addition of cold 10% acetic acid in portions (2 times the volume of the solution). The neutralized solution was dialyzed against 0.01 M hydrochloric acid solution (x3) and distilled water (x3) at 4 ° C. After lyophilization, a white powder was obtained from the dialyzed solution. It was confirmed by gel permeation chromatography (GPC) and ¹ H-NMR measurement that the obtained mPEG-pAsp (DET) had a unimodal molecular weight distribution and almost 100% conversion from PBLA to pAsp (DET). .

Poly (ethylene glycol) -poly (N- {N- [N- (3-propionyl-2-methylmaleamyl) -2-aminoethyl] -2-aminoethyl} aspartamide: mPEG-pAsp (DET-CDM) Synthesis:

mPEG-pAsp (DET) (0.538 mmol of primary amino) was dissolved in 0.5 M bicine buffer (pH 9) (2 ml). CDM anhydride (5 equivalents to primary amine of pAsp (DET)) was slowly added to the solution and stirred at 0 ° C. for 2 hours. The reaction mixture was purified with Amicon Ultra (MWCO = 10,000; manufactured by Millipore) (× 3 with 0.5M NaHCO ₃ (pH 9.1) and × 3 with distilled water). The final product was obtained as a white powder after lyophilization. ^The conversion yield calculated from ¹ H-NMR was over 90%.

Preparation of siRNA-supported calcium phosphate (CaP) nanoparticles as DDS agent (preparation of PEG-CCP / calcium phosphate particles and encapsulation of siRNA)
1 μL of 2.5 M CaCl ₂ solution was diluted with 11.5 μL of 10 mM Tris / HCl buffer (pH 7.5) (first solution, 12.5 μL). A solution containing a charge conversion polymer (CCP), mPEG-pAsp, diluted with 10 mM Tris / HCl buffer (pH 7.7) was added to a 15 μM siRNA solution (in 10 mM Hepes buffer (pH 7.2)) and 1.5 mM Na. _It was mixed with 50 mM Hepes buffer (pH 7.5) containing ₃ PO ₄ and 140 mM NaCl (second solution, 12.5 μL to 13 μL). The first solution was mixed with the second solution with a pipette at a constant speed for about 20 seconds to prepare a solution of siRNA-loaded CaP nanoparticles.

<Example 15> Relationship between PRDM14 gene and differentiation trait For breast cancer cell line HCC1937 expressing PRDM14, PRDM14 is knocked down by shRNA specific for PRDM14 gene, and expression of CD44 and CD24 on the cell surface is flowed. It was analyzed by cytometry. The results are shown in Figure 8-A. Reducing the expression of PRDM14 only reduced the expression of CD44. As shown in Table 1, since CD44 is a molecule that serves as an indicator of the stemness of breast cancer, it is suggested that the CD44 expression shows a differentiation trait as the expression of CD44 decreases.
Moreover, each of a breast cancer cell line (HCC1937) and a pancreatic cancer cell line (PK-1) in which the expression of PRDM14 is reduced by shRNA is subcutaneously transplanted into a mouse, which serves as an index of differentiation of the formed tumor. Immunostaining was performed with antibodies specific for the molecules (CK5 and PDX1). CK5 is a marker of mammary gland undifferentiation, and PDX1 is a marker of pancreatic cell undifferentiation. The obtained results are shown in FIG. 8-B. It was found that when the expression of PRDM14 was decreased, the expression of the undifferentiated marker was decreased in all cells.

[Summary]
(I) PRDM14, which is a transcription factor, was found to have elevated mRNA and protein expression in a cancer tissue-specific manner by examination with clinical multiple samples of breast cancer, ovarian cancer, pancreatic cancer, and renal cancer. Furthermore, although it is a study in about 20 cases, similarly, an increase in expression is observed in lung cancer, esophageal cancer, colon cancer, renal cancer, prostate cancer, urinary tract tumor, and testicular tumor. On the other hand, the expression level of normal tissue is none or extremely low.

  (Ii) A strain overexpressing the PRDM14 gene and a KD strain constitutively constitutive KD strain were established, and the effect on cancer cells was examined: A) In the Side Population (SP) fraction, the PRDM14 gene caused an excess of the same fraction. It was demonstrated in multiple cancer cell lines that expression increased and decreased in KD (FACS Aria). B) In the Tumor sphere assay, the ability to form spheres was increased by introducing the PRDM14 gene. Moreover, when the expression of the stem cell marker was evaluated in the sphere formed at that time, the protein expression of Oct3 / 4, Nanog, SSEA1, and SSEA4 was increased (ICC, confocal). C) When the PRDM14 gene-introduced strain was transplanted into the mammary gland of nude mice (♀, 6 W), a prominent tumor increasing effect was observed. On the contrary, it was found that KD hardly formed tumors. D) On the other hand, when the gene transfer strain was examined in vitro, the proliferation activity of cancer cells was not increased.

  (Iii) Various evaluations were performed using specific siRNA against PRDM14: A) Treatment of cancer cells with siRNA reduced the viability of cancer cells, induced apoptosis, and further eliminated SP fraction. B) The viability of the cancer cells was evaluated by using CDDP, ADM, and DOX together with siRNA for the breast cancer line and GEM together with the pancreatic cancer line, and the viability was further improved as compared with the siRNA alone group. Decreased, and apoptosis prominently occurred. C) A therapeutic model with siRNA was constructed using nude mice, evaluated in combination with DDS by local injection into tumor (PEI) and tail vein injection (micelle). As a result, the antitumor effect and the effect of suppressing lung metastasis in the lung metastasis model were confirmed.

  The molecular marker according to the present invention is extremely promising because it can predict metastasis and recurrence due to cancer stem cell properties in a wide range of cancer types. Moreover, similar therapeutic effects have been observed in different and repeated laboratories in combination with DDS agents, and they are extremely promising as therapeutic targets. The molecular marker according to the present invention is expected to be highly effective for patients with intractable cancer of a wide range of cancer types that are expected to relapse after a long time, and sufficiently satisfies the needs of the people.

Claims (14)

In a subject, the presence or absence of cancer stem cells is determined in vitro using a molecular marker for detecting cancer stem cells from the subject based on the expression of PRDM14 (PR domain-containing protein 14) gene, or cancer A method for determining the degree of stem cell induction in vitro, comprising:
The subject is a cell population derived from one or more cells or tissues selected from the group consisting of breast, lung, pancreas, ovary, kidney, bladder and testis,
The molecular marker consists of the PRDM14 gene product or a fragment thereof, the gene product being mRNA,
A step of detecting the gene product by RT-PCR, wherein the relative expression ratio of the gene product and the internal standard gene is significantly higher than the relative expression ratio of a normal subject, cancer The above determination method, wherein it is determined that there are stem cells or that the degree of induction of cancer stem cells is high.   A method for assisting the determination of the timing of switching from one anticancer agent to another anticancer agent using the determination method according to claim 1, wherein an increase in the expression of PRDM14 gene is used as an index in the anticancer agent treatment. . A method for assisting the prediction of cancer prognosis using the determination method according to claim 1, wherein the PRDM14-positive cancer has a poorer prognosis than the PRDM14-negative cancer. A kit for use in the determination method according to claim 1, comprising at least a reagent for detecting a molecular marker for detecting a cancer stem cell from a subject by expression of PRDM14 gene,
The kit, wherein the reagent for detecting is a probe and / or primer having a base sequence complementary to the PRDM14 gene for detecting mRNA which is a product of the PRDM14 gene. A method for determining in vitro the degree of induction of cancer stem cells in a subject, using a molecular marker for detecting cancer stem cells from the subject, as an index, according to the expression of PRDM14 gene,
The subject is a cell population derived from one or more cells or tissues selected from the group consisting of breast, lung, pancreas, ovary, kidney, bladder and testis,
The molecular marker consists of the PRDM14 gene product or a fragment thereof, the gene product being mRNA,
A step of detecting the gene product by the RT-PCR method using the kit according to claim 4, wherein the relative expression ratio of the gene product and the internal standard gene is compared with the relative expression ratio of a normal subject. In vitro determination method of the degree of induction of cancer stem cells, in which the degree of induction of cancer stem cells is judged to be high when the degree of induction of cancer stem cells is significantly increased.   A pharmaceutical composition for inhibiting the function of a cancer stem cell, which comprises a nucleic acid used for suppressing the expression of the PRDM14 gene in the cancer stem cell.   The nucleic acid is a nucleic acid that inhibits the expression of a polynucleotide encoding a protein or a peptide that is a part of the protein containing an amino acid sequence identical to the amino acid sequence of the PRDM14 gene or an amino acid sequence having 90% or more identity with this The pharmaceutical composition according to claim 6.   The pharmaceutical composition according to claim 7, wherein the nucleic acid is one or more selected from the group consisting of antisense, siRNA and shRNA.   siRNA is continuous from a polynucleotide RNA encoding a protein having an amino acid sequence identical to the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 90% or more identity therewith, or a peptide which is a part thereof. The pharmaceutical composition according to claim 8, which comprises a sense strand sequence of 18 to 28 nucleotides and an antisense strand sequence which is a complementary sequence thereof.   The pharmaceutical composition according to any one of claims 6 to 9, wherein the pharmaceutical composition further comprises an anticancer agent. Pharmaceutical composition, Ri Oh is for cancer treatment,
Cancer, using the determination method according to claim 1, carcinoma cancer stem cells is determined that there or, Ru Gandea that cancer stem cells derived degree is determined to be high, claim 6 11. The pharmaceutical composition according to any one of 10 (except when the cancer is breast cancer, ovarian cancer, and non-small cell lung cancer) . Pharmaceutical composition state, and are not for suppressing metastasis, invasion and / or aging after recurrence of cancer,
Cancer, using the determination method according to claim 1, carcinoma cancer stem cells is determined that there or, Ru Gandea that cancer stem cells derived degree is determined to be high, claim 6 11. The pharmaceutical composition according to any one of 10 (except when the cancer is breast cancer, ovarian cancer, and non-small cell lung cancer) . Cancer stem cells are breast cancer, lung cancer, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cervical cancer, uterine body cancer, ovarian cancer, kidney cancer, prostate cancer , bladder cancer, testicular cancer, thyroid cancer, derived from one or more cells selected from the group consisting of adrenal cancer, and lymphoma, according to any one of claims 6-10 Pharmaceutical composition.   The pharmaceutical composition according to claim 13, wherein the cancer stem cells are derived from one or more types of cells selected from the group consisting of breast cancer, pancreatic cancer, and kidney cancer.