WO2015152477A1 - 유방암 재발 또는 전이 억제용 제제의 스크리닝 방법 - Google Patents
유방암 재발 또는 전이 억제용 제제의 스크리닝 방법 Download PDFInfo
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Definitions
- the present invention relates to a method for screening an agent for inhibiting recurrence or metastasis of breast cancer, and more particularly, to a method for screening a test compound for reducing the expression or activity level of a gene expressing breast cancer stem cell specifically with a breast cancer recurrence or metastasis inhibitor. It is about.
- Prognostic indicators provide many information about the prognosis as well as tumor size, lymph node status and histological grade and include many common factors, such as molecular markers that are likely to respond to a particular therapeutic agent. For example, steroid hormone receptor status measurements of estrogen (ER) and progesterone (PR) are commonly performed to evaluate breast cancer patients. Tumors that are hormone receptor positive must be responsive to hormone therapy, and typically also proliferate less actively, so the prognosis of patients with ER + / PR + tumors is better.
- ER estrogen
- PR progesterone
- HER-2 / neu human epidermal growth factor receptor 2
- trastuzumab Herceptin; Genentech
- an anti-Her-2 / neu antibody therapeutic has been developed using Her2 / neu expression levels in breast tumors.
- tumor suppressor gene p53 which is known to be associated with increased aggressiveness of the disease and poor prognosis.
- Ki-67 a cell proliferation marker with non-histone nuclear proteins, has been demonstrated to correlate with poor prognosis of breast cancer.
- Cancer stem cells refer to primitive cells that trigger cancer in cancer cells, and are known to exist separately from cancer cells and have characteristics of normal stem cells. If there are cancer stem cells that cause breast cancer in the patient's body after treatment of breast cancer, the presence of such breast cancer-derived cancer stem cells may occur because the cancer stem cells may have a poor prognosis such as recurrence or metastasis of breast cancer. By detecting, it is expected that the recurrence and prognosis of breast cancer can be evaluated. However, a method for detecting breast cancer-derived cancer stem cells has not been developed yet, and a method using breast cancer-derived cancer stem cells has not been utilized.
- the present inventors in order to develop a method of utilizing breast cancer-derived cancer stem cells, as a result of intensive studies, to identify marker genes that are specifically expressed in breast cancer-derived cancer stem cells whose expression level is changed by the treatment of Wnt signaling inhibitors, By measuring the change in the expression level of the marker gene it was confirmed that the screening agent for inhibiting breast cancer metastasis or recurrence, and completed the present invention.
- One object of the present invention is to provide a method for screening a cancer metastasis or recurrence inhibitor using a change in the expression level of a marker gene of breast cancer-derived cancer stem cells.
- the method of the present invention can be widely used in the effective treatment of breast cancer because it can be screened for agents that can be quickly, easily and with excellent accuracy and prevent or treat recurrence or metastasis of breast cancer.
- FIG. 1 is a heatmap showing the results of analysis of genes whose expression patterns were changed in 4T1 mouse breast cancer cell lines cultured by stem cell culture method using Ingenuity Pathways Analysis (IPA), compared to general cell cultured cells.
- IPA Ingenuity Pathways Analysis
- Figure 2 shows the results of the analysis of the gene expression is reduced in breast cancer-derived cancer stem cells by treatment with Wnt signaling inhibitors.
- Figure 3 shows the culture of IGF1 gene obtained from the culture of the mouse-derived breast cancer cell line 4T1 and human-derived breast cancer cell line MCF7 treated with or without Wnt signaling inhibitors (CWP232228, Sino pharmaceuticals) in adherent culture and suspension culture It is a photograph showing the result of immunofluorescence staining.
- Wnt signaling inhibitors CWP232228, Sino pharmaceuticals
- the present inventors came to pay attention to genes specifically expressed in cancer stem cells during various studies to develop a method for evaluating the recurrence and prognosis of breast cancer by detecting cancer stem cells derived from breast cancer.
- the cancer cells were cultured by the stem cell culture method, the genes specifically expressed in the cancer stem cells whose expression levels were significantly increased than those in the normal attachment culture could be identified, which is a gene involved in the Wnt signaling pathway. Confirmed.
- Wnt signaling pathway in cancer stem cells, it is expected that the expression level of marker genes that are essentially expressed in cancer stem cells is significantly reduced, and confirmed this.
- IGF1 Insulin-like growth factor 1
- Id2 Inhibitor of DNA binding 2
- MMP2 matrix metalloproteinase-2
- MMP9 matrix metalloproteinase-9
- Wnt5a Wi-type MMTV integration site family, member 5A
- the reduced genes are not expressed in breast cancer cells but specifically expressed in breast cancer-derived cancer stem cells, the five genes are inhibitors capable of inhibiting recurrence or metastasis of breast cancer by breast cancer-derived cancer stem cells. It can be used for screening.
- the present invention is to treat a test compound expected to be able to prevent or treat the recurrence or metastasis of breast cancer caused by breast cancer-derived cancer stem cells to the breast cancer-derived cancer stem cells, the cancer line administered Screening agents for inhibiting recurrence or metastasis of breast cancer, comprising measuring mRNA levels of genes selected from the group consisting of IGF1, Id2, MMP2, MMP9, Wnt5a, and combinations thereof in the stromal cells or the levels of proteins expressed therefrom Provide a way to.
- the screening method of the agent for preventing or treating breast cancer recurrence or metastasis of the present invention (a) a test compound for treating a test compound expected to be able to prevent or treat the recurrence or metastasis of breast cancer to breast cancer-derived cancer stem cells Processing step; (b) Insulin-like growth factor 1 (IGF1), Inhibitor of DNA binding 2 (Id2), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and Wnt5a (Wingless-) in cells treated with the test compound.
- IGF1 Insulin-like growth factor 1
- Id2 Inhibitor of DNA binding 2
- MMP2 matrix metalloproteinase-2
- MMP9 matrix metalloproteinase-9
- Wnt5a Wiless-
- the mRNA level of each gene or the level of the protein expressed therefrom may be measured using an agent that measures the level of mRNA of the gene or the protein expressed therefrom or by using a composition or kit comprising the agent. And when the level measured in the experimental group is significantly reduced compared to the level measured in the control group, it can be determined that the test compound can be used as an agent capable of preventing or treating recurrence or metastasis of breast cancer.
- breast cancer cell refers to a cell derived from cancer tissue of a breast cancer patient, and may be used in the same sense as a breast cancer cell line which is typically immortalized and capable of infinite growth through passage.
- the breast cancer cells may be HeLa cells and the like.
- cancer stem cell when injected into immunosuppressed mice can produce tumors with high efficiency, and the formed tumors clearly show the inherent heterogeneity of the primary tumors. It is a kind of cancer cell with unlimited regenerative capacity.
- general cell culture method refers to a method of culturing general cells exhibiting different characteristics from stem cells, and may be a method of culturing to form a single layer by attaching to the bottom of the culture vessel. have.
- stem cell culture method means a method of culturing stem cells exhibiting different characteristics from ordinary cells, and may be a method of culturing while floating without attaching to the bottom of the culture vessel.
- 4T1 mouse breast cancer cell line was inoculated in DMEM medium containing EGF, bFGF, heparin, and B27, and suspended in culture for 7 days at 37 ° C. and 5% CO 2 , thereby forming a globular form. Cancer stem cells were obtained.
- IGF1 gene refers to a gene encoding Insulin-like growth factor 1, which serves to mediate the body's growth response by growth hormone by acting as a receptor for growth hormone. . Specific sequence and protein information of the gene is known from the NCBI (GenBank: NM_000618).
- Id2 gene refers to a gene encoding DNA-binding protein inhibitor 2, which is used as a transcriptional regulator including a helix-loop-helix (HLH) domain. Specific sequence and protein information of the gene is known from the NCBI (GenBank: NM_002166).
- MMP2 gene refers to a gene encoding matrix metalloproteinase-2, which plays a role in degrading extracellular epilepsy in cell physiological activities such as embryonic development, angiogenesis and bone formation. do. Specific sequence and protein information of the gene is known from the NCBI (GenBank: NM_001127891).
- MMP9 gene refers to a gene encoding Matrix metalloproteinase 9, which plays a role in degrading extracellular epilepsy in cell physiological activities such as embryonic development, angiogenesis, and bone formation. . Specific sequence and protein information of the gene is known from the NCBI (GenBank: NM_004994).
- Wnt5a gene refers to a gene encoding Wnt-5a, which is involved in Wnt signaling. Specific sequence and protein information of the gene is known from the NCBI (GenBank: NM_001256105).
- the term "agent for measuring mRNA level of a gene” refers to an agent used in a method for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of a target gene included in a sample.
- RT-PCR RT-PCR
- Competitive RT-PCR RT-PCR
- Real-time RT-PCR RNase protection assay (RPA)
- Northern blotting It may be a probe or a primer that can specifically bind to a target gene used in a method such as DNA chip analysis, but is not particularly limited thereto.
- primer refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can be initiated for DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.
- the primer may be a primer that can be used for amplification of the IGF1, Id2, MMP2, MMP9 or Wnt5a gene, by complementary binding to the IGF1, Id2, MMP2, MMP9 or Wnt5a gene by PCR method
- the nucleotide sequence of the primer is not limited.
- probe refers to a nucleic acid fragment such as RNA or DNA, which may correspond to a short base to several hundred bases, which may achieve specific binding with a gene or mRNA, and includes an oligonucleotide probe, It may be prepared in the form of single stranded DNA probe, double stranded DNA probe, RNA probe, or the like, and may be labeled for easier detection.
- the probe may be a probe capable of complementarily binding to IGF1, Id2, MMP2, MMP9 or Wnt5a gene, and may complementarily bind to the IGF1, Id2, MMP2, MMP9 or Wnt5a gene.
- the nucleotide sequence of the probe is not limited.
- the term "agent for measuring the level of protein” refers to an agent used in a method for measuring the level of a target protein included in a sample, preferably Western blotting, ELISA (enzyme linked immunosorbent). assay, Radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement It may be an antibody used in methods such as the Complement Fixation Assay, FACS, and Protein Chip Assay.
- the term “antibody” refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody may be obtained by cloning a gene into an expression vector according to a conventional method. The protein encoded by the gene can be obtained and prepared from conventional proteins by conventional methods.
- the form of the antibody is not particularly limited and, if it is a polyclonal antibody, a monoclonal antibody or antigen-binding, a part thereof may be included in the antibody of the present invention and may include all immunoglobulin antibodies, as well as humanized antibodies. It may also contain a special antibody of.
- the antibodies include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
- a functional fragment of an antibody molecule means a fragment having at least antigen binding function and may be Fab, F (ab '), F (ab') 2 and Fv.
- the antibody may be an antibody that can specifically bind to a protein expressed from the IGF1, Id2, MMP2, MMP9 or Wnt5a gene, and preferably can specifically bind to each of the above proteins.
- kits comprising an agent for measuring the mRNA level of each gene or the level of protein expressed therefrom can be used for the mRNA level of the gene or the level of protein expressed therefrom.
- an agent for measuring the mRNA level of each gene or the level of protein expressed therefrom can be used for the mRNA level of the gene or the level of protein expressed therefrom.
- primers, probes or antibodies to measure the activity one or more other component compositions, solutions or devices suitable for the assay method may be included.
- the kit for measuring the mRNA expression level of the IGF1, Id2, MMP2, MMP9 or Wnt5a gene of the present invention may be a kit containing the necessary elements necessary to perform RT-PCR.
- the RT-PCR kit includes a test tube or other appropriate container, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each primer pair specific for the gene.
- reaction buffers pH and magnesium concentrations vary
- dNTPs deoxynucleotides
- Taq-polymerases Taq-polymerases
- reverse transcriptases in addition to each primer pair specific for the gene.
- enzymes DNase, RNAse inhibitors, DEPC-water (DEPC-water)
- DEPC-water DEPC-water
- It may also comprise primer pairs specific for the genes used as quantitative controls.
- kits of the present invention may include the necessary elements necessary to perform DNA chip assays.
- the DNA chip analysis kit may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and a reagent, a preparation, an enzyme, or the like for preparing a fluorescence-labeled probe.
- the substrate may comprise cDNA corresponding to the quantitative control gene or fragment thereof.
- the kit of the present invention may be a protein chip analysis kit for measuring the level of the protein expressed from the IGF1, Id2, MMP2, MMP9 or Wnt5a gene
- the kit is not particularly limited, but the antibody For immunological detection of the substrate, a suitable buffer, a secondary antibody labeled with a coloring enzyme or a fluorescent material, a coloring substrate and the like can be included.
- the substrate is not particularly limited thereto, but a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like may be used.
- peroxidase alkaline phosphatase
- the fluorescent material is not particularly limited, but may be FITC, RITC, and the like, and the colorant substrate solution is not particularly limited thereto.
- a mouse-derived breast cancer cell line is obtained for each culture by a general cell culture method and a stem cell culture method, compared with the expression level of the gene expressed from each culture Wnt signal It was confirmed that the expression level is significantly increased in the culture cultured by the stem cell culture method of delivery-related genes (Example 1), and the genes are reduced in the expression of cancer stem cells when the Wnt signaling is inhibited As a result, IGF1, Id2, MMP2, MMP9 or Wnt5a genes were selected, and the selected genes were not expressed in breast cancer cells but specifically expressed in breast cancer-derived cancer stem cells, and when inhibiting Wnt signaling in cancer stem cells, Since it was confirmed that the expression level is reduced (FIGS. 1 and 2), the five genes may be used as marker genes for cancer stem cells. I could see that.
- the agent for measuring the mRNA level of the IGF1, Id2, MMP2, MMP9 or Wnt5a gene of the present invention or the level of the protein expressed therefrom can suppress the recurrence or metastasis of breast cancer by breast cancer-derived cancer stem cells It was found that it can be used for screening inhibitors.
- DMEM Invitrogen
- FBS fetal bovine serum
- penicillin / streptomycin penicillin / streptomycin
- DMEM medium containing 20ng / ml EGF, 20ng / ml bFGF, 4 ⁇ g / ml heparin, B27 was added to 4T1 mouse breast cancer cell lines, and spheroids were suspended in culture for 7 days at 37 ° C. and 5% CO 2. A culture in the form of a sphere was obtained.
- Example 1-1 Each culture cultured in Example 1-1 was applied to the RNeasy Plus Mini Kit (Qiagen Inc, Valencia, CA) to extract the total RNA from each culture. Each extracted total RNA was applied to a random hexamer and ReverAid H Minus First Strand cDNA Synthesis Kit (Thermo scientific) to synthesize each cDNA. Using the synthesized cDNA, RTQ-PCR (ABI) using Stem cell PCR array (SABioscience (www.sabiosciences.com), cat no: PAMM-405) containing primers of 84 key genes related to stem cells 7300).
- Example 1 As shown in the results of Example 1, it was confirmed that the expression level of genes involved in Wnt signaling is significantly increased in cancer stem cells. When the Wnt signaling is inhibited, the gene whose expression level is decreased is cancer stem cells. Since it was expected to be used as a marker gene that plays an important role in, it was intended to discover the gene.
- 4T1 mouse breast cancer cell lines were cultured for 8 days by the suspension culture method of Example 1-1 under or without treatment with the Wnt signaling inhibitor (CWP232228, FTC). At this time, the Wnt signaling inhibitor was treated a total of four times once every two days at a concentration of 1 ⁇ M.
- Wnt signaling inhibitor CWP232228, FTC
- the data obtained through RTQ-PCR was analyzed to select genes having a difference of 2-fold or more and a P value ⁇ 0.05 based on 2-deltadelta ct values between each cultured cells. As a result, it was confirmed that IGF1, Id2, MMP2, MMP9 and Wnt5a are genes that change significantly as the Wnt inhibitor is treated.
- MMP2 F 5'-TTT CTA TGG CTG CCC CAA GG-3 '(SEQ ID NO: 3)
- MMP2 R 5'-GTC AAG GTC ACC TGT CTG GG-3 '(SEQ ID NO: 4)
- MMP9 F 5'-TGA GTC CGG CAG ACA ATC CT-3 '(SEQ ID NO: 5)
- MMP9 R 5'-CCA GTA CCA ACC GTC CTT GAA-3 '(SEQ ID NO: 6)
- WNT5A F 5'-ACT ATG GCT ACC GCT TCG C-3 '(SEQ ID NO: 7)
- WNT5A R 5'-GCG CTC TCA TAG GAA CCC TT-3 '(SEQ ID NO: 8)
- IGF1 F 5'-GTG GAT GAG TGT TGC TTC CG-3 '(SEQ ID NO: 9)
- IGF1 R 5'-TTT GTA GGC TTC AGT GGG GC-3 '(SEQ ID NO: 10)
- IGF1, Id2, MMP2, MMP9 and Wnt5a gene was confirmed that the expression level significantly decreased as the Wnt inhibitor treatment.
- Example 3 Verification of the decrease in the expression level of the marker gene at the cellular level
- Example 2 The five genes discovered in Example 2 were treated at the cellular level to determine whether expression levels were significantly reduced as the Wnt inhibitors were treated.
- IGF1 gene which is one of the five identified genes, intracellularly, 4T1, a mouse-derived breast cancer cell line, and MCF7, a human-derived breast cancer cell line, were treated with a Wnt signaling inhibitor (CWP232228).
- Each culture was obtained by adhesion culture and suspension culture by the method of Example 1 under untreated conditions.
- An anti-IGF1 mouse antibody (Milipore, cat. # 05-172) was added to each of the cultures obtained above, and the first reaction was performed, followed by an anti-mouse IgG antibody (Invitrogen cat.A11001) to which FITC (fluorescein isothiocyanate) was bound. ) was added to perform a secondary reaction, fluorescence immunostaining was performed for the IGF1 gene, which was observed under a fluorescence microscope (Zeiss LSM 510) to measure the expression level of IGF1 (FIG. 3).
- IGF1 gene was not expressed regardless of whether the Wnt signal transduction inhibitor was treated at the time of adhesion culture, but the expression of IGF1 gene was confirmed during suspension culture. It was found that it can be used as a marker gene specific for breast cancer-derived cancer stem cells.
- Wnt inhibitors were applied to the cultured breast cancer-derived cancer stem cells, expression levels of the genes were decreased in both types of cancer stem cells obtained from the two types of breast cancer cell lines, and the mouse rather than the cancer stem cells derived from human breast cancer cell lines. Inhibition of IGF1 gene expression in breast cancer cell line-derived cancer stem cells was significantly higher.
- the five genes provided by the present invention are marker genes that are not expressed in breast cancer cells but specifically expressed in breast cancer-derived cancer stem cells, a test compound for reducing the expression or activity level of the genes is provided. It can be usefully used for screening as a metastasis and / or relapse inhibitor of breast cancer.
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Description
Claims (2)
- (a) 유방암 유래 암줄기세포에 유방암의 재발 또는 전이를 예방 또는 치료할 수 있을 것으로 예상되는 피검 화합물을 처리하는 피검 화합물 처리단계;(b) 상기 피검 화합물을 처리한 세포 내 IGF1(Insulin-like growth factor 1), Id2(Inhibitor of DNA binding 2), MMP2(matrix metalloproteinase-2), MMP9(matrix metalloproteinase-9) 및 Wnt5a(Wingless-type MMTV integration site family, member 5A)로 구성된 군으로부터 선택되는 1종 이상의 유전자의 mRNA 수준 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 단계; 및(c) 상기 피검 화합물을 처리하지 않은 음성 대조군과 비교하여 상기 유전자의 mRNA 수준 또는 상기 유전자가 코딩하는 단백질 수준을 감소시킨 피검 화합물을 선별하는 단계를 포함하는, 유방암 재발 또는 전이에 대한 예방 또는 치료용 제제의 스크리닝 방법.
- 제1항에 있어서,상기 피검 화합물은 Wnt 신호전달 저해활성을 나타내는 신호전달 저해제인 것인 방법.
Priority Applications (2)
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US14/412,081 US20180128815A1 (en) | 2014-03-31 | 2014-10-02 | Method of screening an agent for inhibiting recurrence or metastasis of breast cancer |
CA2876640A CA2876640A1 (en) | 2014-03-31 | 2014-10-02 | Method of screening an agent for inhibiting recurrence or metastasis of breast cancer |
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KR10-2014-0038031 | 2014-03-31 | ||
KR1020140038031A KR101632628B1 (ko) | 2014-03-31 | 2014-03-31 | 유방암 재발 또는 전이 억제용 제제의 스크리닝 방법 |
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WO2015152477A1 true WO2015152477A1 (ko) | 2015-10-08 |
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PCT/KR2014/009302 WO2015152477A1 (ko) | 2014-03-31 | 2014-10-02 | 유방암 재발 또는 전이 억제용 제제의 스크리닝 방법 |
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US (1) | US20180128815A1 (ko) |
KR (1) | KR101632628B1 (ko) |
CA (1) | CA2876640A1 (ko) |
WO (1) | WO2015152477A1 (ko) |
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KR101950717B1 (ko) * | 2016-11-23 | 2019-02-21 | 주식회사 젠큐릭스 | 유방암 환자의 화학치료 유용성 예측 방법 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2013063321A1 (en) * | 2011-10-25 | 2013-05-02 | The General Hospital Corporation | Wnt/b-catenin inhibitors and methods of use |
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2014
- 2014-03-31 KR KR1020140038031A patent/KR101632628B1/ko active IP Right Grant
- 2014-10-02 US US14/412,081 patent/US20180128815A1/en not_active Abandoned
- 2014-10-02 CA CA2876640A patent/CA2876640A1/en not_active Abandoned
- 2014-10-02 WO PCT/KR2014/009302 patent/WO2015152477A1/ko active Application Filing
Patent Citations (1)
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WO2013063321A1 (en) * | 2011-10-25 | 2013-05-02 | The General Hospital Corporation | Wnt/b-catenin inhibitors and methods of use |
Non-Patent Citations (4)
Title |
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JANG, GYU BEOM: "The study on roles of Wnt signaling in breast cancer stem cells", SCIENCE MASTER THESIS * |
KAKARALA ET AL.: "Implications of the cancer stem- cell hypothesis for breast cancer prevention and therapy", JOURNAL OF CLINICAL ONCOLOGY, vol. 26, no. 17, 2008, pages 2813 - 2820 * |
KAKARALA ET AL.: "Targeting breast stem cells with the cancer preventive compounds curcumin and piperine", BREAST CANCER RESEARCH AND TREATMENT, vol. 122, no. 3, 2010, pages 777 - 785, XP019814409, ISSN: 0167-6806 * |
REYA ET AL.: "Wnt signalling in stem cells and cancer", NATURE, vol. 434, no. 7035, 2005, pages 843 - 850, XP055229280, ISSN: 0028-0836 * |
Also Published As
Publication number | Publication date |
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KR101632628B1 (ko) | 2016-06-23 |
CA2876640A1 (en) | 2016-04-02 |
US20180128815A1 (en) | 2018-05-10 |
KR20150114122A (ko) | 2015-10-12 |
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