WO2019037658A1 - 新型的肿瘤微环境相关靶点tak1及其在抑制肿瘤中的应用 - Google Patents

新型的肿瘤微环境相关靶点tak1及其在抑制肿瘤中的应用 Download PDF

Info

Publication number
WO2019037658A1
WO2019037658A1 PCT/CN2018/101003 CN2018101003W WO2019037658A1 WO 2019037658 A1 WO2019037658 A1 WO 2019037658A1 CN 2018101003 W CN2018101003 W CN 2018101003W WO 2019037658 A1 WO2019037658 A1 WO 2019037658A1
Authority
WO
WIPO (PCT)
Prior art keywords
tak1
tumor
gene
cells
expression
Prior art date
Application number
PCT/CN2018/101003
Other languages
English (en)
French (fr)
Inventor
孙宇
张博逸
Original Assignee
中国科学院上海生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海生命科学研究院 filed Critical 中国科学院上海生命科学研究院
Priority to US16/640,826 priority Critical patent/US11344601B2/en
Publication of WO2019037658A1 publication Critical patent/WO2019037658A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is in the field of pharmacology, and more particularly, the present invention relates to a novel tumor-associated target TAK1 and its use in inhibiting tumors.
  • Cellular senescence is the process by which a cell enters a permanent cell cycle arrest.
  • Cellular aging is often accompanied by a number of pathological features, including local inflammation.
  • Cellular senescence occurs in damaged cells and prevents their proliferation in the organism.
  • cell damage can lead to obvious signs of cell aging; when the damage accumulates and reaches a certain limit, the tissue exhibits various visually recognizable tissue regression changes and physiological aging phenotypes.
  • SASP senescence-associated secretory phenotype
  • SASP includes pro-inflammatory cytokines (such as IL-1 ⁇ , IL-1 ⁇ , IL-6 and IL-8), growth factors (such as HGF, TGF- ⁇ and GM-CSF), and chemokines (such as CXCL1/3 and CXCL10).
  • cytokines such as IL-1 ⁇ , IL-1 ⁇ , IL-6 and IL-8
  • growth factors such as HGF, TGF- ⁇ and GM-CSF
  • chemokines such as CXCL1/3 and CXCL10
  • matrix remodeling enzymes such as MMP1, MMP3, etc.
  • PKC ⁇ promotes senescence by up-regulating the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and enhancing the transcription and secretion of IL-6; while the expression of IL-8 is specifically inhibited by PKC ⁇ .
  • TNF- ⁇ is a major component of certain cell types of SASP.
  • the biotherapeutic drug adalimumab is a monoclonal antibody that directly inhibits TNF- ⁇ , which can attenuate the secretion of SASP and release IL-6. The amount was significantly decreased, while the expression levels of eNOS and miR-126-3p were significantly increased. Adalimumab can also induce epigenetic modifications of senescent cells, thereby attenuating the cancer-promoting effects of SASP.
  • Certain flavonoids can inhibit the development of SASP, and some are related to the NF- ⁇ B subunit p65 and I ⁇ B ⁇ signaling pathway, which can effectively protect or alleviate the chronic low degree in degenerative diseases such as cardiovascular disease and advanced cancer. Inflammation.
  • the natural flavonoids apigenin and kaempferol can strongly inhibit the expression of SASP.
  • These flavonoids up-regulate the expression of I ⁇ B ⁇ through the signaling pathway of IRAK1/I ⁇ B ⁇ , thereby inhibiting the activity of NF- ⁇ B subunit p65.
  • inhibition of I ⁇ B ⁇ expression increases the expression of SASP.
  • oral apigenin can significantly reduce SASP in the kidneys of aged rats, which is closely related to the elevated level of I ⁇ B ⁇ mRNA.
  • JAK inhibitors have been found to reduce the secretion of SASP from adipose precursor cells and umbilical vein endothelial cells (HUVEC), so the JAK pathway may be a potential target for anti-aging-related dysfunction.
  • TRIM28 has a positive regulatory effect on IL-6, IL-8 and other SASP components, while the secretory phenotype is strongly inhibited when TRIM28 is depleted.
  • mTOR inhibitor rapamycin can be a potent SASP inhibitor.
  • mTOR controls SASP by regulating the translation of IL-1 ⁇ and MAPKAPK2.
  • the p38 downstream signal MAPKAPK2 phosphorylates the RNA binding protein ZFP36L1, thereby preventing the degradation of SASP factor transcripts.
  • ZFP36L1 is dephosphorylated, resulting in the degradation of SASP transcripts and the abolition of the IL-1 ⁇ feedback loop. Therefore, small molecule inhibitors of p38MAPK kinase and MK2 can inhibit SASP in human fibroblasts.
  • Rapamycin reduces IL-6 and other cytokine mRNA levels and selectively inhibits translation of the membrane-bound cytokine IL-1 ⁇ , and decreased secretion of IL-1 ⁇ also reduces NF- ⁇ B transcriptional activity, whereas NF- ⁇ B It has more regulatory effects on SASP and reveals the anti-aging effect of the mTOR inhibitor rapamycin.
  • rapamycin can inhibit the ability of senescent fibroblasts to stimulate prostate tumor growth in mice. Therefore, rapamycin may improve age-related diseases, including advanced cancer, by inhibiting aging-related inflammation.
  • Cellular senescence is an effective anti-cancer mechanism that prevents the proliferation of mitotic cells and prevents malignant transformation.
  • the aging therapy has recently become a new treatment for cancer treatment, but this concept conflicts with the SASP of aging tumor cells, because SASP can promote tumors, although aging cells have anti-tumor effects.
  • SASP can promote tumors, although aging cells have anti-tumor effects.
  • activation of the JAK2/STAT3 pathway establishes an immunosuppressive tumor microenvironment that leads to tumor growth and drug resistance development.
  • a downregulator of a TAK1 gene or protein for the preparation of a pharmaceutical composition for inhibiting tumors; said tumor being selected from the group consisting of prostate cancer, breast cancer, and lung cancer.
  • the tumor is a tumor of a chemotherapeutic drug or a radiation therapy treatment (or an ionizing radiation treatment treatment); or the tumor is a tumor expressing TAK1.
  • the chemotherapeutic agent includes, but is not limited to, bleomycin, mitoxantrone, docetaxel, paclitaxel.
  • the pharmaceutical composition is used in combination with a chemotherapeutic agent to inhibit tumors.
  • the down-regulating agent is selected from the group consisting of: a small molecule compound that specifically inhibits TAK1; an interfering molecule that specifically interferes with TAK1 gene expression; or a gene editing reagent that specifically knocks out the TAK1 gene (eg, targets An sgRNA of the TAK1 gene; or an antibody or ligand that specifically binds to a protein encoded by the TAK1 gene.
  • the down-regulator is a small molecule compound that specifically inhibits TAK1, which is selected from the group consisting of: 5Z-7-oxozeaenol (ie, 5Z-7) or LYTAK1.
  • a TAK1 gene or protein for the preparation of a composition for regulating a senescence-associated secretory phenotype (SASP); or for the preparation of a pharmaceutical composition for inhibiting a disease associated with aging.
  • SASP senescence-associated secretory phenotype
  • the aging-related diseases include: atherosclerosis, osteoarthritis, osteoporosis, and other organ degenerative diseases.
  • a method of screening for a potential substance that inhibits a tumor comprising: (1) treating a system expressing a TAK1 gene with a candidate substance; and (2) detecting a TAK1 gene in the system Expression or activity; wherein, if the candidate substance reduces the expression or activity of the TAK1 gene, it indicates that the candidate substance is a potential substance for inhibiting tumor.
  • step (1) comprises: adding a candidate substance to the system expressing TAK1 in the test group; and/or
  • the step (2) comprises: detecting the expression or activity of TAK1 in the system of the test group, and comparing with the control group, wherein the control group is a system expressing TAK1 without adding the candidate substance;
  • TAK1 in the test group is statistically lower than the control group, it indicates that the candidate is a potential substance for inhibiting tumors.
  • the system is selected from the group consisting of a cellular system (such as a cell or cell culture expressing TAK1), a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
  • a cellular system such as a cell or cell culture expressing TAK1
  • a subcellular system such as a cell or cell culture expressing TAK1
  • a solution system such as a cell or cell culture expressing TAK1
  • tissue system such as a cell or cell culture expressing TAK1
  • an animal system such as a cell or cell culture expressing TAK1
  • the statistically lower than preferably is significantly lower than, for example, 20% or more lower, preferably 50% or more lower; more preferably 80% or more lower.
  • the candidate substance includes, but is not limited to, a small molecule compound designed for the TAK1 gene or protein, and an interference molecule designed for a signal pathway involved in the TAK1 gene or protein or an upstream or downstream protein thereof. , a nucleic acid inhibitor, a binding molecule (such as an antibody or a ligand), and the like.
  • the method further comprises performing further cellular experiments and/or animal tests on the obtained potential substances to further select and determine substances useful for inhibiting the tumor from the candidate substances.
  • a pharmaceutical composition for inhibiting tumors comprising: a downregulator of a TAK1 gene or protein; and a chemotherapeutic drug or a radiation therapy (ionizing radiation) therapeutic drug.
  • kits for inhibiting a tumor comprising:
  • the down-regulating agent comprises: a small molecule compound that specifically inhibits TAK1; an interfering molecule that specifically interferes with TAK1 gene expression; or a gene editing reagent that specifically knocks out the TAK1 gene (eg, targets the TAK1 gene) sgRNA); or an antibody or ligand that specifically binds to a protein encoded by the TAK1 gene.
  • the chemotherapeutic agent includes, but is not limited to, bleomycin, mitoxantrone, docetaxel, paclitaxel.
  • an agent that specifically recognizes a TAKl gene or a protein encoded thereby for use in the preparation of a reagent or kit for performing a prognostic evaluation of a tumor.
  • the agent that specifically recognizes the TAK1 gene or the protein encoded thereby is selected from the group consisting of: a primer that specifically amplifies the TAK1 gene; a probe that specifically recognizes the TAK1 gene; or a specific binding to the TAK1 gene.
  • a primer that specifically amplifies the TAK1 gene a probe that specifically recognizes the TAK1 gene
  • a specific binding to the TAK1 gene a specific binding to the TAK1 gene.
  • An antibody or ligand for a protein is selected from the group consisting of: a primer that specifically amplifies the TAK1 gene; a probe that specifically recognizes the TAK1 gene; or a specific binding to the TAK1 gene.
  • kits for tumor prognosis evaluation comprising: a reagent that specifically recognizes a TAK1 gene or a protein encoded thereby is provided.
  • FIG. 1 Phosphorylated ATM (p-ATM) antibody mediated IP analysis.
  • the expression level of TRAF6, p-ATM and ATM was detected by Western blotting.
  • PSC27 was treated with bleomycin (50 ug/ml) and then with the ATM small molecule inhibitor KU55933 (KU, 10 ⁇ M).
  • FIG. 1 PSC27 cells were treated with bleomycin supplemented with 5Z-7 (500 nM), and then subjected to anti-TAK1-mediated IP sedimentation. Western blot analysis of p-TAK1, TRAF6, p-ATM and cells in IP products. Expression levels of p-TAK1, TRAF6 and p-ATM in lysates.
  • FIG. 3 PSC27 control cells and TRAF6 specific shRNA stably transfected with bleomycin. After anti-TRAF6-mediated IP, the product was analyzed by Western blot for post-translational modification of major proteins. The analysis is carried out simultaneously.
  • FIG. 4 After cytoplasmic cells were treated with bleomycin, cytoplasmic proteins and nuclear proteins were specifically isolated and extracted by kits, and ATM, TAK1 activation and NF- ⁇ B nuclear translocation were analyzed. Control cells and 5Z-7 treated cells were analyzed in parallel.
  • PSC27 cells were treated with different clinically available chemotherapeutic drugs and radiotherapy rays.
  • the lysates of the damaged cells were collected for Western blot analysis of p-TAK1 expression, and total TAK1 and GAPDH were used as loading controls.
  • FIG. 6 In the presence of the TAK1 inhibitor 5Z-7, PSC27 cells were treated with bleomycin and the cell lysate was subjected to anti-p-TAK1 mediated IP sedimentation. The IP product was analyzed by an in vitro kinase assay and MKK6 was a TAK1 substrate. Phosphorylation of p38 was analyzed by Western blot and GAPDH was used as a loading control. In addition, IL-1 ⁇ (20 ng/ml) was used to treat stromal cells and analyzed by similar IP and in vitro kinase assays. In the figure, RAD refers to radiotherapy radiation treatment ( ⁇ -radiation at 743 rad/min).
  • PSC27 cells were shRNA-mediated IL-1 alpha knockdown, and p-TAK1 mediated IP, and subsequent Western blot analysis.
  • FIG. 1 Immunofluorescence staining analysis ( ⁇ -H2AX antibody) for DNA damage repair. Ruler, 10 ⁇ m. The lower part, the statistical results of the analysis and comparison.
  • Figure 10 Representative pictures of cell clone formation ability detection. After treatment with bleomycin and/or 5Z-7, PSC27 cells were fixed on the seventh day to determine the number of colonies.
  • FIG. 13 Immunofluorescence staining of DNA foci after treatment of PSC27 cells with satraplatin and/or 5Z-7. The DDR case is counted and compared by category.
  • FIG. 14 Analysis of stromal cell proliferation potential.
  • PSC27 was treated with bleomycin (50 ug/ml) and/or 5Z-7 (500 nM) and then continuously propagated and passaged under in vitro culture conditions, and the multiplication ratio was plotted against the culture time.
  • FIG. 15 Cells were harvested on day 7 after stromal cells were treated with bleomycin and/or 5Z-7. The lysate was analyzed by Western blot for the phosphorylation level of ATM, TAK1, and the activation of JNk and p38 downstream of TAK1. The chemokine IL-8 was used as a marker exocrine factor for SASP, and its expression level was also analyzed.
  • FIG. 16 After treatment with bleomycin, stromal cells were harvested at different time points and analyzed for Akt and mTOR phosphorylation levels by Western blot.
  • FIG. 1 Immunofluorescence assay analysis of p-mTOR expression levels on day 7 after PSC27 cells were treated with bleomycin (50 ug/ml) and/or RAD001 (50 nM).
  • FIG. 18 After treatment of stromal cells with bleomycin and/or RAD001, cells were harvested on day 7 and analyzed for activation of mTOR and its downstream substrate S6K1/4E-BP1 by Western blot.
  • FIG. 19 After treatment of stromal cells with bleomycin and/or RAD001, cells were harvested on day 7 and their BrdU insertion in DNA was examined.
  • Fig. 20 and Fig. 19 show statistical comparison analysis of SA-B-Gal staining of each group of cells.
  • FIG. 21 After stromal cells were treated with bleomycin and/or RAD001, cells were harvested on day 7 and tested for NF-kB complex activation. GAPDH and Histone H3 are cytoplasmic and nuclear protein loading controls, respectively.
  • FIG. 22 After stromal cells pre-transformed into the NF-kB transcriptionally active luciferase reporter vector were treated with bleomycin and/or RAD001, cells were harvested on day 7 and the fluorescent signal intensity of their reporter vector was measured.
  • FIG. 23 After treatment of stromal cells with bleomycin and/or RAD001, cells were harvested on day 7 and the interaction between the mTOR and IKK complex subunits was analyzed by immunoprecipitation. IgG, control antibody.
  • Figure 24 Experimental procedure of drug treatment and in vitro kinase assay in stromal cells in vitro.
  • FIG. 25 Stromal cells were processed through the experimental sequence in Figure 24, followed by anti-Flag mediated IP sedimentation. The IP product was analyzed by Western blot for the expression of p-IKKa and p-mTOR to determine the physical interaction between the two.
  • FIG. 26 Stromal cells were specifically shRNA-mediated knockdown of IKK complex subunits alpha and beta, respectively, and then treated with bleomycin and/or RAD001 to analyze the NF-kB transcriptional activity of the luciferase reporter vector. Signal strength.
  • Figure 27 After stimulation of stromal cells with bleomycin and/or RAD001 and IL-1 ⁇ (20 ng/ml), IKK ⁇ phosphorylation, IkB ⁇ and IRAK1 protein expression, and p65/p50 nuclear import were analyzed by Western blot. --actin and Histone H3 were cytoplasmic and nuclear sample loading controls, respectively.
  • FIG. 28 After shRNA-mediated IL-1 ⁇ knockout, stromal cells were treated with bleomycin and/or RAD001, and their IKK ⁇ , IKK ⁇ phosphorylation, IkB ⁇ and IRAK1 protein expression, and p65/p50 were analyzed by Western blot. Nuclear situation. --actin and Histone H3, protein loading controls.
  • FIG. 29 Stromal cells treated with Akt inhibitor MK2206 were harvested and lysed on day 7 after bleomycin injury, and their activation of p38, Akt and mTOR was analyzed by Western blot.
  • FIG. 30 The catalytic subunit p110 of PI3K was knocked out by shRNA, and the stromal cells were treated with bleomycin. On day 7 after injury, cells were lysed and analyzed for p38, Akt, mTOR activation, and changes in p110 and p85 ⁇ expression levels.
  • FIG. 31 After IKKa and IKK ⁇ were knocked out by shRNA, respectively, stromal cells were treated with bleomycin and/or SB203580 and collected and lysed after 7 days. The activation level of p38 and its substrate HSP27, IKK ⁇ and IKK ⁇ , and the nuclear access of NF-kB subunit were analyzed by Western blot.
  • Figure 32 Detection of NF- ⁇ B transcriptional activity based on reporter vectors.
  • the stromal cells were treated with PI3K small molecule inhibitor LY294002 (1 ⁇ M), Akt inhibitor MK-2206 (100 nM), mTOR inhibitor RAD001 (50 nM), p38 inhibitor SB203580 (10 ⁇ M) and TAK1 inhibitor 5Z-7 (500 nM).
  • Bleomycin treatment cell lysate was used to determine luciferase activity.
  • the extracellular fluid released from each group of cells in Fig. 33 and Fig. 32 was subjected to ELISA to determine the protein expression level of the exogenous factor IL-8.
  • Figure 35 GSEA method comparative analysis of the relative expression of SASP-specific expression tags in stromal cells after bleomycin and/or 5Z-7 treatment.
  • NES normalized enrichment score
  • FDR false discovery rate.
  • Figure 36 similar to Figure 35, GSEA comparative analysis of the difference in expression of SASP tags between bleomycin and/or SB203580.
  • FIG 37 Similar to Figure 35, GSEA compares the difference in expression between the SASP tag and bleomycin and/or RAD001.
  • FIG. 38 The chord diagram shows the relationship between the factors in which stromal cells are significantly down-regulated by 5Z-7, SB or RAD001, respectively (fold change > 2).
  • Meta a set of meta databases from the data generated by the three inhibitors, respectively, to enhance statistical effectiveness and comparison effectiveness between groups.
  • Figure 39 KEGG pathway analysis and assessment of the biological relationship between top 809 genes with 5Z-7 specific downregulation (fold change > 2).
  • An IMEx interactome database was used for protein-protein interaction analysis.
  • Figure 40 Figure 39 shows the network of signal nodes constructed by top 809 genes. Green, canonical SASP factor. Wired, speculative protein-protein interactions.
  • Figure 41 Fluorescence quantitative RT-PCR analysis of multiple SASP factor expression changes after stromal cell treatment with bleomycin and/or 5Z-7.
  • Figure 42 Experimental procedure for stromal cell culture, drug treatment, indirect co-culture with cancer cells, and phenotypic detection in vitro.
  • Figure 43 The tendency of stromal cell extracellular fluid to promote prostate cancer epithelial cell proliferation decreased significantly under the action of 5Z-7.
  • Figure 44 The ability of stromal cell extracellular fluid to promote migration of prostate cancer epithelial cells in vitro was significantly reduced by 5Z-7.
  • Figure 45 The pattern of stromal cell extracellular fluid promoting prostate cancer epithelial cell invasion was significantly attenuated by 5Z-7.
  • Figure 46 Morphological changes of prostate cancer PC3 cell line under drug release conditions in vitro. SASP inhibitors RAD001, SB203580 and 5Z-7 were processed simultaneously with MIT, or the results of MIT alone treatment were compared in parallel.
  • FIG. 47 After several different treatments, PSC27 cells were collected for extracellular fluid, which was then used to culture prostate cancer epithelial cell lines (BPH1, M12, PC3, DU145 and LNCaP), while IC50 concentration was added to the culture medium. Statistical comparison of the number of viable cells after treatment with the drug mitoxantrone.
  • Figure 48 Comparison of apoptotic index under different conditions for PC3 cells cultured in extracellular fluid collected after treatment of PSC27 with drug mitoxantrone (IC50 concentration) and several SASP inhibitors (including 5Z-7). Caspase 3/7 activity assay results were used for direct mapping.
  • Figure 49 Extracellular fluid produced by each subline of PSC27 was used to treat PC3 cells, which was compared to the cell viability of the untreated control group in the presence of different concentrations of mitoxantrone. Dose response curve, nonlinear regression.
  • FIG. 50 Stromal cell extracellular fluid was used to treat prostate cancer epithelial cell lines, which were simultaneously compared in parallel by the microtubule drug pacetaxel (DTX) (IC50 concentration).
  • DTX microtubule drug pacetaxel
  • Figure 51 Comparison of apoptosis index of PC3 under the treatment conditions of several drug combinations. Caspase 3/7 activity values were used for apoptosis testing.
  • Figure 52 Comparison of cell viability under several different drug combination treatment conditions. Dose response curve, nonlinear regression.
  • Figure 53 The tendency of stromal cell extracellular fluid to promote prostate cancer epithelial cell proliferation was significantly reduced by the action of another inhibitor of TAK1, LYTAK1.
  • Figure 54 The ability of stromal cell extracellular fluid to promote migration of prostate cancer epithelial cells in vitro was significantly reduced by the action of LYTAK1.
  • Figure 55 The pattern of stromal cell extracellular fluid promoting prostate cancer epithelial cell invasion is significantly attenuated by the action of LYTAK1.
  • FIG. 56 After treatment with several different drugs, PSC27 cells were collected for extracellular fluid and subsequently used to culture prostate cancer epithelial cell lines (BPH1, M12, PC3, DU145 and LNCaP) with IC50 concentration in the culture medium. Statistical comparison of the number of viable cells after treatment with the drug mitoxantrone.
  • FIG 57 Comparison of apoptotic index under different conditions for the culture of extracellular fluid collected after PC3 cells were treated with mitoxantrone and several SASP inhibitors (including LYTAK1). Caspase 3/7 activity assay results were used for direct mapping.
  • Figure 58 Extracellular fluid produced by each subline of PSC27 was used to treat PC3 cells, which was compared to the cell viability of the untreated control group in the presence of different concentrations of mitoxantrone. Dose response curve, nonlinear regression.
  • FIG 59 Stromal cell extracellular fluid (including LYTAK1 treated group) was used to treat prostate cancer epithelial cell lines, which were simultaneously compared in parallel with the number of cells administered by the microtubule drug pacetaxel (DTX) (IC50 concentration).
  • DTX microtubule drug pacetaxel
  • Figure 60 Comparison of the apoptotic index of PC3 under the treatment conditions of several extracellular fluids of paclitaxel and PSC27. Caspase 3/7 activity values were used for apoptosis testing.
  • Figure 61 Extracellular fluid produced under several conditions of PSC27 was used to treat PC3 cells, which was compared to the cell viability of the untreated control group in the presence of different concentrations of paclitaxel. Dose response curve, nonlinear regression.
  • FIG. 62 Clinically, patients with prostate cancer (PCa) were analyzed for histochemical staining for TAK1 phosphorylation (activation) in tissues before and after chemotherapy. Selected samples (left and right) were representative tissues before chemotherapy and after chemotherapy, respectively. .
  • PCa prostate cancer
  • Figure 63 Statistical analysis of survival of PCa patients based on TAK1 activation. The number of patients with low expression of p-TAK1 was 20, and that of the high expression group was 30.
  • Figure 64 Experimental flow diagram of mice in a preclinical trial subcutaneously inoculated with cancer cells and/or stromal cells, followed by chemotherapeutic drug treatment and pathological analysis.
  • Figure 65 Pre-clinical chemotherapy protocol. The entire process lasted for 8 weeks, and the drug treatment began in the third week after the mouse inoculation tissue reorganization. MIT is injected intraperitoneally every other week, and 5Z-7 or LYTAK1 are administered simultaneously. At the end of the 8th week trial, the mouse tumor volume was measured and histologically analyzed.
  • Figure 66 Comparative analysis of tumor volume statistics in mice. Compared with the fifth group, the volume of the sixth group decreased by 37%; compared with the sixth group, the volume of the eighth group decreased by 60%.
  • the tumor growth and development in vivo were detected by BLI during the course of chemotherapy.
  • the fluorescein signal indicates that the cancer cells are concentrated near the subcutaneous hind legs and there are no signs of distant metastasis.
  • Figure 68 Comparison of median survival curves of mice under several drug or drug combination treatment conditions. The difference between MIT and MIT/5Z-7 was significant, P ⁇ 0.0001.
  • Figure 69 the same as Figure 66, but the mice underwent a combination therapy with the SASP inhibitor RAD001 and MIT.
  • the corresponding tumor volume reductions were 36% and 44%, respectively.
  • Figure 70 the same as Figure 66, but the mice underwent a combination therapy with SASP inhibitor SB and MIT. .
  • the corresponding tumor volume reductions were 36% and 46%, respectively.
  • Figure 71 Parallel comparison of transcript levels of chemokine IL-8 expression levels between groups after microdissection of mouse tumors by laser capture.
  • Figure 72 Parallel comparison of transcript levels of extracellular factor AREG expression levels between groups after microdissection of mouse tumors by laser capture.
  • Figure 73 Parallel comparison of transcript level growth factor SPINK1 expression levels between groups after microdissection of mouse tumors by laser capture.
  • Figure 74 Parallel comparison of transcript levels of extracellular matrix metalloproteinase MMP3 expression levels between groups after microdissection of mouse tumors by laser capture.
  • Figure 75 Comparative analysis of tumor volume in mouse terminal (LYTAK1 instead of 5Z-7 administration). Compared with Group 5, the volume of Group 6 decreased by 37%; compared with Group 6, the volume of Group 8 decreased by 63%.
  • Figure 76 Parallel comparison of transcript level levels of chemokine IL-8 at the transcript level after microdissection of mouse tumors (including the LYTAK1 administration group) after laser capture microdissection.
  • Figure 77 Parallel comparison of transcript levels of extracellular factor AREG expression levels between groups in mouse tumors (including the LYTAK1 administration group) after laser capture microdissection.
  • Figure 78 Parallel comparison of transcript level growth factor SPINK1 expression levels between groups after microdissection of mouse tumors (including LYTAK1 administration group) after laser capture microdissection.
  • Figure 79 Parallel comparison of transcript levels of extracellular matrix metalloproteinase MMP3 expression levels between groups after microdissection of mouse tumors (including the LYTAK1 administration group) after laser capture microdissection.
  • Figure 80 IHC and HE pathology analysis of orthotopic tumor tissue in clinical NSCLC and BCa patients.
  • the upper group of each cancer type was not treated with chemotherapy, and the lower layer was treated with chemotherapy.
  • the left sample is the result of IHC staining based on p-TAK1, in which the selected area of the red frame is magnified in the middle and the right side is the corresponding HE staining result of the intermediate tissue.
  • Figure 81 Statistical analysis of survival of NSCLC patients based on TAK1 activation. The number of patients with low expression of p-TAK1 was 71, and that of the high expression group was 28.
  • Figure 82 Statistical analysis of survival of BCa patients based on TAK1 activation. The number of patients with low expression of p-TAK1 was 25, and that of the high expression group was 37.
  • Figure 83 End-stage tumor volume of NSCLC xenograft mice (NSCLC cell line A549/stromal cell line WI38; LYTAK1 for single administration or synergistic treatment with bleomycin combined with chemotherapy). Compared with the fifth group, the volume of the sixth group decreased by 38%; compared with the sixth group, the volume of the eighth group decreased by 63%.
  • FIG 84 Tumor tumor volume of BCA xenograft mice (BCa cell line MDA-MB-231/stromal cell line HBF1203; LYTAK1 for single administration or synergistic treatment with bleomycin combined with chemotherapy). Compared with the fifth group, the volume of the sixth group decreased by 32%; compared with the sixth group, the volume of the eighth group decreased by 67%.
  • TAK1 may play an important biological role in the development of the SASP phenotype, and it is also closely related to the development of tumors. Therefore, TAK1 can be used as a research target for SASP phenotype regulation, and can be used as a marker for diagnosis and prognosis of tumors, and can also be used as a target to develop drugs for inhibiting tumors.
  • TAK1 Transforming growth factor kinase 1 (TAK1) has a Gene ID of 6885 in GenBank and encodes a protein of 606 amino acids.
  • TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is functionally located upstream of mitogen-dependent protein kinase (MAPK) and IKB kinase. In vivo, it can be activated by a variety of cytokines including IL-1, participate in many important physiological processes of the body, and can be activated by co-expression with the binding protein TAB1. Phosphorylation is thought to be an important regulatory mechanism in TAK1-dependent signal transduction, but the regulatable phosphorylation site of TAK1 protein has not been fully confirmed. The activation mechanism of TAK1 at the molecular level has not been fully elucidated.
  • the present inventors have found that the ATM-TRAF6-TAK1 signal axis regulates the activation of NF- ⁇ B complex in an acute response induced by stromal cell DNA damage, which is closely related to the expression of a chronic SASP downstream effector. TAK1-mediated activation of the p38MAPK signaling pathway under DNA damage conditions, but drug inhibition against its kinase activity does not affect DNA damage response and stromal cell proliferation potential. Moreover, the mTOR pathway downstream of TAK1 plays an important role in the development of the chronic phase of SASP.
  • TAK1 can reverse multiple malignant phenotypes conferred on cancer cells by injured stromal cells under in vitro conditions.
  • Targeting TAK1 can effectively restore the sensitivity of tumors to chemotherapy drugs by interfering with the development of SASP in stromal cells in the microenvironment.
  • the TAK1 down-regulation alone has no significant effect on tumor growth.
  • Simultaneous administration with conventional chemotherapy drugs can cause a significant decrease in tumor volume by blocking the micro-environment SASP secretion phenotype, and thus can be a new example of old drugs. .
  • SASP senescence associated secretory phenotype
  • the aging-related diseases include: atherosclerosis, osteoarthritis, osteoporosis, and other organ degenerative diseases.
  • the present invention provides a use of a downregulator of the TAK1 gene or protein for the preparation of a pharmaceutical composition for inhibiting tumors.
  • the tumor is selected from the group consisting of: a chemotherapeutic drug treated or a radiation treated tumor; or the tumor is a tumor in which stromal cells express TAK1.
  • the chemotherapeutic drug treatment or radiation treatment includes: docetaxel, paclitaxel, bleomycin, mitoxantrone, radiation therapy radiation, and the like.
  • the downregulator of the TAK1 gene or protein includes inhibitors, antagonists, blockers, blockers, and the like.
  • the down-regulation agent of TAK1 gene or protein refers to any activity which can reduce the activity of TAK1 protein, decrease the stability of TAK1 gene or protein, down-regulate the expression of TAK1 protein, reduce the effective action time of TAK1 protein, or inhibit the transcription and translation of TAK1 gene.
  • These substances can be used in the present invention as a substance useful for down-regulating TAK1, and thus can be used for inhibiting tumors.
  • the down-regulator is an interfering RNA molecule or an antisense nucleotide that specifically interferes with TAK1 gene expression; or an antibody or ligand that specifically binds to a protein encoded by the TAK1 gene, and the like.
  • the lowering agent is a small molecule compound for TAK1. Screening of such small molecule compounds can be performed by one of ordinary skill in the art using routine screening methods in the art.
  • the small molecule compound is 5Z-7-oxozeaenol (5Z-7) or LYTAK1.
  • the down-regulator is a TAK1-specific interfering RNA molecule (shRNA), and the inventors observed that with the interfering RNA molecule of the present invention, TAK1 can be significantly down-regulated for tumors. The inhibition is very significant.
  • shRNA TAK1-specific interfering RNA molecule
  • the preparation method of the interfering RNA molecule is not particularly limited, and includes, but is not limited to, chemical synthesis, in vitro transcription, and the like.
  • the interfering RNA can be delivered to the cells by the use of appropriate transfection reagents, or can be delivered to the cells using a variety of techniques known in the art.
  • targeted CRISPR editing can be performed using the CRISPR/Cas9 system to knock out the TAK1 gene in a disease-targeting region.
  • a common method of knocking out the TAK1 gene includes co-transporting a sgRNA or a nucleic acid capable of forming the sgRNA, Cas9 mRNA, or a nucleic acid capable of forming the Cas9 mRNA into a targeting region or a targeting cell. After the target site is determined, known methods can be employed to introduce sgRNA and Cas9 into the cell.
  • the nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector
  • the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and the expression vector is introduced into the cell, thereby thereby Active sgRNA and Cas9 mRNA are formed therein.
  • TAK1 can be used as a marker for tumor prognosis evaluation: (i) performing tumor typing, differential diagnosis, and/or susceptibility analysis; (ii) evaluating tumor treatment drugs in relevant populations, Drug efficacy, prognosis, and the choice of appropriate treatment. For example, a population with abnormal expression of the TAK1 gene can be isolated, thereby enabling more targeted treatment.
  • the tumor prognosis of the subject providing the sample to be evaluated can be predicted by judging the expression or activity of TAK1 in the sample to be evaluated, and the appropriate drug is selected for the treatment.
  • a threshold of TAK1 can be specified, and when the expression of TAK1 is higher than the prescribed threshold, treatment with a regimen that inhibits TAK1 is considered.
  • the threshold is easily determined by those skilled in the art, for example, by comparing the expression of TAK1 in the normal human tissue microenvironment with the expression of TAK1 in the tumor patient's microenvironment, an abnormal expression of TAK1 is obtained. Threshold.
  • the present invention provides the use of a TAK1 gene or protein for the preparation of a reagent or kit for tumor prognosis evaluation.
  • TAK1 gene can be detected by the present invention.
  • existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, and the like can be used, and these methods can be used in combination.
  • the invention also provides reagents for detecting the presence or absence and expression of a TAK1 gene in an analyte.
  • a primer that specifically amplifies TAK1; or a probe that specifically recognizes TAK1 can be used to determine the presence or absence of the TAK1 gene; when the protein level is detected, specificity can be used.
  • the expression of the TAK1 protein is determined by binding to an antibody or ligand of a protein encoded by TAK1.
  • the design of a specific probe for the TAK1 gene is a technique well known to those skilled in the art, for example, to prepare a probe which specifically binds to a specific site on the TAK1 gene, and is not specific to a gene other than the TAK1 gene. Sexually bound, and the probe carries a detectable signal.
  • TAK1 protein in an analyte using an antibody that specifically binds to the TAK1 protein are also well known in the art.
  • the present invention also provides a kit for detecting the presence or absence and expression of a TAK1 gene in an analyte, the kit comprising: a primer for specifically amplifying a TAK1 gene; a probe for specifically recognizing the TAK1 gene; or a specific An antibody or ligand that binds to a protein encoded by the TAK1 gene.
  • the kit may further include various reagents required for extracting DNA, PCR, hybridization, color development, etc., including but not limited to: extract, amplification solution, hybridization solution, enzyme, and control solution. , coloring liquid, washing liquid, etc.
  • instructions for use and/or nucleic acid sequence analysis software and the like may also be included in the kit.
  • the specific tumor is selected from the group consisting of prostate cancer, breast cancer, and lung cancer.
  • the present invention provides a method of screening for a potential substance that inhibits tumors, the method comprising: treating a system expressing TAK1 with a candidate substance; and detecting expression or activity of TAK1 in the system; if the candidate substance is inhibited
  • the expression or activity of TAK1 indicates that the candidate substance is a potential substance for inhibiting tumors.
  • the TAK1-expressing system is a cell (or cell culture) system, and the cell may be a cell that endogenously expresses TAK1; or may be a cell that recombinantly expresses TAK1.
  • a control group in order to make it easier to observe changes in the expression or activity of TAK1, a control group may be provided, and the control group may be a system expressing TAK1 without adding the candidate substance. .
  • the method further comprises performing further cellular experiments and/or animal tests on the obtained potential substances to further select and determine substances that are truly useful for inhibiting the tumor.
  • the invention also provides a potential substance for inhibiting tumors obtained by the screening method.
  • These initially screened materials can constitute a screening library so that one can finally screen out substances that can be useful for inhibiting the expression and activity of TAK1 and thereby inhibiting tumors.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount (e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight) of the TKa1 gene or protein down-regulating agent And a pharmaceutically acceptable carrier.
  • an effective amount e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • a pharmaceutically acceptable carrier e.g., 0.000001 to 50% by weight; preferably 0.00001 to 20% by weight; more preferably 0.0001-10% by weight
  • Any of the aforementioned down-regulators of the TAK1 gene or protein can be used in the preparation of the composition.
  • compositions for inhibiting tumors comprising an effective amount of the interfering RNA molecule of the present invention, and a pharmaceutically acceptable carrier.
  • composition for inhibiting tumors which comprises an effective amount of a down-regulator of a TAK1 gene or protein, and an effective amount of other preparation, said other preparation
  • a genotoxic drug or a DNA-damaging drug for example, a genotoxic drug or a DNA-damaging drug, an ionizing radiation therapy drug.
  • the "effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • the “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, or a buffer.
  • auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the vector may also contain a cell transfection reagent.
  • the down-regulator or its coding gene, or a pharmaceutical composition thereof can be administered to a mammal by a variety of methods well known in the art. These include, but are not limited to, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the administration mode is parenterally administered.
  • the down-regulator of TAK1 can be directly administered to a subject by a method such as injection; or, the expression unit (such as an expression vector or virus, or siRNA) carrying a down-regulator of TAK1 can be delivered to a certain route to The TAK1 down-regulation on the target and expressing it, depending on the type of down-regulation described, is well known to those skilled in the art.
  • the expression unit such as an expression vector or virus, or siRNA
  • the effective amount of the down-regulator of the TAK1 gene or protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the TAK1 gene or protein down-regulator, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's Immunization status, route of administration, etc.
  • the normal human prostate primary stromal cell line PSC27 (obtained from the Fred Harrison Cancer Research Center, USA) was propagated and passaged in PSCC complete medium.
  • Prostate benign epithelial cell line BPH1, prostate cancer epithelial cell line M12, DU145, PC3, LNCaP and VCaP (purchased from ATCC) were all in 5% FBS in RPMI-1640 complete medium at 37 ° C, 5% CO 2 Culture in an incubator.
  • Logarithmic growth phase cells were harvested with 0.25% trypsin, centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in freshly disposed cryopreservation. The cells are dispensed into the indicated sterile cryotubes. Then, the gradient was cooled (4 ° C for 10 min, -20 ° C for 30 min, -80 ° C for 16-18 h), and finally transferred to liquid nitrogen for long-term storage.
  • the frozen cells in liquid nitrogen were taken out and immediately placed in a 37 ° C water bath to allow rapid melting. Add 2 ml of cell culture medium directly to allow the cells to be evenly suspended. After the cells are attached, replace the new medium.
  • PSC27 cells were grown to 80% (referred to as PSC27-Pre) with 100 nM docetaxel (DTX), 100 nM paclitaxel (PTX), and 200 nM vincristine (VCR). 50 ⁇ g/ml bleomycin (BLEO), 1 ⁇ M mitoxantrone (MIT), or 10 Gy 137 Cs ionizing radiation ( ⁇ -radiation at 743 rad/min, RAD).
  • BLEO bleomycin
  • MIT mitoxantrone
  • RAD 10 Gy 137 Cs ionizing radiation
  • the fusion protein of full-length human IKK ⁇ and Flag was cloned between the expression vector pCR3.1 restriction sites BamHI and XbaI.
  • the fusion protein of full-length human mTOR and GST was cloned between a pair of NotI restriction sites of the expression vector pcDNA3.
  • the packaging line 293FT was used for cell transfection and lentiviral production.
  • the sequences of small hairpin RNAs (shRNAs) used to knock out TRAF6 are:
  • shRNAs small hairpin RNAs
  • shRNAs small hairpin RNAs
  • Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) or mouse monoclonal antibody anti-Phosphor-53BP1 (Cat# sc-135748, Santa Cruz), and secondary antibody Alexa 488 (or 594)-conjugated F(ab') 2 was added sequentially to slides coated with fixed cells.
  • the nuclei were counterstained with 2 ⁇ g/ml of 4',6-diamidino-2-phenylindole (DAPI). The most representative image was selected from the three observation fields for data analysis and results display.
  • the FV1000 laser scanning confocal microscope (Olympus) was used to obtain confocal fluorescence images of cells to determine DNA damage.
  • the anti-TAK1 antibody used for IHC staining in clinical prostate cancer, non-small cell lung cancer patients and breast cancer patients was purchased from Proteintech. The specific steps are as follows: conventional dewaxing, incubation with 0.6% H 2 O 2 methanol at 37 ° C for 30 min, then repaired with 0.01 M pH 6.0 citrate buffer for 20 min, and cooled at room temperature for 30 min. The cells were blocked with normal goat serum for 20 min, incubated with TAK1 primary antibody (1:200) for 1 h at 37 ° C, and transferred to a refrigerator at 4 ° C overnight.
  • the cells were washed three times with TBS, and the secondary antibody (HRP-conjugated goat anti-rabbit) was incubated at 37 ° C for 45 min, then washed with TBS for 3 times, and finally developed with DAB.
  • the secondary antibody HRP-conjugated goat anti-rabbit
  • the PSC27 cells were cultured for 3 days with DMEM + 0.5% FBS medium, and then the abundance of the cell population was washed with 1 time PBS. After simple centrifugation, the supernatant was collected and stored as a conditional medium at -80 ° C or used directly.
  • Prostate epithelial cells were cultured in vitro in this conditional medium for 3 days.
  • epithelial cell lines are cultured in low serum DMEM (0.5% FBS) ("DMEM”), or in a conditioned medium, while mitoxantrone (MIT) is used to treat cells for 1 to 3 days, concentrations near IC 50 value of each cell line, and then observed under a bright field microscope.
  • RNA of the growth phase was extracted with Trizol reagent, and 1 ml of Trizol was added to each T25 flask, and the cell layer was scraped off with a cell scraper, transferred to a centrifuge tube, and thoroughly mixed until not thick.
  • RNA status and quality After quantifying the RNA with a spectrophotometer, a small amount of total RNA was taken for 1% agarose electrophoresis to check the RNA status and quality.
  • First strand cDNA synthesis was performed according to the following conditions:
  • the reverse transcription reaction product cDNA was diluted 50-fold as a template.
  • the reaction conditions were: pre-denaturation at 95 ° C for 15 s, then 95 ° C for 5 s, 60 ° C for 31 s, 40 cycles; the melting curve conditions were 95 ° C for 15 s, 60 ° C for 30 s, and 95 ° C for 15 s.
  • the samples were reacted on an ABI ViiA7 (ABI) instrument.
  • the expression of ⁇ -actin was used as an internal reference.
  • the amplification of each gene was analyzed by software analysis, and the corresponding number of domain value cycles were derived.
  • the relative expression of each gene was calculated by the 2- ⁇ Ct method. The peaks and waveforms of the melting surve are analyzed to determine if the resulting amplification product is a specific single-purpose fragment.
  • RIPA cell lysis buffer (Invitrogen) containing 1 mM PMSF (protease inhibitor) was added, and the cells were lysed on ice for 30 min, and the cell lysate was collected with a cell scraper at 1 ° C for 12,000 °C. Centrifuge at rpm for 15 min, take the supernatant, and store at -80 °C.
  • the BCA protein quantification kit (Pierce) was prepared by mixing reagent A and reagent B in a ratio of 1:50 to prepare a working solution for use.
  • the standard protein was diluted to a concentration of 0 ⁇ g/ ⁇ l, 25 ⁇ g/ ⁇ l, 50 ⁇ g/ ⁇ l, 100 ⁇ g/ ⁇ l, 250 ⁇ g/ ⁇ l, 500 ⁇ g/ ⁇ l, 750 ⁇ g/ ⁇ l, 1000 ⁇ g/ ⁇ l, 2000 ⁇ g/ ⁇ l.
  • Add 5 ⁇ l of standard protein or 5 ⁇ l of sample to the plate add 100 ⁇ l of BCA working solution, mix well and then bath at 37 ° C for 30 min, and read the absorbance at 570 nm with a microplate reader.
  • a standard curve is drawn by taking the absorbance value as the ordinate and the standard protein concentration as the abscissa. The concentration of the sample was calculated from the standard curve.
  • Protein samples were mixed in a 5:1 ratio with 6 ⁇ loading buffer (containing 300 mM Tris-HCl, 12% SDS, 600 mM DTT, 60% glycerol, 0.6% bromophenol blue), boiled in water for 10 min, ice bath After cooling for 5 min, combined with protein quantification results, the same amount of protein samples were added to each lane, and electrophoresed by Bio-Rad electrophoresis apparatus. Electrophoresis was carried out at 80 V for about 20 minutes until the front of bromophenol blue entered the separation gel, and the voltage was raised to 120 V. Electrophoresis was continued for about 1 hour until the bromophenol blue band reached the bottom of the separation gel and the electrophoresis was completed.
  • 6 ⁇ loading buffer containing 300 mM Tris-HCl, 12% SDS, 600 mM DTT, 60% glycerol, 0.6% bromophenol blue
  • the nitrocellulose filter was blocked in a blocking solution (TBST (0.1% Tween-20 in TBS) containing 5% skim milk powder) for 1 hour at room temperature. Incubate overnight in a primary anti-hybrid solution at 4 °C. Rinse 3 times with TBST at room temperature for 2 minutes each time. HRP-conjugated secondary antibody hybrids prepared in blocking solution were added and incubated for 0.5 hour at room temperature. The filter was rinsed 3 times with PBST at room temperature for 2 min each time.
  • TBST 0.1% Tween-20 in TBS
  • 5% skim milk powder 5% skim milk powder
  • the anti-viral vector pBabe-Puro-I ⁇ B ⁇ -Mut (super repressor) containing two IKK phosphorylation mutation sites S32A and S34A encoding the I ⁇ B ⁇ protein sequence was used to transfect the lentiviral packaging cell line PHOENIX. Lentivirus was subsequently used to infect the PSC27 stromal cell line, while 1 ⁇ g/ml puromycin was used to screen for positive clones. As another method, 5 ⁇ M of the small molecule inhibitor Bay 11-7082 (purchased from Selleck) was used for NF- ⁇ B activity control.
  • the stromal cells are then exposed to several different forms of cytotoxicity, and the resulting phenotype is recorded in time to analyze the expression of the relevant genes.
  • the cells treated in this manner are collected and the conditioned medium is collected for various detections of epithelial cells.
  • the chemotherapy regimen was based on the pathological features of patients with castration-resistant prostate cancer, relapsed and refractory non-small cell lung cancer (Clinical Trial Registration No. NCT02889666) and patients with osmotic ductal breast cancer (Clinical Trial Registration No. NCT02897700). .
  • Patients with a clinical stage of prostate cancer below T2a and without significant distant metastatic lesions were recruited into the clinical cohort.
  • Patients with primary lung cancer above I subtype A (IA) T1a, N0, M0
  • no significant distant metastatic lesions were enrolled in the clinical cohort.
  • the immunological activity score was classified as 0-1 (negative), 1-2 (weak), 2-3 (middle), 3-4 (strong) four according to the histological staining of each tissue sample. Class (Fedchenko and Reifenrath, 2014). The diagnosis of NSCLC and BCa samples was judged and scored by pathologists independent of each other. The randomized controlled trial (RCT) protocol and all experimental procedures were approved and authorized by the IRB of Shanghai Jiao Tong University School of Medicine and gradually developed in accordance with authoritative guidelines.
  • IACUC Laboratory Animal Care and Use Committee
  • ICR SCID mice body weight about 25 g
  • PSC27 stromal cells and epithelial cells in a 1: 4 ratio mixture, and each graft containing 1.25 ⁇ 10 6 cells, for tissue reconstruction.
  • the transplanted tumor was implanted into the mouse by subcutaneous transplantation, and the animal was euthanized 8 weeks after the end of the transplantation.
  • lung cancer and breast cancer xenografts were composed of A549 (non-small cell lung cancer cell line) and WI38 (lung fibroblast cell line), MDA-MB-231 (triple negative breast cancer cell line) and HBF1203 (mammary fibroblast), respectively. Department) formed by tissue remodeling.
  • mice that were subcutaneously transplanted were fed a standard experimental diet, and two weeks later, the chemotherapy drug mitoxantrone (0.2 mg/kg dose) and/or SASP inhibitor (500 ⁇ l, 10 mg/kg dose, RAD001, SB203580 and 5Z-7 were purchased from TOCRIS; LYTAK1, purchased from Lilly Co (Indianapolis, IN), 5 mg/kg, administered intraperitoneally.
  • the time point was the first day of the 3rd, 5th, and 7th week, and the entire course of treatment was administered in 3 cycles, each cycle being 2 weeks.
  • mouse kidneys were collected for tumor measurement and histological analysis.
  • mice Each mouse cumulatively received mitoxantrone 0.6 mg/kg body weight, and the SASP inhibitor 30 mg/kg body weight (LYTAK1 was 15 mg/kg body weight).
  • Lung cancer and breast cancer xenograft mice received bleomycin (0.3 mg/kg total) and doxorubicin (0.2 mg/kg total), respectively, at the same time and frequency as mitoxantrone.
  • the chemotherapy test was carried out until the end of the 8th week, the mice were dissected immediately after sacrifice, and the transplanted tumors were collected and used for pathological analysis.
  • Example 1 The ATM-TRAF6-TAK1 signal axis regulates the activation of NF- ⁇ B complex in the acute response induced by stromal cell DNA damage, which is closely related to the expression of chronic SASP downstream effector.
  • stromal cells will have a special physiological response in a short period of time, that is, an acute stress-associated phenotype (ASAP).
  • ASAP acute stress-associated phenotype
  • Many exogenous factors in the genome-wide range are highly up-regulated during this phenotypic formation process, and the phenotype will then gradually transition to a senescence-associated secretory phenotype (SASP), the latter being a Chronic, long-term and stable state.
  • SASP senescence-associated secretory phenotype
  • the regulation of which molecular and cellular mechanisms is still unclear.
  • ATM that senses DNA damage signals, whether it plays a key role in the cell is also a question that many scientists in the world have heatedly discussed and answered in recent years.
  • the inventors first analyzed the stromal cell lysate after bleomycin treatment by phosphorylation of ATM (p-ATM) antibody using ChIP, and found that there is an interaction between activated ATM and TRAF6, but can be inhibited by ATM.
  • the agent KU55933 was abolished ( Figure 1). Since ATM binding to TRAF6 activates TRAF6-mediated poly-ubiquitination and leads to some downstream reactions including TAK1 activation, the inventors immediately analyzed whether similar phenomena exist in stromal cells. To this end, cell lysates were detected with anti-TRAF6 using IP after bleomycin treatment of stromal cells, noting the rapid growth of auto-ubiquitination of TRAF6, confirming its ubiquitin in injured cells.
  • TRAF6 interacts with both activated ATM and TAK1, suggesting that TRAF6 can act as an intermediate molecule to deliver ATM signals to TAK1 (Fig. 3).
  • knockout of TRAF6 abolished TAK1 activation in damaged stromal cells, rather than ATM activation, again confirming the specific role of TRAF6 in mediating ASAP acute response signals.
  • TAK1 indirectly activated by upstream DDR signaling, associated with activation of a core transcription factor involved in SASP broad-spectrum expression, the NF- ⁇ B complex?
  • IKK ⁇ I ⁇ B kinase subunit gamma
  • NF- ⁇ B is an event downstream of the signaling pathway mediated by TAK1 in the cytoplasm of stromal cells (Fig. 4).
  • Example 2 TAK1-mediated activation of the p38MAPK signaling pathway under DNA damage conditions, but drug inhibition against its kinase activity does not affect DNA damage response and stromal cell proliferation potential
  • TAK1 can activate MAPK family members such as p38, Jnk and Erk in a variety of physiological processes including local inflammation and tissue homeostasis. The inventors hypothesized that TAK1 is involved in the chronic progression of SASP.
  • the present inventors analyzed changes in the activity of TAK1 in the presence or absence of 5Z-7-oxozeaenol (hereinafter referred to as 5Z-7, which is called dihydroxybenzoic acid lactone) after bleomycin treatment.
  • 5Z-7 which is called dihydroxybenzoic acid lactone
  • the stromal cell lysate collected under in vitro conditions was detected by IP and in vitro kinase assay, and it was found that DNA damage activated TAK1, leading to phosphorylation, which was confirmed by the interaction between TAK1 and MKK6 (Fig. 5-6). ).
  • the increasing 5Z-7 gradually attenuated the activation of TAK1 in damaged stromal cells, while 5Z-7 at 500 nM substantially abolished TAK1 activation.
  • the present inventors treated PSC27 cells with IL-1 ⁇ , and found significantly enhanced TAK1/MKK6 interaction and a greatly increased p38 kinase activity, which is very similar to the change of PSC27 cells under the action of bleomycin (Fig. 5 to 6).
  • the inventors knocked out IL-1 ⁇ using shRNA prior to bleomycin administration.
  • deletion of IL-1 ⁇ resulted in a significant decrease in the activation of TAK1 in damaged stromal cells and activation of p38MAPK, suggesting that TAK1 activation is regulated by IL-1 ⁇ in these virulence-treated cells (Fig. 7). .
  • TAK1 activation did not alter the DNA damage response.
  • the DDR foci at the single cell level did not change significantly with the presence or absence of 5Z-7 ( Figure 8, Figure 9).
  • the ability of PSC27 cells to clone was determined by DNA damage, but not with the inhibition of TAK1 activity (Fig. 10, Fig. 11).
  • the inventors subsequently used the other two chemotherapeutic drugs to treat the same batch of stromal cells, including mitoxantrone (MIT, a DNA topoisomerase inhibitor) and satraplatin. (Satraplatin, SAT, a platinum analogue), which can cause DNA damage through different mechanisms.
  • mitoxantrone MIT, a DNA topoisomerase inhibitor
  • satraplatin SAT, a platinum analogue
  • TAK1 deficiency does not affect the proliferative potential of cells under in vitro conditions, as evidenced by the cell population doubling curve, which is specifically used to evaluate the expression of cells in continuous passage under culture conditions.
  • Maximum multiplication capacity Figure 14
  • the present inventors also examined whether the phosphorylation state of ATM was changed when TAK1 activity was inhibited after the cells were subjected to genotoxic damage, that is, whether the inhibition of TAK1 affects the intensity of DNA damage reaction from another angle.
  • Western blot data showed that the phosphorylation status of ATM induced by DNA damage did not depend on whether TAK1 activity was inhibited by 5Z-7, although the degree of phosphorylation of JNk and p38 MAPK was significantly decreased when TAK1 was inhibited (Fig. 15).
  • the typical marker effector IL-8 which develops the SASP phenotype, is also significantly down-regulated when DNA damage persists.
  • the inventors' experimental data consistency indicates that maintenance of TAK1 activity is essential for the chronic development of SASP.
  • Example 3 the mTOR pathway downstream of TAK1 plays an important role in the development of the chronic phase of SASP
  • DNA damage promotes the formation of senescent cells, which remain metabolically active and physiologically active within a few months, while exhibiting significantly increased lysosomal mass and enhanced SA-B-Gal enzyme activity. Since molecules such as TAK have a critical signal-mediated role in the acute phase of ASAP following DNA damage, the inventors asked if other molecules were also activated during the acute cellular reaction and promoted the chronic development of SASP.
  • Akt/mTOR activation of Akt/mTOR began to occur in the late stage of acute reaction following DNA damage, which was confirmed by post-translational modification of two sites, Akt (Ser473) and mTOR (Ser2448), both of which were Phosphorylation started 24 hours after bleomycin treatment and entered the plateau after 7 days (Figure 16).
  • Immunofluorescence experiments showed that activated mTOR appeared in the cytoplasm.
  • the inventors then examined changes in pathways and key molecules upstream and downstream of mTOR. For example, phosphorylation of two substrates S6K1 downstream of mTOR and its catalytic subunits S6 and 4E-BP1, while indicating functional activation of mTOR (Fig. 17).
  • rapamycin can reduce the phosphorylation of S6K1 and 4E-BP1 occurring in senescent fibroblasts under irradiation conditions, and can negatively regulate the translation of mRNA with stable secondary structure by intracellular helicase machinery.
  • RAD001 a rapamycin analogue
  • S6K1 and 4E-BP1 activation was also observed in the case where mTOR phosphorylation was blocked, confirming that rapamycin was inhibited as SASP.
  • the effectiveness of the agent ( Figure 18). Despite this, the DDR foci caused by bleomycin remained unchanged in damaged stromal cells (Fig. 17), while cell cycle arrest and SA- ⁇ -Gal activity were not affected in the presence of RAD001, suggesting Cellular senescence and metabolic activity were maintained (Fig. 19, Fig. 20).
  • the inventors will next ask whether mTOR is directly or indirectly related to the activation of the NF- ⁇ B complex.
  • the present inventors analyzed stromal cells after bleomycin treatment, and found that degradation of I ⁇ B ⁇ in the cytoplasm and stabilization of the NF- ⁇ B subunit p65 (Rel A), both of which indicate that the NF- ⁇ B complex is DNA activates the state of activation in cells ( Figure 21).
  • data from reporter vector transfection experiments also confirmed a significant up-regulation of NF- ⁇ B transcriptional activity, but was significantly attenuated in the presence of RAD001 ( Figure 22).
  • mTOR can regulate the expression of SASP broad-spectrum effector factors by restricting the translation of the cytokine IL-1 ⁇ , in which NF- ⁇ B complex is inhibited.
  • IL-1 ⁇ controls NF- ⁇ B transcriptional activity and which IKK subunit specifically mediates signaling pathways for IL-1 ⁇ stimulation.
  • the inventors found that phosphorylation of IKK ⁇ , degradation of IRAK1 and I ⁇ B ⁇ , and entry of NF- ⁇ B complex subunits p65 and p50 occur after DNA damage ( FIG. 27 ). Although these changes were substantially abolished after RAD001 was added to the medium, the addition of IL-1 ⁇ reversed it.
  • TAK1 inhibition can reverse multiple malignant phenotypes conferred on cancer cells by injured stromal cells under in vitro conditions
  • the experimental data of the present inventors confirmed that the formation of SASP can be effectively intervened from the signal pathway upstream thereof, and whether the activity control of TAK1 can have certain biological effects, especially the cancer cells which damage the stromal cells in the microenvironment.
  • a series of phenotypes First, the inventors examined which genes were significantly down-regulated in the case where SASP was inhibited. Comparative transcriptomic data showed that most of the SASP effectors were significantly inhibited when bleomycin was treated alone and in combination with 5Z-7 ( Figure 34). In contrast to the combination treatment of bleomycin/SB20580 and bleomycin/RAD001, 5Z-7-mediated inhibition of TAK1 activity appears to be more effective in down-regulating the expression of most exogenous proteins of SASP. Although there is some volatility between the different SASP factor declines, this trend of overall agreement indicates that broad-spectrum SASP is basically controlled.
  • GSEA Gene Set Enrichment Analysis
  • the inventors evaluated the effect of stromal cell TAK1 inhibition on the proliferation of cancer cells.
  • the inventors collected their extracellular fluid and immediately used it for the culture of prostate cancer cells (Fig. 42).
  • the rate of proliferation of epithelial cancer cells decreased significantly when TAK1 was inhibited (Fig. 43).
  • the extracellular fluid of the injured stromal cells significantly increased the mobility and invasion rate of the cancer cells, these changes were significantly down-regulated when the stromal cells TAK1 were inhibited (Fig. 44, Fig. 45).
  • stromal cells confer acquired viability on cancer cells when damaged (Fig. 45, Fig. 46).
  • the increase in the apoptotic index exhibited by the latter when mitoxantrone was treated with cancer cells was confirmed by the caspase 3/7 activity test data (Fig. 47).
  • the great change in the drug resistance potential of cancer cells is confirmed by the non-linear curve of cell viability caused by mitoxantrone in the concentration range of 0.1-1 ⁇ M.
  • This concentration range is basically the same as that of serum in patients with prostate cancer under clinical conditions.
  • the levels match each other ( Figure 48). Therefore, in either case, the decrease in cancer cell malignant data caused by inhibition of stromal cell TAK1 activity was more significant than that caused by RAD001 and SB203580 (Fig. 49).
  • the present inventors treated the cancer cells under the action of paclitaxel using the same set of CM, and found that the extracellular fluid produced by stromal cells when TAK1 is inhibited can increase the cytotoxicity of paclitaxel to cancer cells, resulting in a decrease in cell survival rate.
  • the apoptotic index increased and the cancer cell response curve shifted (Fig. 50, Fig. 51, Fig. 52). Therefore, the inhibition of stromal cell TAK1 activity caused by 5Z-7 can attenuate the acquired resistance of stromal cells to cancer cells for various chemotherapeutic drugs.
  • LYTAK1-mediated TAK1 inhibition can significantly reduce the proliferation, migration and invasion of cancer cells under the action of stromal cell extracellular fluid (Fig. 53, Fig. 54, Fig. 55).
  • stromal cell extracellular fluid Fig. 53, Fig. 54, Fig. 55.
  • LYTAK1-mediated TAK1 inhibition can significantly reduce the proliferation, migration and invasion of cancer cells under the action of stromal cell extracellular fluid (Fig. 53, Fig. 54, Fig. 55).
  • the extracellular fluid of stromal cells damaged by bleomycin showed a significant decrease in drug resistance to cancer cells in the presence of LYTAK1, ie, the matrix caused by LYTAK1.
  • Inhibition of intracellular TAK1 activity was able to counteract the acquired viability of cancer cells caused by stromal cell damage (Fig. 56, Fig.
  • LYTAK1 can significantly reduce the drug resistance or anti-apoptotic ability of cancer cells obtained under the action of extracellular fluid of stromal cells ( Figure 59, Figure 60, Figure 61).
  • Example 5 targeting TAK1 can effectively restore the sensitivity of tumors to chemotherapy drugs by interfering with the development of SASP in stromal cells in the microenvironment.
  • the broad-spectrum expression of SASP in the microenvironment can accelerate many malignant events, including tumorigenesis, local inflammation, and therapeutic resistance. However, whether this trend toward malignant progression can be avoided by specifically controlling the formation of SASP in the microenvironment, and how to effectively inhibit SASP in the in vivo microenvironment activated by anticancer therapy has been a difficult problem in the scientific community. It should be noted that after clinical chemotherapy, the activation of TAK1, which is closely related to the development of SASP, is common in the tissue microenvironment (the phosphorylation level is significantly increased compared with the pre-treatment period) (Fig. 62). More importantly, the activation status of TAK1 in the tumor microenvironment was significantly negatively correlated with the survival of patients with prostate cancer at the post-treatment stage ( Figure 63).
  • the present inventors inoculated a subcutaneous site of immunodeficient mice with a mixed cell population of prostate-derived stromal cell line PSC27 and epithelial cancer cell line PC3, and then the mice underwent an 8-week pre-clinical chemotherapy.
  • the protocol which included 3 single or dual drug treatments based on a series of pre-experimental data ( Figure 64, Figure 65).
  • PC3 cells still form tumors under the screening pressure caused by chemotherapeutic drugs, although the volume is smaller than that produced by stromal cells and cancer cells simultaneously, this difference is also objectively confirmed.
  • the tumor-promoting effect of the microenvironment (Fig. 66).
  • mice in the mitoxantrone (MIT)/5Z-7 combination treatment group achieved a significantly prolonged median survival, and the animals in this group were less than the mitoxantrone alone in the chemotherapy group.
  • the survival time of the disease was extended by about 50% (Fig. 68, comparing the green and blue groups).
  • the use of 5Z-7 alone only slightly extended mouse survival (Fig. 6586, versus purple and red groups). The above results show that the joint use of MIT/TAK1 is ideal.
  • the inventors systematically compared the difference between the effect of TAK1 inhibition and the result of decreased mTOR or p38 activity under the conditions in which SASP was developed in the microenvironment.
  • the combination treatment of MIT/RAD001 and MIT/SB203580 can significantly reduce the terminal volume of the subcutaneous tumor at the end of the chemotherapy treatment.
  • the combination of MIT/RAD001 resulted in a further 44% reduction in MIT monotherapy, while MIT/SB203580 was further reduced by 46% ( Figure 69, Figure 70).
  • mTOR or p38 is a targeted combination therapy can significantly delay tumor growth, the anti-tumor effect caused by TAK1 inhibition is generally more impressive.
  • the inventors used laser-captured fibers to dissecting the stromal cells in the tumor and performing transcript level analysis, and found that IL-8 was included.
  • SASP effectors including AREG, SPINK1 and MMP3, were significantly reduced ( Figure 71, Figure 72, Figure 73, Figure 74).
  • the inventors further systematically analyzed the pathological association between TAK1 and clinical patient survival. Results from clinical data indicate that there is a significant negative association between TAK1 activity and survival of patients with NSCLC and breast cancer (BCa) (Fig. 80, Fig. 81, Fig. 82). In order to demonstrate the association between TAK1 expression and these two cancer types under in vivo conditions, the inventors further performed pre-clinical experiments using transplanted tumor mice based on tissue remodeling.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

公开了新型的肿瘤微环境相关靶点TAK1及其在抑制肿瘤中的应用。TAK1作为SASP表型调控的研究靶点，可作为肿瘤的诊断、预后评估标志物，还可作为肿瘤微环境的特异靶点开发抑制肿瘤药物。

Description

新型的肿瘤微环境相关靶点TAK1及其在抑制肿瘤中的应用 技术领域
本发明属于药物学领域，更具体地，本发明涉及新型的肿瘤相关靶点TAK1及其在抑制肿瘤中的应用。
背景技术
1.衰老与衰老相关分泌表型
细胞衰老是一个细胞进入永久性细胞周期停滞的过程。细胞衰老常伴随许多病理学特征，包括局部炎症。细胞衰老发生于受损细胞，并防止其在生物体增殖。在各种外界刺激和内部因素影响下，细胞损伤可以导致明显的细胞衰老迹象；当损伤累积和达到一定的限度，组织中呈现各种肉眼可辨的组织退行变化和生理上的衰老表型。
衰老相关分泌表型(senescence-associated secretory phenotype，SASP)的概念是由Coppe等人在2008年首次提出。他们发现衰老细胞能通过分泌胞外基质蛋白、炎症相关因子及癌细胞生长因子促进邻近癌前细胞发生癌变，并称这些蛋白为SASP因子。体内条件下，衰老细胞在各个器官积累的同时伴随一系列复杂的SASP，它是不同种类细胞因子的胞内表达和胞外释放明显增强的一种表型，是衰老细胞的一个重要生化和细胞学特征。SASP包括促炎细胞因子(如IL-1α、IL-1β、IL-6和IL-8)，生长因子(如HGF、TGF-β和GM-CSF)，趋化因子(如CXCL1/3和CXCL10)和基质重塑酶(如MMP1、MMP3)等，SASP的构成成分诱导出的不同的生物学活性表明它与其他细胞作用并构成一个特殊的多配基-多受体信号机制调节组织局部微环境，并具有导致或加剧衰老相关疾病包括癌症等恶性病理现象的多重潜力。
2.SASP的分子机制与药物调节
研究显示，一些理化因素或生物刺激会上调细胞SASP因子的合成与分泌。经过LPS处理的骨髓基质血管细胞也出现SASP，使得TNF-α、IL-1β、IL-6、MCP-1和VEGF的表达均有所升高。HuR不仅调节复制寿命，也调节小鼠成纤维细胞SASP相关细胞因子的表达，RNAi介导的HuR抑制导致SASP相关细胞因子表达的上升。果蝇成虫上皮细胞中，线粒体功能障碍或激活Ras可以导致细胞衰老和SASP。PKCη通过上调细胞周期抑制剂p21Cip1和p27Kip1的表达和增强IL-6的转录和分泌来促进衰老；而IL-8的表达则受PKCη的特殊抑制。
至今报道的基于SASP的大部分数据均为关于促进慢性炎症、旁分泌相关衰老及加速肿瘤恶性进展等病理作用，而有关如何抑制SASP的发展和分泌，实现 延缓衰老和衰老相关疾病的研究则相对较少。
TNF-α是某些细胞类型SASP因子中的主要成分，生物治疗药物阿达木单抗(adalimumab)是可以直接抑制TNF-α的单克隆抗体，它可以减弱SASP的分泌，使IL-6的释放量明显下降，而eNOS和miR-126-3p的表达水平明显增高。阿达木单抗还可以诱导衰老细胞的表观遗传修饰，从而减弱SASP的促癌作用。
某些特定的黄酮类化合物可以抑制SASP的发生，部分和NF-κB亚单位p65以及IκBζ信号通路有关，可以有效地保护或减轻退行性疾病(如心血管疾病和晚期癌症)中的慢性低度炎症。天然黄酮类化合物芹黄素和山柰酚可以强烈抑制SASP的表达，这些类黄酮通过IRAK1/IκBα的信号通路上调IκBζ表达,从而抑制NF-κB亚基p65活性。相反，抑制IκBζ表达则会提高SASP的表达。体内实验中,口服芹黄素可以使老年大鼠肾脏中SASP明显降低，这同IκBζmRNA的水平升高密切关联。
近年发现JAK抑制剂可以减少脂肪前体细胞和脐静脉内皮细胞(HUVEC)SASP的分泌，因此JAK通路可能是对抗衰老相关功能障碍的一个潜在靶点。TRIM28对IL-6、IL-8和其它SASP的成分有正向调节的作用，而当TRIM28耗尽时分泌表型则受到强烈的抑制。
新的数据表明，mTOR抑制剂雷帕霉素可以是一个有效的SASP抑制剂。mTOR通过调节IL-1α和MAPKAPK2的翻译来控制SASP。反过来，在衰老过程中，p38下游信号MAPKAPK2磷酸化RNA结合蛋白ZFP36L1，从而防止SASP因子转录物的降解。当mTOR被药物抑制时，ZFP36L1去磷酸化，导致SASP转录本降解和IL-1α反馈环路的废除。因此，p38MAPK激酶和MK2的小分子抑制剂可以抑制人成纤维细胞的SASP。雷帕霉素降低IL-6和其他细胞因子mRNA水平，并选择性地抑制膜结合细胞因子IL-1α的翻译,分泌减少的IL-1α对NF-κB转录活性刺激也降低，而NF-κB对SASP有着更多的调控作用，揭示了mTOR抑制剂雷帕霉素的抗衰老作用。同时，雷帕霉素可以抑制衰老成纤维细胞刺激小鼠前列腺肿瘤生长的能力，因此，雷帕霉素可能通过抑制衰老相关炎症，改善年龄相关的疾病，包括晚期癌症。
细胞衰老是一个有效的抗癌机制，阻止有丝分裂感受态细胞的增殖，从而防止恶性转化。促衰老疗法最近成为一个治疗癌症的新治疗思路，但这一观念与衰老肿瘤细胞的SASP相冲突，因为SASP可以促瘤，尽管衰老细胞具有抑瘤的效果。在PTEN基因缺失的前列腺癌细胞中，JAK2/STAT3通路的激活，建立了免疫抑制肿瘤微环境，导致肿瘤生长和抗药性发展。通过下调蛋白质酪氨酸磷酸酶PTPN11/SHP2，激活PTEN缺失肿瘤中JAK2/STAT3信号通路，该激活通路是稳定的，这为存在一个新的PTEN/SHP2轴提供了证据。更重要的是，用多烯紫杉醇 联合JAK2抑制剂作用于PTEN缺失肿瘤，将改变SASP并提高多烯紫杉醇诱导衰老的效率。这些说明衰老肿瘤细胞的免疫监视可以在特殊遗传背景下被抑制，但是也可能被药物治疗所激活。衰老细胞产生的外泌蛋白功能，往往取决于衰老肿瘤细胞的基因背景。尽管SASP对肿瘤生物学具有重要的意义，但是它如何调控肿瘤仍然不甚明确。
发明内容
本发明的目的在于提供新型的肿瘤微环境相关靶点TAK1及其在抑制肿瘤中的应用。
在本发明的第一方面，提供一种TAK1基因或蛋白的下调剂的用途，用于制备抑制肿瘤的药物组合物；所述的肿瘤选自：前列腺癌，乳腺癌，肺癌。
在一个优选例中，所述的肿瘤是化疗药物或放疗射线处理(或电离辐射治疗处理)的肿瘤；或所述的肿瘤是表达TAK1的肿瘤。
在另一优选例中，所述化疗药物包括(但不限于)：博来霉素，米托蒽醌，紫杉萜，紫杉醇。
在另一优选例中，所述的药物组合物与化疗药物联合应用于抑制肿瘤。
在另一优选例中，所述的下调剂选自：特异性抑制TAK1的小分子化合物；特异性干扰TAK1基因表达的干扰分子；或特异性敲除TAK1基因的基因编辑试剂(如靶向于TAK1基因的sgRNA)；或特异性与TAK1基因编码的蛋白结合的抗体或配体。
在另一优选例中，所述的下调剂是特异性抑制TAK1的小分子化合物，其选自：5Z-7-oxozeaenol(即5Z-7)或LYTAK1。
在本发明的另一方面，提供一种TAK1基因或蛋白的用途，用于制备调控衰老相关分泌表型(SASP)的组合物；或用于制备抑制衰老相关疾病的药物组合物。
在一个优选例中，所述的衰老相关疾病包括：动脉粥样硬化，骨关节炎，骨质疏松，以及其它器官退行性疾病。
在本发明的另一方面，提供一种筛选抑制肿瘤的潜在物质的方法，所述方法包括：(1)用候选物质处理表达TAK1基因的体系；和(2)检测所述体系中TAK1基因的表达或活性；其中，若所述候选物质可降低TAK1基因的表达或活性，则表明该候选物质是抑制肿瘤的潜在物质。
在一个优选例中，步骤(1)包括：在测试组中，将候选物质加入到表达TAK1的体系中；和/或
步骤(2)包括：检测测试组的体系中TAK1的表达或活性，并与对照组比较， 其中所述的对照组是不添加所述候选物质的表达TAK1的体系；
如果测试组中TAK1的表达或活性在统计学上低于对照组，就表明该候选物是抑制肿瘤的潜在物质。
在另一优选例中，所述的体系选自：细胞体系(如表达TAK1的细胞或细胞培养物)、亚细胞体系、溶液体系、组织体系、器官体系或动物体系。
在另一优选例中，所述的统计学上低于优选显著低于，如低20％以上，较佳的低50％以上；更佳的低80％以上。
在另一优选例中，所述的候选物质包括(但不限于)：针对TAK1基因或蛋白设计的小分子化合物，针对TAK1基因或蛋白所参与的信号通路或其上游或下游蛋白设计的干扰分子、核酸抑制物、结合分子(如抗体或配体)等。
在另一优选例中，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以从候选物质中进一步选择和确定对于抑制肿瘤有用的物质。
在本发明的另一方面，提供一种用于抑制肿瘤的药物组合物，所述的药物组合物中包括：TAK1基因或蛋白的下调剂；和化疗药物或放疗射线(电离辐射)治疗药物。
在本发明的另一方面，提供一种用于抑制肿瘤的药盒，所述的药盒中包括：
容器1，以及包装于容器1中的TAK1基因或蛋白的下调剂；和
容器2，以及包装于容器2中化疗药物或放疗射线(电离辐射)治疗药物。
在一个优选例中，所述的下调剂包括：特异性抑制TAK1的小分子化合物；特异性干扰TAK1基因表达的干扰分子；或特异性敲除TAK1基因的基因编辑试剂(如靶向于TAK1基因的sgRNA)；或特异性与TAK1基因编码的蛋白结合的抗体或配体。
在另一优选例中，所述化疗药物包括(但不限于)：博来霉素，米托蒽醌，紫杉萜，紫杉醇。
在本发明的另一方面，提供一种特异性识别TAK1基因或其编码的蛋白的试剂的用途，用于制备进行肿瘤预后评估的试剂或试剂盒。
在一个优选例中，所述的特异性识别TAK1基因或其编码的蛋白的试剂选自：特异性扩增TAK1基因的引物；特异性识别TAK1基因的探针；或特异性结合TAK1基因编码的蛋白的抗体或配体。
在本发明的另一方面，提供一种用于肿瘤预后评估的试剂盒，所述的试剂盒中含有：特异性识别TAK1基因或其编码的蛋白的试剂。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、磷酸化ATM(p-ATM)抗体介导的IP分析。IP沉淀产物经Western blot检测TRAF6，p-ATM，ATM的表达水平。TRAF6，上样对照。PSC27经由博来霉素(50ug/ml)处理，再以ATM小分子抑制剂KU55933(KU，10μM)作用。
图2、PSC27细胞经过博来霉素辅之以5Z-7(500nM)处理之后，再经anti-TAK1介导的IP沉降，Western blot分析IP产物中p-TAK1，TRAF6，p-ATM以及细胞裂解物中p-TAK1，TRAF6和p-ATM的表达水平。
图3、PSC27对照组细胞和TRAF6 specific shRNA稳转系经过博来霉素处理之后，经anti-TRAF6介导的IP，产物再经Western blot分析各主要蛋白的翻译后修饰水平，细胞裂解物中的分析同时进行。
图4、基质细胞经过博来霉素处理之后胞质蛋白和核蛋白经试剂盒特异性分离提取，分析其中ATM，TAK1活化和NF-κB核转位情况。对照细胞和经过5Z-7处理的细胞进行平行分析。
图5、使用不同临床常用化疗药物和放疗射线处理PSC27细胞，收集受损细胞的裂解液经Western blot分析p-TAK1表达水平，同时以总TAK1和GAPDH作为上样对照。
图6、有TAK1抑制剂5Z-7存在的情况下，PSC27细胞经博来霉素处理，细胞裂解物再经anti-p-TAK1介导的IP沉降。IP产物经in vitro kinase assay分析，MKK6为TAK1底物。p38磷酸化修饰经Western blot分析，GAPDH为上样对照。此外，IL-1α(20ng/ml)用于处理基质细胞，再经类似的IP和in vitro kinase assay分析。图中RAD指放疗射线处理(γ-radiation在743rad/min)。
图7、PSC27细胞经过shRNA介导的IL-1α敲除，和p-TAK1介导的IP，和随后的Western blot分析。
图8、免疫荧光染色分析(γ-H2AX抗体)DNA损伤修复情况。标尺，10μm。下半部分，统计结果分析对比。
图9、图8中细胞染色结果在各组中的统计结果对比分析。
图10、细胞克隆形成能力检测代表性图片。PSC27细胞经博来霉素和/或5Z-7处理之后，第七天经固定，确定集落数量。
图11、图10中的集落数量统计结果对比分析。
图12、PSC27细胞经过米托蒽醌和/或5Z-7处理之后，免疫荧光染色DNA foci。将DDR情况进行计数和按类别统计比较。
图13、PSC27细胞经过沙铂和/或5Z-7处理之后，免疫荧光染色DNA foci。将DDR情况进行计数和按类别统计比较。
图14、基质细胞增殖潜力分析。PSC27经过博来霉素(50ug/ml)和/或5Z-7(500nM)处理，然后在体外培养条件下连续增殖和传代，将增殖倍数对培养时间进行作图。
图15、基质细胞经博来霉素和/或5Z-7处理之后，第7天收集细胞。裂解物经Western blot分析其ATM，TAK1的磷酸化水平，以及TAK1下游底物JNk和p38的活化情况。趋化因子IL-8作为SASP的标记性外泌因子，同时得以分析其表达水平。
图16、基质细胞经过博来霉素处理之后，于不同时间节点被收集并以Western blot分析Akt和mTOR磷酸化水平。
图17、免疫荧光检测分析PSC27细胞经过博来霉素(50ug/ml)和/或RAD001(50nM)处理之后，第7天时的p-mTOR表达水平。
图18、基质细胞经博来霉素和/或RAD001处理之后，第7天收集细胞并以Western blot分析其mTOR及其下游底物S6K1/4E-BP1的活化情况。
图19、基质细胞经博来霉素和/或RAD001处理之后，第7天收集细胞并检测其BrdU在DNA中的嵌入情况。
图20、图19中各组细胞的SA-B-Gal染色情况统计对比分析。
图21、基质细胞经博来霉素和/或RAD001处理之后，第7天收集细胞并检测其NF-kB复合物活化情况。GAPDH和Histone H3分别为胞质和胞核蛋白上样对照。
图22、预先转入NF-kB转录活性荧光素酶报告载体的基质细胞经博来霉素和/或RAD001处理之后，第7天收集细胞并检测其报告载体的荧光信号强度。
图23、基质细胞经博来霉素和/或RAD001处理之后，第7天收集细胞并以免疫共沉淀方法分析mTOR和IKK复合物亚单位之间的相互作用。IgG，对照抗体。
图24、基质细胞体外条件下经药物处理和in vitro kinase assay的实验流程。
图25、基质细胞经过图24中的实验顺序处理，再以anti-Flag介导的IP沉降。IP产物经过Western blot分析其中p-IKKa和p-mTOR的表达情况，从而确定二者之间的物理性相互作用。
图26、基质细胞分别经过shRNA介导的IKK复合物亚单位α和β特异性敲除之后，再经博来霉素和/或RAD001处理，分析其NF-kB转录活性荧光素酶报告载体的信号强度。
图27、基质细胞经博来霉素和/或RAD001处理、IL-1α(20ng/ml)刺激之后，以Western blot分析其IKKβ磷酸化,IkBα和IRAK1蛋白表达，以及p65/p50入核情况。β-actin和Histone H3分别为胞质和胞核样本上样对照。
图28、经过shRNA介导的IL-1α敲除，基质细胞经博来霉素和/或RAD001 处理，再以Western blot分析其IKKα、IKKβ磷酸化，IkBα和IRAK1蛋白表达，以及p65/p50入核情况。β-actin和Histone H3，蛋白上样对照。
图29、经过Akt抑制剂MK2206处理的基质细胞，被博来霉素损伤之后的第7天，收集并裂解，以Western blot分析其p38,Akt和mTOR的活化情况。
图30、以shRNA敲除PI3K的催化亚基p110，基质细胞再被博来霉素处理。损伤之后的第7天，细胞被裂解并分析其p38，Akt，mTOR活化情况，及p110和p85α表达水平变化。
图31、IKKa和IKKβ分别经shRNA敲除之后，基质细胞经过博来霉素和/或SB203580处理，7天之后收集并裂解。以Western blot分析其p38及其底物HSP27，IKKα和IKKβ的活化水平，及NF-kB亚单位的入核情况。
图32、基于报告载体的NF-κB转录活性检测。基质细胞分别经过PI3K小分子抑制剂LY294002(1μM)，Akt抑制剂MK-2206(100nM)，mTOR抑制剂RAD001(50nM)，p38抑制剂SB203580(10μM)和TAK1抑制剂5Z-7(500nM)跟博来霉素处理，细胞裂解物用于测定荧光素酶活性。
图33、图32中各组细胞释放的胞外液经过ELISA检测之后，确定外泌因子IL-8的蛋白表达水平。
图34、PSC27细胞经过博来霉素和/或5Z-7，SB203580(SB)and RAD001同时处理之后，总RNA经过表达谱芯片分析，热图显示几组样本之间的表达谱差异。
图35、GSEA方法对比分析SASP特异性表达标签在博来霉素和/或5Z-7处理之后基质细胞中的相对表达情况。NES，normalized enrichment score；FDR，false discovery rate。
图36、同图35相似，GSEA对比分析SASP标签在博来霉素和/或SB203580之间造成的表达差异。
图37、同图35相似，GSEA对比分析SASP标签在博来霉素和/或RAD001之间造成的表达差异。
图38、弦图显示基质细胞分别被5Z-7，SB or RAD001所显著下调的因子之间的相互关系(fold change>2)。Meta，从三种抑制剂分别产生的数据合并而成的一套meta数据库，以增强统计效力和组间比较有效性。
图39、KEGG通路分析和评估5Z-7特异性下调的top 809个基因之间的生物学关系(fold change>2)。一个IMEx interactome database被用于蛋白-蛋白之间相互作用分析。
图40、图39中top 809个基因构建的信号节点网络。绿色，canonical SASP因子。连线，推测性的蛋白-蛋白相互作用。
图41、荧光定量RT-PCR分析基质细胞在博来霉素和/或5Z-7处理之后的多 个SASP因子表达变化。
图42、体外条件下基质细胞培养、药物处理、间接与癌细胞共培养和表型检测的实验流程。
图43、基质细胞胞外液促进前列腺癌上皮细胞增殖的趋势在5Z-7作用下显著下降。
图44、基质细胞胞外液促进前列腺癌上皮细胞体外迁移的能力在5Z-7作用下显著降低。
图45、基质细胞胞外液促进前列腺癌上皮细胞侵袭的模式在5Z-7作用下显著弱化。
图46、体外条件下前列腺癌PC3细胞系在药物出料条件下的形态学变化。SASP抑制剂RAD001，SB203580和5Z-7与MIT同时处理，或MIT单独处理的结果平行比较。
图47、PSC27细胞经过几种不同方式的药物处理之后，收集其细胞胞外液，随后用于培养前列腺癌上皮细胞系(BPH1，M12，PC3，DU145和LNCaP)，同时培养液中加入IC50浓度的药物米托蒽醌处理之后的存活细胞数量统计比较。
图48、PC3细胞经过药物米托蒽醌(IC50浓度)和几种SASP抑制剂(包括5Z-7)处理PSC27之后收集的胞外液培养，不同条件下凋亡指数的比较。Caspase3/7活性检测结果用于直接作图。
图49、PSC27各个亚系产生的胞外液用于处理PC3细胞，在不同浓度的米托蒽醌存在的情况下，其相比于未经药物处理的对照组的细胞存活率。剂量反应曲线，非线性回归。
图50、基质细胞胞外液用于处理前列腺癌上皮细胞系，后者同时被微管毒药物紫杉醇(docetaxel，DTX)(IC50浓度)作用时的细胞数量平行对比。
图51、PC3在几种药物组合的处理条件下，细胞凋亡指数对比。Caspase3/7活性数值用于凋亡检验。
图52、几种不同的药物组合处理条件下的细胞存活率比较。剂量反应曲线，非线性回归。
图53、基质细胞胞外液促进前列腺癌上皮细胞增殖的趋势在TAK1另一抑制剂LYTAK1作用下显著下降。
图54、基质细胞胞外液促进前列腺癌上皮细胞体外迁移的能力在LYTAK1作用下显著降低。
图55、基质细胞胞外液促进前列腺癌上皮细胞侵袭的模式在LYTAK1作用下显著弱化。
图56、PSC27细胞经过几种不同方式的药物处理之后，收集其胞外液，随后 用于培养前列腺癌上皮细胞系(BPH1，M12，PC3，DU145和LNCaP)，同时培养液中加入IC50浓度的药物米托蒽醌处理之后的存活细胞数量统计比较。
图57、PC3细胞经过药物米托蒽醌和几种SASP抑制剂(包括LYTAK1)处理PSC27之后收集的胞外液培养，不同条件下凋亡指数的比较。Caspase3/7活性检测结果用于直接作图。
图58、PSC27各个亚系产生的胞外液用于处理PC3细胞，在不同浓度的米托蒽醌存在的情况下，其相比于未经药物处理的对照组的细胞存活率。剂量反应曲线，非线性回归。
图59、基质细胞胞外液(包括LYTAK1处理组)用于处理前列腺癌上皮细胞系，后者同时被微管毒药物紫杉醇(docetaxel，DTX)(IC50浓度)作用时的细胞数量平行对比。
图60、PC3在紫杉醇和PSC27几种胞外液组合的处理条件下，细胞凋亡指数对比。Caspase3/7活性数值用于凋亡检验。
图61、PSC27几种条件下产生的胞外液用于处理PC3细胞，在不同浓度的紫杉醇存在的情况下，其相比于未经药物处理的对照组的细胞存活率。剂量反应曲线，非线性回归。
图62、前列腺癌(PCa)临床患者在经过化疗前后的组织经过组化染色分析TAK1的磷酸化水平(活化)变化，所选样本(左、右)分别为化疗之前与化疗之后的代表性组织。
图63、基于TAK1活化情况的PCa患者生存期统计分析。低表达p-TAK1的患者数量为20，高表达组为30。
图64、预临床试验中的小鼠皮下接种癌细胞和/或基质细胞，然后经过化疗药物处理和病理分析的实验流程图。
图65、预临床化疗试验方案。整个过程为期8周，小鼠接种组织重组之后的第3周开始接受药物处理。MIT每隔一周腹腔注射一次，5Z-7或LYTAK1同时给药。第8周末试验结束时，测量小鼠肿瘤体积并进行组织学分析。
图66、小鼠终端肿瘤体积统计比较分析。同第5组相比，第6组体积下降37％；同第6组相比，第8组体积降低60％。
图67、图66中各组动物在化疗进行过程中，以BLI检测其体内肿瘤生长和发展情况。荧光素信号表明癌细胞集中位于皮下后腿附近，没有远端转移迹象。
图68、几种药物或药物组合处理条件下，小鼠的中位生存曲线比较。在MIT和MIT/5Z-7之间的差异显著，P<0.0001。
图69、同图66，但小鼠经历SASP抑制剂RAD001同MIT组合式治疗。相应的肿瘤体积下降幅度分别为36％和44％。
图70、同图66，但小鼠经历SASP抑制剂SB同MIT组合式治疗。。相应的肿瘤体积下降幅度分别为36％和46％。
图71、小鼠肿瘤在经过激光俘获显微解剖之后，转录本水平的趋化因子IL-8表达水平在各组之间的平行比较。
图72、小鼠肿瘤在经过激光俘获显微解剖之后，转录本水平的胞外因子AREG表达水平在各组之间的平行比较。
图73、小鼠肿瘤在经过激光俘获显微解剖之后，转录本水平的生长因子SPINK1表达水平在各组之间的平行比较。
图74、小鼠肿瘤在经过激光俘获显微解剖之后，转录本水平的胞外基质金属蛋白酶MMP3表达水平在各组之间的平行比较。
图75、小鼠终端肿瘤体积统计比较分析(LYTAK1代替5Z-7给药)。同第5组相比，第6组体积下降37％；同第6组相比，第8组体积降低63％。
图76、小鼠肿瘤(包括LYTAK1给药组)在经过激光俘获显微解剖之后，转录本水平的趋化因子IL-8表达水平在各组之间的平行比较。
图77、小鼠肿瘤(包括LYTAK1给药组)在经过激光俘获显微解剖之后，转录本水平的胞外因子AREG表达水平在各组之间的平行比较。
图78、小鼠肿瘤(包括LYTAK1给药组)在经过激光俘获显微解剖之后，转录本水平的生长因子SPINK1表达水平在各组之间的平行比较。
图79、小鼠肿瘤(包括LYTAK1给药组)在经过激光俘获显微解剖之后，转录本水平的胞外基质金属蛋白酶MMP3表达水平在各组之间的平行比较。
图80、临床NSCLC和BCa患者原位肿瘤组织的IHC和HE病理分析。每种癌型上层一组样本为未经化疗，下层为经历化疗患者组织。左侧样本为基于p-TAK1的IHC染色结果，其中红框所选区域在中间放大，右侧为中间组织的相应HE染色结果。
图81、基于TAK1活化情况的NSCLC患者生存期统计分析。低表达p-TAK1的患者数量为71，高表达组为28。
图82、基于TAK1活化情况的BCa患者生存期统计分析。低表达p-TAK1的患者数量为25，高表达组为37。
图83、NSCLC移植瘤小鼠终端肿瘤体积(NSCLC细胞系A549/基质细胞系WI38；LYTAK1用于单独给药或协同博来霉素联合化疗处理)统计比较分析。同第5组相比，第6组体积下降38％；同第6组相比，第8组体积降低63％。
图84、BCa移植瘤小鼠终端肿瘤体积(BCa细胞系MDA-MB-231/基质细胞系HBF1203；LYTAK1用于单独给药或协同博来霉素联合化疗处理)统计比较分析。同第5组相比，第6组体积下降32％；同第6组相比，第8组体积降低67％。
具体实施方式
本发明人经过广泛而深入的研究，首次揭示了TAK1在SASP表型发生发展的过程中可能发挥重要的生物学作用，并且其与肿瘤的发展也密切相关。因此，TAK1可作为SASP表型调控的研究靶点，可作为肿瘤的诊断、预后评估标志物，还可作为靶点开发抑制肿瘤的药物。
TAK1
转化生长因子激酶1(TAK1)在GenBank中的Gene ID为6885，编码606个氨基酸的蛋白质。
TAK1是丝裂源激活蛋白激酶激酶激酶(MAP3K)家族成员之一，在功能上位于丝裂源蛋白激酶(MAPK)和IKB激酶的上游。在体内可以既可以被包括IL-1在内的多种细胞因子激活，参与机体的许多重要生理过程，又可以通过与结合蛋白TAB1的共表达而被激活。磷酸化修饰被认为是TAK1依赖的信号转导过程中重要调节机制，但是TAK1蛋白的可调节性磷酸化位点并没有被完全确认。TAK1在分子水平的激活机制也没有被完全阐明。
本发明人发现，ATM-TRAF6-TAK1信号轴在基质细胞DNA损伤引发的急性反应中调控NF-κB复合物活化，后者与慢性SASP下游效应因子的表达密切相关。DNA损伤条件下TAK1介导p38MAPK信号通路的活化，但针对其激酶活性的药物抑制并不影响DNA损伤反应和基质细胞增殖潜力。并且，TAK1下游的mTOR通路在SASP慢性阶段的发展中发挥重要作用。
本发明人还发现，抑制TAK1可以在体外条件下逆转损伤基质细胞赋予癌细胞的多个恶性表型。靶向TAK1可以通过干预微环境中基质细胞的SASP发生发展，有效恢复肿瘤对于化疗药物的敏感性。然而，TAK1下调剂单独用药时对于肿瘤的生长并无显著影响，同常规化疗药物同时给药可以通过阻滞微环境SASP分泌表型而造成肿瘤体积大幅下降，因而可以成为老药新用的范例。
本领域技术人员了解，细胞衰老的同时常伴随着衰老相关分泌表型(senescence associated secretory phenotype，SASP)的发生。鉴于TAK1与衰老相关分泌表型的密切相关性，而SASP已知为可以以TAK1作为靶点，研究、开发或制备抑制衰老相关疾病的药物组合物。作为本发明的优选方式，所述的衰老相关疾病包括：动脉粥样硬化，骨关节炎，骨质疏松，以及其它器官退行性疾病。
TAK1下调剂
基于本发明人的上述新发现，本发明提供了一种TAK1基因或蛋白的下调 剂的用途，用于制备抑制肿瘤的药物组合物。所述的肿瘤选自：化疗药物处理或放射处理的肿瘤；或所述的肿瘤是基质细胞表达TAK1的肿瘤。所述化疗药物处理或放射处理包括：紫杉萜，紫杉醇，博来霉素，米托蒽醌，放疗射线等等。
如本文所用，所述的TAK1基因或蛋白的下调剂包括了抑制剂、拮抗剂剂、阻滞剂、阻断剂等。
所述的TAK1基因或蛋白的下调剂是指任何可降低TAK1蛋白的活性、降低TAK1基因或蛋白的稳定性、下调TAK1蛋白的表达、减少TAK1蛋白有效作用时间、或抑制TAK1基因的转录和翻译的物质，这些物质均可用于本发明，作为对于下调TAK1有用的物质，从而可用于抑制肿瘤。例如，所述的下调剂是：特异性干扰TAK1基因表达的干扰RNA分子或反义核苷酸；或是特异性与TAK1基因编码的蛋白结合的抗体或配体，等等。
作为本发明的一种选择方式，所述的下调剂是针对TAK1的小分子化合物。本领域技术人员可以采用本领域的常规筛选方法，来进行这类小分子化合物的筛选。例如，所述的小分子化合物为5Z-7-oxozeaenol(5Z-7)或LYTAK1。
作为本发明的一种选择方式，所述的下调剂是一种TAK1特异性的干扰RNA分子(shRNA)，本发明人观察到，采用本发明的干扰RNA分子，可显著地下调TAK1，对于肿瘤的抑制作用非常显著。
本发明对干扰RNA分子的制备方法没有特别的限制，包括但不限于：化学合成法，体外转录法等。所述的干扰RNA可通过采用适当的转染试剂被输送到细胞内，或还可采用本领域已知的多种技术被输送到细胞内。
作为本发明的另一种选择，可采用CRISPR/Cas9系统进行靶向的基因编辑，从而在靶向疾病的区域敲除TAK1基因。常见的敲除TAK1基因的方法包括：将sgRNA或能形成所述sgRNA的核酸、Cas9 mRNA或能形成所述Cas9 mRNA的核酸共转到靶向区域或靶向细胞中。在确定了靶位点之后，可以采用已知的方法来使得sgRNA及Cas9被引入到细胞内。所述的能形成所述sgRNA的核酸为核酸构建体或表达载体，或所述的能形成所述Cas9 mRNA的核酸为核酸构建体或表达载体，将这些表达载体导入到细胞内，从而在细胞内形成有活性的sgRNA及Cas9 mRNA。
肿瘤预后评估的试剂或试剂盒
基于本发明人的上述新发现，可以将TAK1作为肿瘤预后评估的标志物：(i)进行肿瘤的分型、鉴别诊断、和/或易感性分析；(ii)评估相关人群的肿瘤治疗药物、药物疗效、预后，以及选择合适的治疗方法。比如，可分离出由TAK1基因表达异常的人群，从而可进行更有针对性地治疗。
根据本发明人的新发现，可以通过判断待评估样本中TAK1的表达情况或活性情况，来预测提供该待评估样本的受试者的肿瘤预后情况，选择合适的药物实施治疗。通常，可以规定一个TAK1的阈值，当TAK1的表达情况高于所规定的阈值时，考虑采用抑制TAK1的方案进行治疗。所述的阈值对于本领域技术人员而言是易于确定的，例如可以通过将正常人组织微环境中的TAK1的表达情况与肿瘤患者微环境中的TAK1的表达情况进行比较后，获得TAK1表达异常的阈值。
因此，本发明提供了TAK1基因或蛋白的用途，用于制备肿瘤预后评估的试剂或试剂盒。
可采用各种本领域已知的技术来检测TAK1基因的存在与否以及表达情况，这些技术均包含在本发明中。例如可用已有的技术如Southern印迹法、Western印迹法、DNA序列分析、PCR等，这些方法可结合使用。
本发明还提供了用于在分析物中检测TAK1基因的存在与否以及表达情况的试剂。优选的，当进行基因水平的检测时，可以采用特异性扩增TAK1的引物；或特异性识别TAK1的探针来确定TAK1基因的存在与否；当进行蛋白水平的检测时，可以采用特异性结合TAK1编码的蛋白的抗体或配体来确定TAK1蛋白的表达情况。
针对TAK1基因的特异性探针的设计是本领域人员熟知的技术，例如，制备一种探针，其可与TAK1基因上特定位点发生特异性结合，而不与TAK1基因以外的其它基因特异性结合，且所述探针带有可检测信号。
利用特异性结合TAK1蛋白的抗体来检测分析物中TAK1蛋白表达情况的方法也是本领域人员熟知的技术。
本发明还提供了用于在分析物中检测TAK1基因的存在与否以及表达情况的试剂盒，该试剂盒包括：特异性扩增TAK1基因的引物；特异性识别TAK1基因的探针；或特异性结合TAK1基因编码的蛋白的抗体或配体。
此外，所述的试剂盒中还可包括用于提取DNA、PCR、杂交、显色等所需的各种试剂，包括但不限于：抽提液、扩增液、杂交液、酶、对照液、显色液、洗液等。
此外，所述的试剂盒中还可包括使用说明书和/或核酸序列分析软件等。
药物筛选
在得知了TAK1的过表达可促进特定肿瘤细胞的生长，而抑制TAK1的表达可抑制这些肿瘤细胞的生长这一特征后，可以基于该特征来筛选抑制TAK1的表达或活性的物质。可从所述的物质中找到对于抑制肿瘤真正有用的药物。 优选地，所述的特定的肿瘤选自：前列腺癌，乳腺癌，肺癌。
因此，本发明提供一种筛选抑制肿瘤的潜在物质的方法，所述的方法包括：用候选物质处理表达TAK1的体系；和检测所述体系中TAK1的表达或活性；若所述候选物质可抑制TAK1的表达或活性，则表明该候选物质是抑制肿瘤的潜在物质。所述的表达TAK1的体系较佳的是细胞(或细胞培养物)体系，所述的细胞可以是内源性表达TAK1的细胞；或可以是重组表达TAK1的细胞。
在本发明的优选方式中，在进行筛选时，为了更易于观察到TAK1的表达或活性的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的表达TAK1的体系。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于抑制肿瘤真正有用的物质。
另一方面，本发明还提供了采用所述筛选方法获得的抑制肿瘤的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于抑制TAK1的表达和活性，进而抑制肿瘤有用的物质。
药物组合物
本发明还提供了一种药物组合物，它含有有效量(如0.000001-50wt％；较佳的0.00001-20wt％；更佳的，0.0001-10wt％)的所述的TAK1基因或蛋白的下调剂，以及药学上可接受的载体。任何前述的TAK1基因或蛋白的下调剂均可用于组合物的制备。
作为本发明的一种优选方式，提供了一种用于抑制肿瘤的组合物，所述的组合物含有有效量的本发明所述的干扰RNA分子，以及药学上可接受的载体。
作为本发明的一种优选方式，提供了一种用于抑制肿瘤的组合物，所述的组合物含有有效量的TAK1基因或蛋白的下调剂，以及有效量的其它制剂，所述的其它制剂例如是基因毒药物或DNA损伤性药物，电离辐射治疗药物。
如本文所用，所述“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。所述“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
在得知了所述TAK1基因或蛋白的下调剂的用途后，可以采用本领域熟知 的多种方法来将所述的下调剂或其编码基因、或其药物组合物给药于哺乳动物。包括但不限于：皮下注射、肌肉注射、经皮给予、局部给予、植入、缓释给予等；优选的，所述给药方式是非肠道给予的。
优选的，可采用基因治疗的手段进行。比如，可直接将TAK1的下调剂通过诸如注射等方法给药于受试者；或者，可通过一定的途径将携带TAK1的下调剂的表达单位(比如表达载体或病毒等，或siRNA)递送到靶点上，并使之表达活性的TAK1下调剂，具体情况需视所述的下调剂的类型而定，这些均是本领域技术人员所熟知的。
本发明所述的TAK1基因或蛋白的下调剂的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的TAK1基因或蛋白的下调剂的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
I.材料和方法
1.细胞培养
(1)细胞系维持
正常人源前列腺原代基质细胞系PSC27(获自美国弗雷德哈青森癌症研究中心)于PSCC完全培养液中增殖和传代。前列腺良性上皮细胞系BPH1，前列腺癌症上皮细胞系M12，DU145，PC3，LNCaP和VCaP(购自ATCC)均在5％FBS的RPMI-1640完全培养液中、于37℃、5％CO 2条件的培养箱中培养。
(2)细胞冻存与复苏
a细胞冻存
以0.25％胰蛋白酶收集对数生长期细胞，1000rpm离心2min，弃去上清，重新悬浮细胞于新鲜配置的冻存液中。分装细胞于已标示的无菌冻存管中。然后经梯度降温(4℃10min，-20℃30min，-80℃16-18h)，最后转入液氮中长期储存。
b细胞复苏
取出液氮中冻存的细胞，立即放入37℃水浴，使其快速融化。直接加入2ml 细胞培养液，使细胞均匀悬浮。待细胞贴壁后，更换新的培养液。
(3)体外实验处理
为造成细胞损伤，PSC27细胞生长至80％(简称PSC27-Pre)时培养液中加入100nM紫杉萜(docetaxel，DTX)，100nM紫杉醇(paclitaxel，PTX)，200nM长春新碱(vincristine，VCR)，50μg/ml博来霉素(bleomycin，BLEO)，1μM米托蒽醌(mitoxantrone，MIT)，或10Gy  137Cs电离辐射(γ-radiation at 743rad/min，RAD)。药物处理6小时后，细胞被PBS简单洗过3次，留置于培养液中7～10天，然后进行后续实验。
2.质粒制备和慢病毒转染
全长人源IKKα与Flag的融合蛋白克隆在表达载体pCR3.1酶切位点BamHI和XbaI之间。全长人源mTOR与GST的融合蛋白克隆在表达载体pcDNA3的一对NotI酶切位点之间。包装系293FT被用于细胞转染和慢病毒制造。用于敲除TRAF6的small hairpin RNAs(shRNAs)序列分别为：
5’-gccacgggaaatatgtaatat-3’(SEQ ID NO:1)；和
5’-cggaatttccaggaaactatt-3’(SEQ ID NO:2)。
用于敲除TAK1的small hairpin RNAs(shRNAs)序列分别为：
5’-cccgtgtgaaccatcctaata-3’(SEQ ID NO:3)；和
5’-cgcccttcaatggaggaaatt-3’(SEQ ID NO:4)。
用于敲除IL-1α的small hairpin RNAs(shRNAs)序列分别为：
5’-gccaaagttccagacatgttt-3’(SEQ ID NO:5)；和
5’-gaatgacgccctcaatcaaag-3’(SEQ ID NO:6)。
3.免疫荧光和组化分析
小鼠单克隆抗体anti-phospho-Histone H2A.X(Ser139)(clone JBW301，Millipore)或小鼠单克隆抗体anti-Phosphor-53BP1(Cat#sc-135748，Santa Cruz)，及二级抗体Alexa
Figure PCTCN2018101003-appb-000001
488(或594)-conjugated F(ab’) 2按顺序加入到覆有固定细胞的载玻片上。细胞核用2μg/ml的4’，6-diamidino-2-phenylindole(DAPI)进行复染。从3个观察视野中选取最具代表性的一张图像进行数据分析和结果展示。FV1000激光扫描共聚焦显微镜(Olympus)用于获取细胞共聚焦荧光图像确定DNA损伤情况。
临床前列腺癌、非小细胞肺癌患者和乳腺癌患者组织IHC染色所用抗体anti-TAK1同上，购自Proteintech。具体步骤如下：常规脱蜡，用0.6％H 2O 2甲醇在37℃孵育30min，然后用0.01M pH6.0的柠檬酸缓冲液修复20min，室温冷却 30min。用正常羊血清封闭20min，用TAK1一抗(1:200)在37℃孵育1h，移至4℃冰箱过夜。第二天用TBS洗三次，以二抗(HRP偶联的羊抗兔)在37℃孵育45min，再用TBS洗3次，最后用DAB显色。
4.基质-上皮共培养和体外实验
用DMEM+0.5％FBS的培养液培养PSC27细胞3天，然后以1倍PBS清洗满丰度的细胞群。简单离心后收集上清作为条件性培养基存放-80℃或直接使用。前列腺上皮细胞在这种条件性培养基中连续培养3天的时间里开展体外实验。对于化疗抗性，上皮细胞系在低血清DMEM(0.5％FBS)(简称“DMEM”)中，或条件性培养基中培养，同时米托蒽醌(MIT)用于处理细胞1至3天，浓度接近各个细胞系的IC 50数值，随后在亮场显微镜下进行观察。
5.全基因组范围表达芯片分析(Agilent expression microarray)
对正常人源前列腺原代基质细胞系PSC27进行全基因组范围表达芯片(4x44k)分析的程序和方法参考Sun，Y.等，Nat.Med.2012.18:1359-1368。
6.定量PCR(RT-PCR)测定基因表达
(1)细胞总RNA的提取
以Trizol试剂抽提生长期细胞总RNA，每T25培养瓶细胞加入1ml Trizol，用细胞刮刀刮下细胞层后将其转移至离心管中，充分混匀至不粘稠。每1ml Trizol加0.2ml氯仿，剧烈震荡15秒，室温孵育5-10min；4℃，11,000g离心15分钟；将无色上清液移入一新的离心管中，按每1ml Trizol加0.5ml异丙醇，室温孵育10分钟，11,000g，4℃离心10分钟；倒掉上清，用75％乙醇(每1ml Trizol至少用1ml 75％乙醇)洗涤，4℃，7,500g离心5分钟；室温干燥RNA沉淀5-10分钟(RNA不能干燥)，用DEPC-H 2O溶解沉淀。
分光光度计定量RNA之后，取少量总RNA进行1％琼脂糖电泳，检查RNA状态和质量。
(2)逆转录反应
OligodT 23VN(50uM)      1ul
Total RNA             1-2ug
RNase Free ddH 2O      to 8ul
65℃加热5分钟，迅速置于冰上骤冷，并静置2分钟。
配置第一链cDNA合成液
2x RT Mix             10ul
HiScript II Enzyme Mix 2ul
按照以下条件进行第一链cDNA合成：
25℃                  5min
50℃                  45min
85℃                  5min
(3)实时定量PCR反应
将逆转录反应产物cDNA稀释50倍作为模板。
Figure PCTCN2018101003-appb-000002
按照以上标准加样，反应条件为：95℃预变性15s，然后95℃5s，60℃31s，40个循环；融解曲线条件为95℃15s，60℃30s，95℃15s。样品于ABI ViiA7(ABI)仪上进行反应。以β-actin的表达作内参。反应完成后，经软件分析查看每个基因的扩增情况，导出相应的域值循环数，采用2-ΔΔCt方法，计算每个基因的相对表达量。对融解曲线(melting surve)的波峰和波形进行分析以确定得到的扩增产物是否为特异性单一目的片段。
7.Western blot分析
(1)细胞总蛋白抽提
细胞经冰预冷的PBS缓冲液简单洗涤后，加入含1mM PMSF(蛋白酶抑制剂)的RIPA细胞裂解缓冲液(Invitrogen)，置冰上裂解细胞30min，用细胞刮刀收集细胞裂解液，4℃12,000rpm离心15min，取上清，-80℃保存。
(2)BCA法蛋白定量
BCA蛋白定量试剂盒(Pierce)，将试剂A和试剂B按1：50的比例混合，制成工作液待用。稀释标准蛋白，使其浓度依次为0μg/μl，25μg/μl，50μg/μl，100μg/μl，250μg/μl，500μg/μl，750μg/μl，1000μg/μl，2000μg/μl。在酶标板中加入5μl标准蛋白或5μl样品，再加入100μl BCA工作液，混合均匀后37℃水浴30min，用酶标仪读取570nm波长的吸光度值。以吸光度值为纵坐标，标准蛋白浓度为横坐标，绘制标准曲线。根据标准曲线，计算样品的浓度。
(3)SDS-PAGE电泳
配制12％SDS-PAGE(5ml体系包含30％丙烯酰胺2ml，ddH2O 1.6ml，1.5M  pH8.8 Tris-HCl 1.3ml，10％SDS 50μl，10％过硫酸铵50μl，TEMED 2μl)，迅速混匀后注入干净的预置玻璃板(Bio-Rad)空隙中，并在顶层加入适量去离子水以促进凝胶聚合，室温下静置30min，待分离胶完全凝聚后，弃上层去离子水并用滤纸吸净残留液体。配制浓缩胶(2ml体系中包含30％丙烯酰胺0.33ml，1.0M pH6.8 Tris-HCl0.25ml，10％SDS 20μl，10％过硫酸铵20μl，TEMED 2μl)，混匀后立即加到分离胶上层，插入干净的10齿梳子，室温下静置30min，待浓缩胶凝固完全后拔去梳子，用ddH 2O洗涤加样槽数次，将凝胶置于电泳槽(Bio-Rad)，加入电泳缓冲液(含25mM pH8.0 Tris，0.25M Glycine，0.1％SDS)。蛋白样品按5:1比例加入6×上样缓冲液(含300mM pH6.8 Tris-HCl，12％SDS，600mM DTT，60％甘油，0.6％溴酚蓝)混匀，沸水浴10min，冰浴冷却5min，结合蛋白定量结果，各泳道加入等量蛋白样品，用Bio-Rad电泳仪进行电泳，先以80V电压进行电泳约20分钟至溴酚蓝前沿进入分离胶后，把电压提高至120V，继续电泳约1小时至溴酚蓝条带到达分离胶底部，电泳结束。
(4)蛋白转膜
SDS-PAGE电泳后，切除浓缩胶及无样品区域，将硝酸纤维素滤膜以电泳转移缓冲液短暂浸泡。在电转移装置(Bio-Rad)上由阳极至阴极依次放Bio-Rad 3mm滤纸、硝酸纤维素滤膜、凝胶、Bio-Rad 3mm滤纸。以100V电压进行电转移1.5h。转膜结束后，通过预染Marker和用0.1％丽春红染色(Ponceau Stain)判断转移效果，并用ddH 2O脱色5min。
(5)抗体标记和ECL检测
将硝酸纤维素滤膜在封闭液(含5％脱脂奶粉的TBST(0.1％Tween-20 in TBS))中室温封闭1小时。在一抗杂交液中4℃孵育过夜。用TBST室温漂洗3次，每次2分钟。加入以封闭液配制的有HRP偶联的相应二抗杂交液，室温孵育0.5小时。用PBST室温漂洗滤膜3次，每次2min。
将SuperSignal West Pico试剂盒(Pierce)中等比例的底物及增强剂混合，均匀滴加到滤膜上，室温孵育1分钟，曝光X光片，显影、定影后，扫描X光片并保存用以分析。
8.NF-κB调控分析
含有编码IκBα蛋白序列上两个IKK磷酸化突变位点S32A和S34A的反病毒载体pBabe-Puro-IκBα-Mut(super repressor)，被用于转染慢病毒包装细胞系PHOENIX。慢病毒随后用于侵染PSC27基质细胞系，而1μg/ml嘌呤霉素(puromycin)则被用于筛选阳性克隆。作为另外一种方法，5μM的小分子抑制剂Bay 11-7082(购自Selleck)被用于NF-κB活性控制。基质细胞随后被暴露于几种 不同形式的细胞毒，及时记录由此产生的表型，分析相关基因表达情况。经过这种方式处理过的细胞，产生出来的条件性培养液被收集起来，用于针对上皮细胞的各种检测。
10.临床前列腺癌、非小细胞肺癌和乳腺癌患者组织样本获取和分析
化疗药物方案是根据去势抵抗性前列腺癌、复发和难治性非小细胞肺癌患者(临床试验注册号NCT02889666)及渗透性导管型乳腺癌患者(临床试验注册号NCT02897700)的病理学特征指定的。前列腺癌临床分期在T2a以下且没有明显远端转移病灶的患者招募至临床队列中。原发肺癌在I subtype A(IA)(T1a，N0，M0)以上但没有明显远端转移病灶的患者被招募至临床队列中。同时，年龄75岁以上经临床确诊为NSCLC，或者年龄大于18岁在组织上被证实有渗透性BCa的患者方被招募。所有患者均被提供知情同意书并签字确认。有关肿瘤大小，组织类型，肿瘤渗透，淋巴结转移和病理TNM疾病阶段的数据从病理记录系统获取。肿瘤加工为FFPE样本并处理成组织学切片以供评估。OCT冰冻切片经LCM选择性分离，用于基因表达分析。特别地，根据先前报道的方法(Sun et al.，2012)，化疗前后的腺体相关基质细胞经LCM分离。免疫活性评分(IRS)根据每一组织样本的组化染色呈色深浅分别归类于0-1(阴性)，1-2(弱)，2-3(中)，3-4(强)四类(Fedchenko and Reifenrath，2014)。NSCLC和BCa样本的诊断由彼此独立的病理学医生进行判断和评分。随机对照试验(RCT)方案和所有实验程序均经上海交通大学医学院IRB批准和授权，并根据权威指导原则逐步开展。
11.小鼠移植瘤试验和预临床化疗程序
所有实验小鼠实验均严格遵循中国科学院上海生命科学研究院实验动物照护和使用委员会(IACUC)的有关规章进行。年龄6周左右的免疫缺陷型小鼠ICR SCID mice(体重约25g)用于本专利相关动物实验。基质细胞PSC27和上皮细胞以1:4的比例混合，而每一移植体包含1.25×10 6细胞，用于组织重构。移植瘤通过皮下移植方式植入小鼠体内，移植手术结束之后8周末动物被执行安乐死。肿瘤体积按照如下公式计算：V＝(π/6)x((l+w)/2) 3(V，体积；l，长度；w，宽度)。类似地，肺癌和乳腺癌移植瘤分别由A549(非小细胞肺癌细胞系)和WI38(肺成纤维细胞系)，MDA-MB-231(三阴性乳腺癌细胞系)和HBF1203(乳腺成纤维细胞系)通过组织重构形成。
在预临床化疗试验中，经过皮下移植的小鼠被供给标准实验食谱，2周之后实施化疗药物米托蒽醌(0.2mg/kg剂量)和/或SASP抑制剂(500μl，10mg/kg剂量，RAD001,SB203580和5Z-7均购自TOCRIS；LYTAK1，购自美国Lilly Co (Indianapolis，IN).，5mg/kg)腹腔给药。时间点为第3，5，7周的第一天，整个疗程共进行3次循环给药，每个循环为2周。疗程结束后，小鼠肾脏被收集用于肿瘤测量和组织学分析。每只小鼠累积性共接受米托蒽醌0.6mg/kg体重，SASP抑制剂30mg/kg体重(LYTAK1为15mg/kg体重)。肺癌和乳腺癌移植瘤小鼠分别接受博来霉素(共0.3mg/kg)和阿霉素(共0.2mg/kg)，时间点和频率同米托蒽醌给药方式。化疗试验进行到第8周末结束，小鼠处死之后立即解剖，其移植瘤被收集并用于病理系统分析。
12.生物统计学方法
本专利申请中所有涉及细胞增殖率，迁移性，侵袭性和存活性等的体外实验和小鼠移植瘤及化疗处理的体内试验均重复3次以上，数据以均值±标准误的形式呈现。统计学分析建立在原始数据的基础上，通过one-way analysis of variance or a two-tailed Student’s t-test进行计算，而P<0.05的结果认作具有显著性差异。
II.实施例
实施例1、ATM-TRAF6-TAK1信号轴在基质细胞DNA损伤引发的急性反应中调控NF-κB复合物活化，后者与慢性SASP下游效应因子的表达密切相关
以往曾有文献报道在DNA损伤之后的48小时内，基质细胞在短期内会出现一种特殊生理反应，即呈现急性胁迫相关表型(acute stress-associated phenotype，ASAP)。全基因组范围内有不少外泌因子均在这种表型形成过程中高度上调，并且该表型随后会逐渐过渡为衰老相关分泌表型(senescence-associated secretory phenotype，SASP)，后者是一个慢性、长期而稳定的状态。然而，从ASAP(一般1-2天)到SASP(需要6-8天)的几天时间里，细胞在DNA损伤的作用下发生了哪些变化，什么信号通路发挥了关键性的作用，经过了哪些分子和细胞机制的调控，至今并不清楚。此外，作为感应DNA损伤信号的重要激酶ATM，在细胞内是否起到关键作用，也是近年来国际上不少科学家热烈探讨和回答的问题。
为此，本发明人首先使用ChIP通过磷酸化ATM(p-ATM)抗体分析了博来霉素处理之后的基质细胞裂解液，发现活化的ATM和TRAF6之间存在相互作用，但可被ATM抑制剂KU55933所废除(图1)。因为ATM结合至TRAF6上可以激活TRAF6介导的poly-ubiquitination(多泛素化)并导致一些包括TAK1活化在内的下游反应，本发明人随即分析了基质细胞中是否存在类似的现象。为此，在博来霉素处理基质细胞之后使用IP以anti-TRAF6检测了细胞裂解物，注意到迅速增长的TRAF6的auto-ubiquitination(单泛素化)，证实了其在损伤细胞中的ubiquitin ligase(泛素连接酶)活性。本发明人随后使用phosphorylated TAK1(p-TAK1)抗体进 行了进一步的IP分析，发现TAK1和TRAF6之间存在着物理性相互作用，这在在DNA损伤之后随即出现，但可被TAK1抑制剂5Z-7-oxozeaenol(此后简写为5Z-7)所废除(图2)。相比之下，在TAK1和ATM之间则不存在这种相互作用，尽管这两个分子在DNA损伤之后均被很快地活化。同时，这一数据也暗示着TAK1-TRAF6之间作用的特殊性(图2)。作为支持性证据，anti-TRAF6抗体介导的IP实验表明TRAF6可以同活化状态的ATM和TAK1均发生相互作用，暗示TRAF6可以作为中间分子而传递ATM信号给TAK1(图3)。同时，TRAF6的敲除可以在受损基质细胞中废除TAK1活化，而非ATM激活，再次证实TRAF6在介导ASAP急性反应信号中的特殊作用。
即便如此，被上游DDR信号所间接激活的TAK1是否同SASP广谱表达所涉及的一个核心转录因子即NF-κB复合物的活化有关？而后者曾被报道是被胞质中IκB kinase subunitγ(IKKγ)的mono-ubiquitination所介导。在使用试剂盒将细胞核同细胞质蛋白进行分离之后，本发明人发现TAK1磷酸化是同NF-κB复合物的主要两个亚基p50和p65的核转位有关(图4)。然而在5Z-7存在的情况下，TAK1的磷酸化和p50/p65的入核均被显著抑制，证明NF-κB是基质细胞的胞质中TAK1所介导的信号通路下游的一个事件(图4)。
实施例2、DNA损伤条件下TAK1介导p38MAPK信号通路的活化，但针对其激酶活性的药物抑制并不影响DNA损伤反应和基质细胞增殖潜力
既然影响DNA损伤反应(DNA damage response，DDR)信号被磷酸化的ATM从细胞核传递至细胞质，并随后经被TRAF6/TAK1信号轴的放大，最终导致NF-κB复合物的激活，本发明人质问这些信号是否以及怎样激活同SASP发展相关的多个分子。大量数据已经证明TAK1可以在多个生理过程包括局部炎症发生和组织稳态维持中激活p38，Jnk和Erk等MAPK家族成员，本发明人推测TAK1同SASP的慢性进展有关。首先，本发明人分析了基质细胞在博来霉素处理之后5Z-7-oxozeaenol(以下简作5Z-7，名为二羟基苯甲酸内脂)存在与否的情况下，TAK1的活性变化。体外条件下收集的基质细胞裂解液经过IP和in vitro kinase assay检测，发现DNA损伤激活了TAK1，导致其发生磷酸化，这一点可被TAK1和MKK6之间的相互作用所证实(图5～6)。然而，不断上升的5Z-7使得TAK1在损伤基质细胞中的活化逐渐减弱，而500nM条件下的5Z-7则可以将TAK1活化基本废除。同时，western blot结果显示p38MAPK在损伤细胞中也被磷酸化，这跟TAK1的活化彼此平行，暗示着这两个激酶在基因毒背景下的基质细胞中具有一定关联(图5～6)。既然IL-1α是已知的TAK1的一个上游调控因子，本发明人推测TAK1在这种条件下可以被细胞因子IL-1α所激活，后者同时也是SASP 的一个效应因子，一旦被上调和释放之后即能够进一步强化SASP表型。因此，本发明人使用IL-1α处理了PSC27细胞，发现显著增强的TAK1/MKK6相互作用和大幅上升的p38激酶活性，这跟PSC27细胞在跟博来霉素作用条件下的变化非常相似(图5～6)。为了进一步核实IL-1α在TAK1活化过程中的生物学作用，本发明人在博来霉素加药之前使用shRNA敲除了IL-1α。有趣的是，IL-1α的缺失导致TAK1在受损基质细胞中的活化和p38MAPK的激活显著下降，说明在这些基因毒药物处理过的细胞中TAK1的活化受到IL-1α的调控(图7)。然而，值得注意的是，TAK1活化并未改变DNA损伤反应情况。一旦细胞遭受博来霉素处理，单细胞水平的DDR foci并不随着5Z-7的存在与否而发生显著改变(图8，图9)。同时，PSC27细胞的克隆形成能力取决于DNA损伤情况，而跟TAK1的活性抑制与否并不相关(图10，图11)。
为了排除博来霉素的脱靶效应，本发明人随后使用了另外两种化疗药物处理同样一批基质细胞，包括米托蒽醌(mitoxantrone，MIT，一种DNA拓扑异构酶抑制剂)和沙铂(satraplatin，SAT，一种铂类似物)，二者可以通过不同的机制造成DNA损伤。结果，本发明人得到了同博来霉素处理组类似的DNA损伤结果(图12，图13)。这再次说明DNA损伤反应在客观上是取决于基因毒药物所造成的损伤程度，而非TAK1活性受抑制与否。进而，本发明人发现TAK1功能缺陷并不影响细胞在体外条件下的增殖潜力，这一点可被细胞群体倍增曲线所证实，后者专门用于评价细胞在培养条件下连续传代时所表现出来的最大倍增能力(图14)。
此外，本发明人还检测了细胞遭受基因毒损伤之后TAK1活性被抑制时，是否改变ATM的磷酸化状态，即从另外一个角度分析TAK1被抑制与否是否影响DNA损伤反应强度。Western blot数据表明DNA损伤所激发的ATM磷酸化状态并不取决于TAK1活性是否被5Z-7所抑制，尽管JNk和p38MAPK的磷酸化程度在TAK1被抑制时出现显著下降(图15)。同时，SASP表型发生发展的一个典型标志性效应因子IL-8，蛋白表达水平也在DNA损伤持续存在时被显著下调。综上，本发明人的实验数据一致性说明TAK1活性的维持对于SASP的慢性发展是必不可少的。
实施例3、TAK1下游的mTOR通路在SASP慢性阶段的发展中发挥重要作用
DNA损伤促进衰老细胞的形成，后者在数月之内均能保持代谢活跃并具有生理活性，同时表现出明显增加的溶酶体质量和增强的SA-B-Gal酶活性。既然TAK等分子在DNA损伤之后的ASAP急性阶段具有关键的信号介导作用，本发明人要问是否还有其它分子也在急性细胞反应过程中被激活并促进SASP的慢性 发展。本发明人的数据显示在DNA损伤之后的急性反应后期开始出现Akt/mTOR的激活，这可通过Akt(Ser473)和mTOR(Ser2448)这两个位点的翻译后修饰得以证实，二者均在博来霉素处理后的24小时开始磷酸化并在7天之后进入平台期(图16)。免疫荧光实验结果显示活化后的mTOR出现于细胞质。本发明人随后检测了mTOR上下游的通路和关键分子的变化。例如，mTOR下游两个底物S6K1及其催化亚基S6和4E-BP1的磷酸化，同时表明mTOR的功能性活化(图17)。本发明人近年报道过雷帕霉素可以降低辐射条件下造成的衰老成纤维细胞中出现的S6K1和4E-BP1磷酸化，能够负调细胞内螺旋酶机器抑制具有稳定二级结构的mRNA的翻译。在本发明人中，使用了RAD001，一种雷帕霉素类似物，发现在mTOR磷酸化被阻滞的情况下同样可以造成S6K1和4E-BP1活化的抑制，证实雷帕霉素作为SASP抑制剂的有效性(图18)。尽管如此，博来霉素造成的DDR foci在受损基质细胞中仍然保持不变(图17)，而细胞循环阻滞和SA-β-Gal活性在RAD001存在的情况下也未受影响，暗示细胞衰老和代谢活动均得以维持(图19，图20)。
因细胞衰老相关转录组被包括NF-κB在内的几个主要转录机器所调控，本发明人下面要问mTOR是否同NF-κB复合物的活化直接或者间接相关。为此，本发明人分析了博来霉素处理之后基质细胞，发现其胞质中IκBα的降解和NF-κB亚单位p65(Rel A)的稳定化，二者均表明NF-κB复合物在DNA损伤细胞中的活化状态(图21)。同时，报告载体转染实验的数据也证实NF-κB转录活性的显著上调，但可在RAD001存在时被大幅减弱(图22)。本发明人随后使用mTOR抗体进行了IP实验，发现在IKKα和Raptor之间存在着相互作用(图23)，而以IKKα抗体介导的反向IP结果显示IKKα同mTOR，而非Raptor之间的关联(图23)。因此，一系列实验结果表明IKKα可能在IKK复合物中起到不同于其它亚基的作用，因其跟mTOR之间的物理性互作具有激活NF-κB复合物的特殊潜力。
为了进一步验证mTOR跟IKK复合物之间的相互关系，本发明人使用了PP242，一种第二代小分子mTOR抑制剂，其通过竞争性结合ATP位点而靶向mTOR激酶活性，即以一种区别于雷帕霉素及其类似物的方式发挥抑制作用。In vitro kinase assay数据显示DNA损伤之后的基质细胞中IKKα作为一个mTOR的下游底物被磷酸化(图24，图25)。尽管mTOR同IKKα之间存在直接相互作用，IKK复合物中的另一催化亚基IKKβ在这个过程中的作用却一直未明。为了澄清这一点，本发明人使用shRNA来分别敲除以上两个亚基，并在药物处理之前将NF-κB活性报告载体转染入细胞中。有趣的是，IKKα的清除显著降低了NF-κB的核内活性，但IKKβ的缺失则造成更大幅度的信号下降；当两个亚基同时被敲除时，仅有最低强度的NF-κB活性留存在核内(图26)。这些数据表明，IKKα和 IKKβ均在DNA损伤后的基质细胞中被活化。
本发明人近期发现mTOR可以通过限制细胞因子IL-1α的翻译来调控SASP广谱效应因子的表达，在此过程中NF-κB复合物受到抑制。然而，IL-1α如何控制NF-κB转录活性，以及哪一个IKK亚单位特异性介导IL-1α激发的信号通路，并不清晰。在这里，本发明人发现IKKβ的磷酸化，IRAK1和IκBα的降解，以及NF-κB复合物亚单位p65与p50的入核，均在DNA损伤后发生(图27)。尽管这些变化均在RAD001加入培养基之后被基本废除，IL-1α的加入却能够将其逆转。另一方面，敲除IL-1α能够降低IKKβ活化程度，而IKKα活性却保持不变(图28)。虽然IRAK1蛋白水平大致不变，IκBα蛋白总量却显著降低，暗示IKKα在其中介导NF-κB信号。此外，p65和p50在细胞核内的信号仍然大量保持，即便在IL-1α被敲除的情况下，表明仍然持续激活的NF-κB复合物(图28)。
下面，本发明人分析了PI3K/Akt这一经典信号通路在DNA损伤背景下的基质细胞中是否同mTOR活化相关。在博来霉素和MK-2206(一种Akt小分子抑制剂)同时处理的条件下，基质细胞中mTOR活性被显著下调(图29)。此外，PI3K催化亚基p110的下调，基本阻止了Akt和mTOR在损伤PSC27细胞中的活化(图30)。相比之下，p38在博来霉素处理后的基质细胞中始终被激活，不论PI3K/Akt通路的完整性如何(图30)，暗示p38在这些基质细胞中作为PI3K/Akt/mTOR上游的一个因子发挥作用。进一步的结果显示，无论IKKα还是IKKβ的缺失，均未能降低p38的活性，尽管p38抑制剂SB203580可以大幅下调该激酶磷酸化水平(图31)。DNA损伤所诱导的NF-kB活化和趋化因子IL-8蛋白水平的表达，均被显著降低，当小分子抑制剂LY294002，MK-2206，SB203580或5Z-7(分别靶向PI3K，Akt，p38或TAK1)被加入培养基时，虽然5Z-7看上去似乎可以造成更强的抑制效果(图32，图33)。既然5Z-7主要靶向控制TAK1这一激酶，以上数据证明SASP这一表型可从网络中较为上游的信号节点进行特异性的激酶控制，最终可以获得更高的总体抑制效率。
实施例4、TAK1抑制可以在体外条件下逆转损伤基质细胞赋予癌细胞的多个恶性表型
本发明人实验数据证实SASP的形成可以从其上游的信号通路进行有效干预，那么针对TAK1的活性控制是否可以具有一定的生物学效应，尤其是损伤基质细胞在微环境中所增强的癌细胞的一系列表型？首先，本发明人检测了在SASP被抑制的情况下有哪些基因的表达被显著下调。比较转录组学数据显示在博来霉素单独处理和跟5Z-7联合用药的情况下比较，后者可以造成大多数SASP效应因子被显著抑制(图34)。相较于博来霉素/SB20580和博来霉素/RAD001两种组合式 处理的情况，5Z-7介导的TAK1活性抑制似乎可以更为有效的造成SASP大多数外泌蛋白的下调表达。尽管在不同的SASP因子下降幅度之间存在一定波动性，总体一致的这种趋势表明广谱SASP基本被控制住。
在TAK1活性抑制可以导致SASP总体表达被衰减的前提下，本发明人使用Gene Set Enrichment Analysis(GSEA)评估了SASP的发展程度。根据近年本发明人已发表的人类基质细胞SASP表型密切相关的外泌因子所提前定义的一个SASP特异性表达标签，在TAK1被药物抑制时出现显著下调(图35)。虽然SASP这一表达标签在p38或者mTOR被抑制时也会被明显弱化，两种情况下的控制效果均不如TAK1活性受抑时的数据(图36，图37)。除此之外，本发明人将研究结果进一步延伸，使用基于meta-analysis的人体蛋白相互作用网络分析导出的数据表明，TAK1抑制时所下调的多种蛋白同细胞因子/趋化因子/胞外基质受体之间的相互作用和信号通路交错对话呈现系统关联(图38，图39，40)。以上结果进一步核实了TAK1抑制所产生的生物学效应(图41)。
随后，本发明人评估了基质细胞TAK1抑制对于癌细胞的增殖造成的影响。PSC27在受到博来霉素处理之后的第7天，本发明人收集了其胞外液并立即用于前列腺癌细胞的培养(图42)。有趣的是，上皮癌细胞的增殖速度在TAK1被抑制时出现显著下降(图43)。尽管损伤基质细胞的胞外液可以明显提高癌细胞的迁移率和侵袭率，这些变化均在基质细胞TAK1受到抑制时发生显著下调(图44，图45)。更为重要的是，出现SASP时的基质细胞胞外液所赋予癌细胞在米托蒽醌作用下的耐药性，被显著削弱，暗示5Z-7所导致的基质细胞内TAK1活性控制可以抵消基质细胞受损时赋予癌细胞的获得性存活力(图45，图46)。伴随存活力下降的，是米托蒽醌处理癌细胞时后者所表现出来的凋亡指数的上升，后者可被caspase 3/7活性检测数据所证实(图47)。癌细胞耐药潜力上的巨大变化，更被0.1-1μM浓度范围作用下的米托蒽醌造成的细胞存活性非线性曲线所证实，这一浓度范围基本同临床条件下前列腺癌患者体内的血清水平相互吻合(图48)。因此，任意一种情况下，基质细胞TAK1活性抑制造成的癌细胞获得性恶性数据的下降均比RAD001和SB203580导致的结果更为显著(图49)。
为了进一步扩展以上发现，本发明人使用同样一套CM处理了紫杉醇作用下的癌细胞，发现TAK1抑制时的基质细胞产生的胞外液能够提高紫杉醇对于癌细胞的细胞毒，使得细胞存活率下降，凋亡指数上升，癌细胞反应曲线偏移(图50，图51，图52)。因此，5Z-7所造成的基质细胞TAK1活性抑制可以衰减基质细胞所赋予癌细胞对于多种化疗药物的获得性耐药。
同时，本发明人使用TAK1的另一小分子抑制剂LYTAK1进行了体外实验。结果表明，经过LYTAK1介导的TAK1抑制作用可以显著降低癌细胞在基质细胞 胞外液作用下的增殖、迁移和侵袭(图53，图54，图55)。通过检测体外条件下癌细胞对于米托蒽醌的抵抗，发现被博来霉素损伤的基质细胞胞外液在LYTAK1存在情况下，赋予癌细胞的耐药性显著降低，即LYTAK1所导致的基质细胞内TAK1活性抑制能够抵消基质细胞受损时造成的癌细胞获得性存活力(图56，图57，图58)。除了米托蒽醌，在对另一化疗药物紫杉醇的细胞毒实验中也发现了类似作用，即LYTAK1可以显著降低癌细胞在基质细胞胞外液作用下获得的耐药性或抗凋亡能力(图59，图60，图61)。
实施例5、靶向TAK1可以通过干预微环境中基质细胞的SASP发生发展，有效恢复肿瘤对于化疗药物的敏感性
微环境中SASP的广谱表达可以加速许多恶性事件，包括肿瘤发生，局部炎症和治疗性抵抗。然而，这种向恶性进展的趋势是否可以通过特异性控制微环境中SASP的形成而得以避免，以及如何有效抑制抗癌疗法所激活的体内微环境中的SASP，一直是科学界的难题。需要注意的是，前列腺癌患者在经过临床化疗之后，组织微环境中普遍出现同SASP发生发展密切相关的TAK1的活化(相比治疗前时期，磷酸化水平明显上升)(图62)。更重要的在于，肿瘤微环境中TAK1的活化状态同前列腺癌患者疗后阶段的生存呈现显著负相关(图63)。
为了尽可能模拟临床条件，本发明人向免疫缺陷型小鼠皮下部位接种了前列腺来源的基质细胞系PSC27和上皮癌细胞系PC3混合细胞群，随后小鼠经历了一个为期8周的预临床化疗方案，后者包括根据一系列预实验数据确定的3次单药或双药治疗(图64，图65)。在没有基质细胞的情况下，PC3细胞仍然在化疗药物造成的筛选压下形成了肿瘤，尽管其体积比基质细胞和癌细胞同时接种情况下生成的肿瘤要小，这一差异客观上也印证了微环境的促瘤效果(图66)。尽管5Z-7自身似乎并不改变肿瘤生长趋势，但米托蒽醌给药时却可以造成肿瘤终端体积下降37％(图65，第5组-第6组)。值得注意的是，MIT和5Z-7的联合用药，造成肿瘤体积进一步收缩了60％(图66，第6组-第8组)。同时，为确保皮下接种的肿瘤在整个化疗过程中没有发生远端转移，后者会使得实验数据难以解释，本发明人另外接种了一批整合有荧光素酶报告基因载体片段的PC3和PSC27细胞，后者使得肿瘤生长可以通过生物荧光素成像技术(bioluminescence imaging，BLI)进行实时监控。本发明人发现这些肿瘤产生的信号强度同最终检测到的肿瘤体积大致相互对应，因而从另一角度证实了各组之间的差异(图67)。
尽管没有出现癌细胞的异位转移，本发明人质问了微环境是否造成除了肿瘤生长和药物抵抗之外的其它病理后果。为此，本发明人评估了一批时间延伸性的预临床治疗条件下的多组动物的存活期，以确定肿瘤进展的客观结果。所有动物 被施以肿瘤体积监控，并在肿瘤体积达到一定上限(size≥2000mm 3)时即被认作形成严重疾病。在这种方式下，本发明人发现米托蒽醌(MIT)/5Z-7联合治疗组的小鼠获得了显著延长的中位生存期，该组动物比米托蒽醌单独化疗组在无病生存时间上延长了约50％(图68，对比绿色和蓝色两组)。然而，单独使用5Z-7仅轻微延长了小鼠生存期(图6856，对比紫色和红色组)。以上结果表明，MIT/TAK1联合使用较为理想。
接下来本发明人系统比较了微环境中SASP发生发展的条件下TAK1抑制的效果同mTOR或p38活性降低造成的结果之间的差异。通过开展类似于5Z-7给药的两组体内实验，本发明人发现MIT/RAD001和MIT/SB203580联合给药处理，均能显著降低皮下肿瘤在化疗疗程结束时的终端体积。其中MIT/RAD001合并用药造成比MIT单药给药进一步下降44％的效果，而MIT/SB203580则进一步降低了46％(图69，图70)。尽管mTOR或p38为靶向的联合治疗均能显著延迟肿瘤生长，TAK1抑制造成的抑瘤效果总体上则更可观。为进一步确证治疗过程中体内微环境的SASP表达被抑制剂所有效控制，本发明人使用激光俘获纤维解剖特异性分离了肿瘤中的基质细胞并进行转录本水平的分析，发现包括IL-8，AREG，SPINK1和MMP3在内的几个典型SASP效应因子均被显著降低(图71，图72，图73，图74)。
此外为了进一步验证TAK1靶向抑制剂与化疗药物对于肿瘤的影响，申请人使用LYTAK1进行了类似预临床实验。发现LYTAK1与米托蒽醌联合给药可以在米托蒽醌单独使用的基础上再次降低肿瘤体积63％(图75)。在LYTAK1的抑制作用下，几个典型SASP效应因子的表达也被普遍性地显著降低(图76，图77，图78，图79)。
除了动物实验数据支持TAK1可以作为控制微环境中SASP表型的一个有效靶点，本发明人进而系统分析了TAK1同临床患者生存期之间的病理关联。从临床数据得到的结果表明，TAK1活性跟(非小细胞肺癌)NSCLC和乳腺癌(BCa)患者生存期之间普遍存在显著的负性关联(图80，图81，图82)。为了在体内条件下证明TAK1表达同这两种癌型之间的关联，本发明人进而使用基于组织重构的移植瘤小鼠，分别进行了预临床实验。小鼠治疗数据表明，LYTAK1分别协同博来霉素和阿霉素给药，可以显著降低肿瘤的终端体积(NSCLC为63％，BCa为67％)(图83，图84)。因此，本发明人得出结论，即同时清除恶性增长的癌细胞群和通过靶向TAK1而控制因药物而被动活化的微环境相关SASP，可以显著降低肿瘤生长趋势，而TAK1则可以成为将来临床治疗中一个行之有效的、用于阻断微环境SASP发生发展的新药物靶点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种TAK1基因或蛋白的下调剂的用途，用于制备抑制肿瘤的药物组合物；所述的肿瘤选自：前列腺癌，乳腺癌，肺癌。
  2. 如权利要求1所述的用途，其特征在于，所述的肿瘤是化疗药物或放疗射线处理的肿瘤；或所述的肿瘤是表达TAK1的肿瘤。
  3. 如权利要求2所述的用途，其特征在于，所述化疗药物包括：博来霉素，米托蒽醌，紫杉萜，紫杉醇。
  4. 如权利要求2或3所述的用途，其特征在于，所述的药物组合物与化疗药物联合应用于抑制肿瘤。
  5. 如权利要求1所述的用途，其特征在于，所述的下调剂选自：
    特异性抑制TAK1的小分子化合物；
    特异性干扰TAK1基因表达的干扰分子；或
    特异性敲除TAK1基因的基因编辑试剂；或
    特异性与TAK1基因编码的蛋白结合的抗体或配体。
  6. 如权利要求5所述的用途，其特征在于，所述的下调剂是特异性抑制TAK1的小分子化合物，其选自：5Z-7-oxozeaenol或LYTAK1。
  7. 一种TAK1基因或蛋白的用途，用于制备调控衰老相关分泌表型的组合物；或用于制备抑制衰老相关疾病的药物组合物。
  8. 一种筛选抑制肿瘤的潜在物质的方法，所述的肿瘤选自：前列腺癌，乳腺癌，肺癌，所述方法包括：
    (1)用候选物质处理表达TAK1基因的体系；和
    (2)检测所述体系中TAK1基因的表达或活性；
    其中，若所述候选物质可降低TAK1基因的表达或活性，则表明该候选物质是抑制肿瘤的潜在物质。
  9. 如权利要求8所述的方法，其特征在于，步骤(1)包括：在测试组中，将候选物质加入到表达TAK1的体系中；和/或
    步骤(2)包括：检测测试组的体系中TAK1的表达或活性，并与对照组比较，其中所述的对照组是不添加所述候选物质的表达TAK1的体系；
    如果测试组中TAK1的表达或活性在统计学上低于对照组，就表明该候选物是抑制肿瘤的潜在物质。
  10. 一种用于抑制肿瘤的药物组合物，所述的肿瘤选自：前列腺癌，乳腺癌，肺癌，其特征在于，所述的药物组合物中包括：
    TAK1基因或蛋白的下调剂；和
    化疗药物或放疗射线治疗药物。
  11. 一种用于抑制肿瘤的药盒，所述的肿瘤选自：前列腺癌，乳腺癌，肺癌，其特征在于，所述的药盒中包括：
    容器1，以及包装于容器1中的TAK1基因或蛋白的下调剂；和
    容器2，以及包装于容器2中化疗药物或放疗射线治疗药物。
  12. 如权利要求10所述的药物组合物或权利要求11所述的药盒，其特征在于，所述的下调剂包括：特异性抑制TAK1的小分子化合物；特异性干扰TAK1基因表达的干扰分子；或特异性敲除TAK1基因的基因编辑试剂；或特异性与TAK1基因编码的蛋白结合的抗体或配体。
  13. 如权利要求10所述的药物组合物或权利要求11所述的药盒，其特征在于，所述化疗药物包括：博来霉素，米托蒽醌，紫杉萜，紫杉醇。
  14. 一种特异性识别TAK1基因或其编码的蛋白的试剂的用途，用于制备进行肿瘤预后评估的试剂或试剂盒；所述的肿瘤选自：前列腺癌，乳腺癌，肺癌。
  15. 如权利要求14所述的用途，其特征在于，所述的特异性识别TAK1基因或其编码的蛋白的试剂选自：
    特异性扩增TAK1基因的引物；
    特异性识别TAK1基因的探针；或
    特异性结合TAK1基因编码的蛋白的抗体或配体。
  16. 一种用于肿瘤预后评估的试剂盒，所述的肿瘤选自：前列腺癌，乳腺 癌，肺癌，其特征在于，所述的试剂盒中含有：特异性识别TAK1基因或其编码的蛋白的试剂。
PCT/CN2018/101003 2017-08-25 2018-08-17 新型的肿瘤微环境相关靶点tak1及其在抑制肿瘤中的应用 WO2019037658A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/640,826 US11344601B2 (en) 2017-08-25 2018-08-17 Tumor microenvironment-related target TAK1 and application thereof in inhibition of tumor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710741005.3A CN109420170B (zh) 2017-08-25 2017-08-25 肿瘤微环境相关靶点tak1及其在抑制肿瘤中的应用
CN201710741005.3 2017-08-25

Publications (1)

Publication Number Publication Date
WO2019037658A1 true WO2019037658A1 (zh) 2019-02-28

Family

ID=65439940

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/101003 WO2019037658A1 (zh) 2017-08-25 2018-08-17 新型的肿瘤微环境相关靶点tak1及其在抑制肿瘤中的应用

Country Status (3)

Country Link
US (1) US11344601B2 (zh)
CN (1) CN109420170B (zh)
WO (1) WO2019037658A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114377067A (zh) * 2020-10-22 2022-04-22 中国科学院上海营养与健康研究所 葡萄籽提取物在制备靶向清除肿瘤微环境衰老细胞及抑制肿瘤的药物中的用途

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109999184A (zh) * 2019-03-15 2019-07-12 南京恒健文化传播有限公司 Tak1蛋白在制备食管癌预后评估试剂或试剂盒中的应用
CN109856351B (zh) * 2019-04-03 2022-05-06 广东省人民医院(广东省医学科学院) 一种预测tak1与药物协同影响细胞凋亡的方法
CN112553330B (zh) * 2019-09-26 2022-07-26 首都医科大学附属北京友谊医院 新型肿瘤相关转录因子zscan16及其在抑制肿瘤中的应用
CN112501239B (zh) * 2020-12-14 2021-12-24 南通大学 Tak1抑制剂抑制pdl1的检测方法及其在制备抗pdl1药物中的应用
WO2023088356A1 (zh) * 2021-11-22 2023-05-25 宁波康柏睿格医药科技有限公司 Rip2抑制剂联合化疗药物的应用
CN117205198B (zh) * 2022-09-26 2024-04-26 中国科学院上海营养与健康研究所 丹参酚酸c(sac)作为新型抗衰老药物原料在细胞衰老、肿瘤治疗与延长寿命中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1921864A (zh) * 2003-12-24 2007-02-28 西奥斯公司 使用TGF－β抑制剂治疗神经胶质瘤
CN101490279A (zh) * 2006-07-10 2009-07-22 阿斯利康(瑞典)有限公司 用taki抑制剂治疗癌症的方法
WO2012166722A1 (en) * 2011-06-03 2012-12-06 The General Hospital Corporation Treating colorectal, pancreatic, and lung cancer

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009097351A2 (en) * 2008-01-28 2009-08-06 The Board Of Regents Of The University Of Texas System TAK1-D MEDIATED INDUCTION OF CELL DEATH IN HUMAN CANCER CELLS BY SPECIFIC SEQUENCE SHORT DOUBLE STRANDED RNAs
CN106138045B (zh) * 2015-04-16 2019-06-21 中国科学院上海生命科学研究院 通过靶向mTOR操纵肿瘤微环境以消除肿瘤耐药性的方法及制剂
CN104911183B (zh) * 2015-06-25 2018-01-09 中山大学孙逸仙纪念医院 抑制胰腺癌TAK1基因表达的shRNA的转录模板

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1921864A (zh) * 2003-12-24 2007-02-28 西奥斯公司 使用TGF－β抑制剂治疗神经胶质瘤
CN101490279A (zh) * 2006-07-10 2009-07-22 阿斯利康(瑞典)有限公司 用taki抑制剂治疗癌症的方法
WO2012166722A1 (en) * 2011-06-03 2012-12-06 The General Hospital Corporation Treating colorectal, pancreatic, and lung cancer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114377067A (zh) * 2020-10-22 2022-04-22 中国科学院上海营养与健康研究所 葡萄籽提取物在制备靶向清除肿瘤微环境衰老细胞及抑制肿瘤的药物中的用途
CN114377067B (zh) * 2020-10-22 2023-04-18 汤臣倍健股份有限公司 葡萄籽提取物在制备靶向清除肿瘤微环境衰老细胞及抑制肿瘤的药物中的用途

Also Published As

Publication number Publication date
US20210030836A1 (en) 2021-02-04
US11344601B2 (en) 2022-05-31
CN109420170A (zh) 2019-03-05
CN109420170B (zh) 2021-03-02

Similar Documents

Publication Publication Date Title
Liu et al. LncRNA CAIF inhibits autophagy and attenuates myocardial infarction by blocking p53-mediated myocardin transcription
US11344601B2 (en) Tumor microenvironment-related target TAK1 and application thereof in inhibition of tumor
Mazur et al. Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma
Wang et al. Thrombospondin enhances RANKL-dependent osteoclastogenesis and facilitates lung cancer bone metastasis
Fang et al. MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression
Lee et al. Endothelial‐to‐mesenchymal transition induced by W nt 3a in keloid pathogenesis
Balli et al. Endothelial cell–specific deletion of transcription factor FoxM1 increases urethane-induced lung carcinogenesis
Zhong et al. Long noncoding RNA NEAT1 promotes the growth of human retinoblastoma cells via regulation of miR‐204/CXCR4 axis
US20140323551A1 (en) Targeting micrornas mir-409-5p, mir-379 and mir-154* to treat prostate cancer bone metastasis and drug resistant lung cancer
Ma et al. AGTR1 promotes lymph node metastasis in breast cancer by upregulating CXCR4/SDF-1α and inducing cell migration and invasion
Clementi et al. Persistent DNA damage triggers activation of the integrated stress response to promote cell survival under nutrient restriction
Martin et al. Role of endothelial cells in pulmonary fibrosis via SREBP2 activation
Shen et al. KDM5D inhibit epithelial‐mesenchymal transition of gastric cancer through demethylation in the promoter of Cul4A in male
Ye et al. Altered ratios of pro‐and anti‐angiogenic VEGF‐A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma
Sun et al. Characterization of cellular senescence in doxorubicin-induced aging mice
Gu et al. miR‐370 inhibits the angiogenic activity of endothelial cells by targeting smoothened (SMO) and bone morphogenetic protein (BMP)‐2
Zhu et al. NT5DC2 knockdown inhibits colorectal carcinoma progression by repressing metastasis, angiogenesis and tumor-associated macrophage recruitment: A mechanism involving VEGF signaling
Chen et al. High expression of Rad51c predicts poor prognostic outcome and induces cell resistance to cisplatin and radiation in non-small cell lung cancer
CN111040032B (zh) 双向调节素在制备细胞衰老及肿瘤的诊断或调控制剂中的应用
Nandi et al. Coordinated activation of c-Src and FOXM1 drives tumor cell proliferation and breast cancer progression
Fan et al. Curcumin mitigates the epithelial‐to‐mesenchymal transition in biliary epithelial cells through upregulating CD109 expression
CN112011614A (zh) Kmt5a在调控胶质瘤干细胞特性及胶质瘤诊治中的应用
Liu et al. Mitofusin1 is a major mediator in glucose-induced epithelial-to-mesenchymal transition in lung adenocarcinoma cells
US11510911B2 (en) Method for prediction of susceptibility to sorafenib treatment by using SULF2 gene, and composition for treatment of cancer comprising SULF2 inhibitor
Wang et al. METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18848098

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18848098

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 27.11.2020)

122 Ep: pct application non-entry in european phase

Ref document number: 18848098

Country of ref document: EP

Kind code of ref document: A1