CN108796071A - Metastatic breast cancer marker and its application in diagnosing and treating - Google Patents
Metastatic breast cancer marker and its application in diagnosing and treating Download PDFInfo
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- CN108796071A CN108796071A CN201710283739.1A CN201710283739A CN108796071A CN 108796071 A CN108796071 A CN 108796071A CN 201710283739 A CN201710283739 A CN 201710283739A CN 108796071 A CN108796071 A CN 108796071A
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
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- 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
Abstract
The present invention relates to a kind of metastatic breast cancer marker and its applications in diagnosing and treating.Specifically, the present invention provides the purposes of a kind of DKK1 genes, mRNA, cDNA or albumen or its detection reagent, (i) are used as the marker of detection metastatic breast cancer；And/or (ii) is used to prepare the diagnostic reagent or kit of detection metastatic breast cancer.The present invention the experimental results showed that；DKK1 can be used as it is a new class of and meanwhile predict Bone of Breast Cancer transfer and Lung metastases biological marker, fully assess patient more target organs shift risk and guiding treatment in terms of have huge application prospect.
Description
Technical field
The present invention relates to oncology and diagnostic field.More particularly it relates to a kind of metastatic breast cancer marker
And its application in diagnosing and treating.
Background technology
The far-end transfer of breast cancer is the main reason for leading to breast cancer treatment failure and death.Breast cancer is main
Far-end transfer target organ is bone and lung.An important phenomenon clinically be breast cancer transfer have organ specificity, i.e., one
Bone tumour easily occurs for the tumour cell that invasion migration occurs from primary tumor for some patients and lung occurs for the then easy of another part patient
Transfer.So development shifts risk and really to prediction patient for the biological marker (biomarker) of transfer organ specificity
Determine therapeutic scheme to play an important roll.Past research is found that some bones or the special associated gene of Lung metastases1-3, but only
It cannot still meet while predict the clinical demand that the more target organs of patient shift risk for the special associated gene of single target organ.
Also, even if these genes, as biological marker, the invasive detection methods such as their more dependences puncture easily cause patient attached
The injury added.
Although in addition, having the pharmaceutical admixtures of some treating organs transspecifics in the past, just for single target organ.
In conclusion the new metastatic breast cancer marker with diagnosis or Combining diagnosis value is found, and specific aim
The research and development that ground carries out targeted drug are the task of top priority.Therefore, there is an urgent need in the art to develop to can be used for detecting or judge metastatic
The specific markers of breast cancer.
Invention content
Can be used for metastatic breast cancer specific markers it is an object of the invention to provide one kind and its in clinical diagnosis and
Purposes in oncotherapy.
First aspect present invention provides the purposes of a kind of DKK1 genes, mRNA, cDNA or albumen or its detection reagent,
(i) it is used as detection metastatic breast cancer or the marker of Metastasis in Breast Cancer risk；And/or (ii) is used to prepare detection metastatic breast
The diagnostic reagent or kit of gland cancer or Metastasis in Breast Cancer risk.
In another preferred example, the diagnostic reagent includes antibody, primer, probe, sequencing library, nucleic acid chip (such as DNA
Chip) or protein-chip.
In another preferred example, the albumen includes full-length proteins or protein fragments.
In another preferred example, the DKK1 genes, mRNA, cDNA or albumen source are preferably come in mammal
Derived from rodent (such as mouse, rat), Primate and people, more preferably, from the patient for being diagnosed with breast cancer or
It is diagnosed with the patient of metastatic breast cancer.
In another preferred example, the DKK1 genes, mRNA, cDNA or albumen source are in patient with breast cancer.
In another preferred example, the accession number of the DKK1 genes is 22943.
In another preferred example, the accession number of the DKK1mRNA is NM_012242.
In another preferred example, the accession number of the DKK1 albumen is AAQ89364.1.
In another preferred example, the detection is body fluid sample detection.
In another preferred example, the detection is blood sample detection and/or serum sample detection.
In another preferred example, the detection reagent include the specific antibody of DKK1, DKK1 specific binding molecules,
Specificity amplification primer, probe or chip.
In another preferred example, the DKK1 albumen or the coupling of its specific antibody or specific binding molecules have or band
There is detectable label.
In another preferred example, the detectable label is selected from the group：Chromophore, chemiluminescent groups, fluorogen, same to position
Element or enzyme.
In another preferred example, the specific antibody of the DKK1 is monoclonal antibody or polyclonal antibody.
In another preferred example, the Metastasis in Breast Cancer is selected from the group：Bone tumour, Lung metastases, brain metastes, hepatic metastases or
A combination thereof.
In another preferred example, the Metastasis in Breast Cancer is selected from the group：Bone of Breast Cancer transfer, breast cancer Lung metastases or
A combination thereof.
In another preferred example, the metastatic breast cancer includes Bone tumour breast cancer and/or Lung metastases breast cancer.
In another preferred example, described shift risk refers to shifting risk for n, and the Any Digit that wherein n is 1-5 (wraps
Include decimal).
Second aspect of the present invention provides a kind of diagnosis examination for detecting metastatic breast cancer or Metastasis in Breast Cancer risk
Agent box, the kit contain a container, contain the detection for detecting DKK1 gene, mRNA, cDNA or albumen in the container
Reagent；And label or specification, the label or specification indicate the kit for detecting metastatic breast cancer or breast
Gland cancer shifts risk.
In another preferred example, the detection metastatic breast cancer or Metastasis in Breast Cancer risk refer to detection Metastasis in Breast Cancer
The metastasis site whether occurred, occurred, and/or
Judge possibility (neurological susceptibility) size of generation Metastasis in Breast Cancer.
In another preferred example, the Metastasis in Breast Cancer includes Bone of Breast Cancer transfer, breast cancer Lung metastases, breast cancer patients with brain
Transfer, breast cancer hepatic metastases, or combinations thereof.
In another preferred example, the Metastasis in Breast Cancer includes Bone of Breast Cancer transfer and/or breast cancer Lung metastases.
In another preferred example, described to judge to include prejudging (prediction).
In another preferred example, the detection reagent of the detection DKK1 genes, mRNA, cDNA or albumen includes：
(a) specific antibody of the anti-DKK1 albumen of；And/or
(b) specific primer of the mRNA or cDNA of specific amplifications DKK1.
In another preferred example, the detection is body fluid sample detection.
In another preferred example, the detection is blood sample detection and/or serum sample detection.
In another preferred example, the following contents is indicated in the label or specification：
(i) when concentration≤2.3ng/ml of the serum DKK1 of detection object (subject) is (preferably, 0.05-2.3ng/
Ml or 0.5-2.0ng/ml), then prompt the risk that breast cancer Lung metastases occur for the detection object high (risk >=30% in 3 years),
And the risk that Bone of Breast Cancer transfer occurs is low (risk≤10% in 3 years)；
In another preferred example, the concentration of serum DKK1 also prompts, which occurs breast cancer Lung metastases probability
Higher than general patient with breast cancer；
(ii) when detecting the concentration > 2.3ng/ml and≤3.0ng/ml of serum DKK1 of object (preferably, 2.3-
It 3.0ng/ml) then prompts the risk that breast cancer Lung metastases occur for the detection object relatively low (risk≤20% in 3 years), and breast occurs
The risk of gland cancer Bone tumour is higher (risk >=20% in 3 years)；
In another preferred example, the concentration of serum DKK1 also prompts, which occurs Bone of Breast Cancer and shift probability
Higher than general patient with breast cancer；
(iii) as the concentration > 3ng/ml (such as 3.0-5.5ng/ml) of the serum DKK1 of detection object, then the detection is prompted
The risk that Bone tumour occurs for object is high (risk >=30% in 3 years), the low (risk in 3 years of the probability for occurring Lung metastases<10%)；
In another preferred example, the concentration of serum DKK1 also prompts, which occurs Bone of Breast Cancer and shift probability
Higher than general patient with breast cancer.
In another preferred example, the following contents is indicated in the label or specification：
(i) as concentration≤2.3ng/ml of the serum DKK1 of detection object, then prompt the detection object that Lung metastases occur
Probability is higher than general patient with breast cancer, and the probability that Bone tumour occurs is less than general patient with breast cancer；
(ii) when detection object serum DKK1 concentration between 2.3-3.0ng/ml, then prompt the detection object occur
The probability of Lung metastases is slightly below general patient with breast cancer；
(iii) when detection object serum DKK1 concentration in > 3.0ng/ml, then prompt the detection object occur lung turn
The probability of shifting is less than general patient with breast cancer, and the probability that Bone tumour occurs is higher than general patient with breast cancer.
In another preferred example, detection object behaviour or non-human mammal.
In another preferred example, the kit is additionally operable to the life span or pre- of prediction metastatic breast cancer patient
Afterwards.
Third aspect present invention provides a kind of method detecting metastatic breast cancer or Metastasis in Breast Cancer risk, the side
Method includes：
A) test sample from subject is provided；
B) concentration of DKK1 albumen in test sample is detected；With
C) concentration of the DKK1 albumen measured in step b) is compared with control,
Wherein compared with the control, the concentration of DKK1 albumen is less than reference value in the sample, shows subject
The probability of Lung metastases is higher than general patient with breast cancer crowd (control group crowd)；And/or the concentration of DKK1 albumen is higher than reference value,
Show that the probability of Bone tumour occurs for subject higher than general patient with breast cancer crowd (control group crowd).
It is in another preferred example, described that subject is a human or non-human mammal.
In another preferred example, the test sample is the body fluid sample of patient with breast cancer.
In another preferred example, the test sample includes the blood sample and/or serum sample of patient with breast cancer.
In another preferred example, the method is non-diagnostic and non-therapeutic.
In another preferred example, the reference value is cutoff value (cut-off values).
In another preferred example, the reference value is 2.3ng/ml and 3.0ng/ml.
Third aspect present invention provides a kind of method of determining therapeutic scheme, including：
A) test sample from subject is provided；
B) concentration of DKK1 albumen in test sample is detected；With
C) therapeutic scheme is determined based on the concentration of the DKK1 albumen in the sample.
It is in another preferred example, described that subject is a human or non-human mammal.
In another preferred example, when concentration≤2.3ng/ml of the DKK1 albumen in the sample is (preferably, 0.05-
2.3ng/ml or 0.5-2.0ng/ml), then prompt the probability that breast cancer Lung metastases occur for the subject to suffer from higher than general breast cancer
Person, the therapeutic scheme include jnk inhibitor therapy.
In another preferred example, when the probability that breast cancer Lung metastases occur for subject is higher than general patient with breast cancer, institute
State the combination that therapeutic scheme further includes TGF beta inhibitors therapy and jnk inhibitor therapy.
In another preferred example, as the concentration > 2.3ng/ml of the DKK1 albumen in the sample and≤3.0ng/ml
(preferably, 2.3-3.0ng/ml) then prompts the probability that Bone of Breast Cancer transfer occurs for the subject to be higher than general patient with breast cancer,
The therapeutic scheme includes the combination of TGF beta inhibitors therapy or TGF beta inhibitors therapy and jnk inhibitor therapy.
In another preferred example, as the concentration > 3ng/ml (such as 3.0-5.5ng/ml) of the DKK1 albumen in the sample,
It then prompts the subject that Bone of Breast Cancer transfer probability occurs and is higher than general patient with breast cancer, the therapeutic scheme includes that TGF β inhibit
The combination of agent therapy or TGF beta inhibitors therapy and jnk inhibitor therapy.
In another preferred example, the therapeutic scheme further includes that the transfer of other Bone of Breast Cancer, and/or breast cancer Lung metastases are treated
Method.
In another preferred example, other Bone of Breast Cancer transfer, and/or breast cancer Lung metastases therapy are selected from the group：
(1) DKK1 neutralizing antibodies therapy；And/or
(2) bisphosphonates therapy；And/or
(3) RANKL targeting antibodies or other inhibitor therapies；And/or
(4) TGF β targeting antibodies drug or other inhibitor therapies；And/or
(5) PTHrP targeting antibodies or other inhibitor therapies；And/or
(6) integrin inhibitors therapy；And/or
(7) inhibitor therapy of targeting mTOR, Src protease, cysteine proteinase equimolecular and signal path；With/
Or
(8) breast cancer hormonotherapy therapy；And/or
(9) people's skins give birth to Long factor acceptor 2 inhibitor therapies；And/or
(10) chemotherapeutics such as alkylating agent, methotrexate (MTX), anthracene nucleus medicament, taxane anticancer agents are applied alone and are combined treatment
Method.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 show DKK1 and Bone of Breast Cancer, Lung metastases be associated with and its function.(a) different transfer group breast cancer patients
Serum DKK1 is horizontal；(b) apneumia/Bone tumour survival rate of high DKK1 groups and low DKK1 groups patient；(c) different transfer group breast cancer
The expression of DKK2 during DKK3, DKK4 be horizontal in patients serum and tumor tissues；(d) tail vein injection DKK1 is overexpressed
With the lung's optical signal and schematic diagram of mouse after control group breast cancer cell；(e) left ventricle injection DKK1 overexpressions and control group
The hind leg optical signal and X-ray imaging schematic diagram of mouse after breast cancer cell.*P<0.05.
Fig. 2 shows that the inhibiting effect to bone, Lung metastases is used in combination in SP600125 and SB431542.(a) DKK1 strikes low
The monocyte (left side) and medullary system inhibitory cells (MDSC, right) (right side) that group is induced with control group breast cancer cell conditioned medium
Migration；(b) content of DKK1 overexpressions group and TGF β in control group breast cancer cell conditioned medium；(c-e) fat pad is in situ
The tumor size (c) formed after mouse mammary carcinoma cell line is injected, pulmonary nodule number (d) is transferred to and is transferred to hind leg bon e formation
Osteoclastic transfer stove area (e) and drug-treated influence.SP：SP600125；SB：SB431542.*, P<0.05；*, P<
0.01；* *, P<0.001；Ns, no significant difference.
Fig. 3 shows that the inhibiting effect to bone, Lung metastases is used in combination in other jnk inhibitors and TGF beta inhibitors.(a)
After the processing of JNK and TGF beta inhibitors in breast cancer cell downstream JUN and SMAD2 albumen phosphorylation level；(b-d) JNK and TGF
Beta inhibitor processing after breast cancer cell conditioned medium induction monocyte emigration (b), osteoclast cell maturation (c) and
The mature osteoclast (d) (black arrow meaning cell) of the multinuclear of the TRAP positives.SP：SP600125；SB：SB431542；
Gal：Galunisertib.*, P<0.05；*, P<0.01；* *, P<0.001；Ns, no significant difference.
Fig. 4 shows DKK1 action models.(1) serum DKK1 levels it is higher in Bone tumour patient with breast cancer and lung turn
It is relatively low in shifting；(2) DKK1 is by inhibiting JNK the and NF- κ B signal accesses in downstream that macrophage/MDSC is inhibited to recruit with TGF β's
Secretion；(3) combination of the inhibitor of JNK and TGF β effectively can alleviate Lung metastases and Bone tumour simultaneously.
Specific implementation mode
The present inventor after extensive and in-depth study, for the first time it was unexpectedly observed that the blood of the patient with breast cancer of Bone tumour group
Clear DKK1 protein levels are significantly higher than the control group of no transfer, and the DKK1 of Lung metastases group patient is then substantially less than control group, because
This DKK1 can be used as biological marker that is a new class of while predicting Bone of Breast Cancer transfer and Lung metastases, be fully assessed to play
Shifting risk for more target organs of patient and guides the effect for the treatment of.On this basis, the present inventor completes the present invention.
Experimental result is shown, as concentration≤2.3ng/ml of the serum DKK1 of patient with breast cancer, then shows that the breast cancer is suffered from
Average probability of the probability higher than general patient with breast cancer crowd of Lung metastases occurs for person, and the probability that Bone tumour occurs is less than ordinary people
Group；As the concentration > 2.3ng/ml for the serum DKK1 that breast cancer is suffered from, preferably, 2.3-3.0ng/ml, more preferably, > 3ng/ml,
Then show that average probability of the probability higher than general patient with breast cancer crowd of Bone tumour occurs for the patient with breast cancer, and lung occurs
The probability of transfer is less than the average probability of general patient with breast cancer crowd.
In addition, the experimental result of the present invention also further displays, according to the concentration level of DKK1 in blood serum of patients with human breast carcinoma
Different therapeutic schemes is selected, for example, concentration≤2.3ng/ml of the serum DKK1 as patient with breast cancer, then select JNK to inhibit
The therapeutic scheme or jnk inhibitor of agent and therapeutic scheme associated with TGF beta inhibitors, when the concentration for the serum DKK1 that breast cancer is suffered from
> 2.3ng/ml, preferably, 2.3-3.0ng/ml, more preferably, > 3ng/ml, then select TGF beta inhibitors or TGF beta inhibitors with
Therapeutic scheme associated with jnk inhibitor.
Term
As used herein, term " metastatic breast cancer " refers to the breast cancer for having and shifting risk, including does not occur or occurred
The breast cancer type of transfer, specifically, including Bone tumour breast cancer, Lung metastases breast cancer, brain metastes breast cancer, liver turn
The breast cancer of the transfer class such as shifting property breast cancer.
Sample
Term " sample " used herein or " sample " refer to subject's specifically associated material, from wherein may be used
With determination, calculate or be inferred to specific information related with subject.Sample can be completely or partially by the life from subject
Object material is constituted.Sample can also be the material contacted in some way with subject, and this way of contact makes to sample
The test of progress can provide information related with subject.Sample can also be the material contacted with other materials,
This other materials are not subjects, but the first material can be made then tested to determine letter related with subject
Breath, such as sample can be the cleaning solution of probe or scalpel.Sample can be the biomaterial source except contact subject, only
The professional of the art is wanted to remain able to determine information related with subject just from sample.
Expression
As used herein, term " expression " includes generations of the mRNA from gene or Gene Partial, and includes by RNA or base
The generation of cause or the encoded protein of Gene Partial further includes and expresses the relevant appearance for detecting substance.For example, cDNA,
Binding partner (such as antibody) and gene or other oligonucleotides, the combination of protein or protein fragments and binding partner it is aobvious
Color part is included in the range of term " expression ".Therefore, in the increase of immunoblotting such as western traces upper half dot density
It is also in the range of the term based on biological molecule " expression ".
Reference value
As used herein, term " reference value " refers to relevant with particular result statistics when compared with analysis result
Value.In preferred embodiments, reference value is the research according to expression and known clinical effectiveness to comparing DKK1 albumen
The statistical analysis of progress determines.Some such researchs are shown in the embodiments herein part.But from text
The user experience of the research and method disclosed herein offered can also be used for producing or adjusting reference value.Reference value can also be by examining
The situation and result especially relevant with the medical history of patient, science of heredity, age and other factors are considered to determine.
In the present invention, the reference value refers to cutoff value (cut-off values), preferably 2.3ng/ml and 3ng/ml (breast cancer
The concentration of DKK1 in patients serum).
Non-breast cancer sample
As used herein, term " non-breast cancer sample " includes but not limited to the crowd for not suffering from breast cancer, and breast cancer is suffered from
The non-breast cancer tissue of person.
DKK1 albumen and polynucleotides
In the present invention, term " albumen of the present invention ", " DKK1 albumen ", " DKK1 polypeptides " are used interchangeably, and all referring to has
The albumen or polypeptide of DKK1 amino acid sequences.They include the DKK1 albumen with or without initial methionine.In addition, the art
Language further includes the DKK1 and its segment of overall length.Signified DKK1 albumen of the invention includes its complete amino acid sequence, its secretion
Albumen, its mutant and its functionally active segment.
DKK1 is that WNT signal paths inhibit one of molecule dickkopf family members, also have in family DKK2,3,4 its
He is three members, and DKK1 albumen is a kind of glycoprotein of secreting type, mainly by secreting signal peptide, two conservative rich cysteines
Structural domain and catenation sequence composition.DKK1 is found to inhibit classical WNT first in the head growth research of Africa xenopus
Signal path.
The DKK1 full length proteins of people are 226 amino acid (accession number AAQ89364.1).The DKK1 full length proteins of mouse are
272 amino acid (accession number NP_034181.2).
In the present invention, term " DKK1 genes ", " DKK1 polynucleotides " are used interchangeably, and are all referred to DKK1 nucleotide
The nucleic acid sequence of sequence.
The full-length genome 3377bp (NCBI GenBank accession number is 22943) of people's DKK1 genes, transcription product
MRNA sequence overall length 1815bp (NCBI GenBank accession number is NM_012242).
The full-length genome 3621bp (NCBI GenBank accession number is 13380) of mouse DKK1 genes, transcription product
MRNA sequence overall length 2245bp (NCBI GenBank accession number is NM_010051).
People and mouse DKK1 are 75.8% in the similitude of DNA level, protein sequence similarity 80.1%.
It is to be understood that when encoding identical amino acid, the substitution of codon nucleotide is acceptable.In addition it needs
Understand, when being replaced by nucleotide and generating conservative amino acid substitution, the transformation of nucleotide is also can be received.
In the case where having obtained the amino acid fragment of DKK1, it can be constructed according to it and encode its nucleic acid sequence, and
Specific probe is designed according to nucleotide sequence.Nucleotide full length sequence or its segment can usually use PCR amplification method, recombination
Method or artificial synthesized method obtain.It, can be according to DKK1 nucleotide sequences disclosed in this invention, especially for PCR amplification method
It is that open reading frame sequence carrys out design primer, the commercially available libraries cDNA is used in combination or by conventional method institute well known by persons skilled in the art
The libraries cDNA of preparation expand as template and obtain related sequence.When sequence is longer, it is often necessary to carry out twice or multiple PCR
The segment that each time amplifies, is then stitched together by amplification by proper order again.
Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical
After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.
At present, it is already possible to obtain encoding albumen of the present invention (or its segment, derivative) by chemical synthesis completely
DNA sequence dna.Then the DNA sequence dna can be introduced into various existing DNA moleculars (such as carrier) as known in the art and cell.
By the recombinant dna technology of routine, it can be used to express or produce recombination using the polynucleotide sequence of the present invention
DKK1 polypeptides.In general there are following steps：
(1) polynucleotides (or variant) of the encoding human DKK1 polypeptides of the present invention, or with containing the polynucleotide
Recombinant expression carrier converts or suitable host cell of transduceing；
(2) host cell that is cultivated in suitable culture medium；
(3) be separated from culture medium or cell, protein purification.
In the present invention, DKK1 polynucleotide sequences can be plugged into recombinant expression carrier.As long as in short, can be in host
It replicates and stablizes, any plasmid and carrier can be used.One important feature of expression vector is to usually contain replication orgin, open
Mover, marker gene and translation control element.
Method well-known to those having ordinary skill in the art can be used to build DNA sequences encoding containing DKK1 and suitably transcribe/turn over
Translate the expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..
The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression vector also wraps
Include the ribosome bind site and transcription terminator of translation initiation.
In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion
The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg
(GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.
The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable
When host cell, allow it to expression protein.
Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high
Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, the bacterial cell of streptomyces；Fungal cell is such as
Yeast；Plant cell；Insect cell；Zooblast etc..
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original
When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl2Method processing, institute
With the step of it is generally well-known in the art.Another method is to use MgCl2.If desired, conversion can also use the side of electroporation
Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as
Microinjection, electroporation, liposome packaging etc..
The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used
Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth
It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction)
Cell is further cultured for a period of time by the promoter for inducing selection.
Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as
Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This
A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used
Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales
The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.
JNK signal paths and TGF signal beta accesses
JNK signal paths are the members of mitosis member activated protein kinase (MAPK) signal path, by environmental pressure, inflammation
Disease reacts and the activation such as growth factor, participates in the significant process such as cell Proliferation, differentiation and apoptosis.After stimulated activation, phosphorylation
JNK protein deliveries to nucleus in, further activate a series of (the JUN, ATF-2 etc.) transcription regulaton factors in downstream, to adjust
Save gene expression.
Transforming growth factor β (TGF β) belongs to transforming growth factor superfamily, and TGF β are in cell Proliferation, differentiation and apoptosis etc.
It plays an important role, plays an important role in ontogeny, tumour generation and metastases in the process.During Bone tumour, TGF
β accesses to promote the maturation of osteoclast, and then can accelerate breast by promoting cancer cells secrete to regulate and control the factor of osteoclast
The osteoclastic property of gland cancer shifts.The present invention the study found that DKK1 passes through non-classical WNT signal paths-JNK and NF- κ B accesses
Adjust the recruitment of the microenvironment cell (macrophage and medullary system source immunosuppressant cell, MDSC) in Pulmonary metastasis focuses and important thin
The secretion of intracellular cytokine TGF β influences the growth of Pulmonary metastasis focuses.DKK1 can inhibit the phosphorus of JNK passage downstream transcription regulaton factors JUN
Acidification level causes downstream gene expression to decline to reduce its transcriptional activity, inhibits tumour cell to generate and attracts macrophage thin
The ability (Fig. 2 a) of born of the same parents/MDSC factors；DKK1 can also inhibit the phosphoric acid of the important transcription regulaton factor RELA of NF- κ B accesses simultaneously
Change level is to reduce the secretion (Fig. 2 b) of downstream TGF β, the common growth for inhibiting breast cancer Pulmonary metastasis focuses.
Specific antibody
In the present invention, term " antibody of the present invention " and " specific antibody of anti-DKK1 " are used interchangeably.
The invention also includes the polyclonal antibodies and monoclonal antibody, especially Dan Ke to people's DKK1 polypeptides with specificity
Grand antibody.Here, " specificity ", which refers to antibody, can be incorporated into people DKK1 gene outcomes or segment.Preferably, referring to those energy and people
DKK1 gene outcomes or segment combination but nonrecognition and the antibody for being incorporated into other non related antigen molecules.Antibody packet in the present invention
Those molecules that can be combined and inhibit people's DKK1 albumen are included, also include the antibody that those have no effect on people's DKK1 protein functions.
The invention also includes the antibody that those can be combined with people's DKK1 gene outcomes of modification or unmodified form.
The present invention includes not only complete monoclonal or polyclonal antibody, but also includes having immunocompetent antibody piece
Section, such as Fab ' or (Fab)2Segment；Heavy chain of antibody；Antibody light chain；Genetically engineered Single Chain Fv Molecule A (Ladner et al.,
United States Patent (USP) No.4,946,778)；Or chimeric antibody, such as there is mouse antibody binding specificity but still retain the antibody portion from people
The antibody divided.
The antibody of the present invention can be prepared by various technologies known to a person skilled in the art.For example, purifying
People DKK1 gene outcomes or its with antigenic segment, animal can be applied to induce the generation of polyclonal antibody.
Similar, it expresses people DKK1 albumen or its cell with antigenic segment can be used to immune animal to produce antibody.
The antibody of the present invention can also be monoclonal antibody.Such monoclonal antibody can using hybridoma technology come prepare (see
Kohler et al.,Nature256；495,1975；Kohler et al.,Eur.J.Immunol.6：511,1976；Kohler etc.
People,Eur.J.Immunol.6：292,1976；Hammerling et al.,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981).The antibody of the present invention includes that can block resisting for people's DKK1 protein functions
Body and the antibody for not influencing people's DKK1 protein functions.All kinds of antibody of the present invention can utilize the segment of people's DKK1 gene outcomes
Or functional areas, it is obtained by common immunological techniques.These segments or functional areas can utilize recombination method to prepare or utilize polypeptide
Synthesizer synthesizes.The antibody combined with the unmodified form of people's DKK1 gene outcomes can be used in prokaryotic cell (such as E.Coli)
The gene outcome of production generates animal is immunized；The antibody combined with posttranslational modification form is (such as glycosylation or phosphorylation
Albumen or polypeptide), animal can be immunized with the gene outcome generated in eukaryocyte (such as yeast or insect cell) and obtain
?.
The antibody of anti-human DKK1 albumen can be used in immunohistochemistry technology, detection sample (especially tissue samples or
Serum sample) in people's DKK1 albumen.Since there are extracellular regions for DKK1 albumen, falls off in extracellular region and enter the feelings of blood
Under condition, these soluble DKK1 extracellular regions can become the target object of Virus monitory.
Detection method
Be present in the body fluid (preferably serum or blood) of breast cancer using DKK1, and with Metastasis in Breast Cancer it is closely related this
One feature, the present invention also provides the methods of detection metastatic breast cancer.
The present invention a preference in, the present invention provide it is a kind of detection DKK1 ELISA method and time resolution exempt from
Epidemic disease fluorescence method (TRFIA).
Detection kit
Correlation based on DKK1 with Metastasis in Breast Cancer, i.e. DKK1 are present in breast cancer body fluid (preferably blood or serum)
In, and the concentration of DKK1 is different, and the transfering type of patient with breast cancer is different, therefore DKK1 can be as metastatic breast cancer
A kind of diagnosis marker.
The present invention also provides a kind of kit of detection metastatic breast cancer, it contain detection DKK1 genes, mRNA,
The detection reagent of cDNA or albumen；And label or specification, the label or specification indicate the kit for detecting
Metastatic breast cancer；
Wherein, the label or specification indicate the following contents：
(i) when concentration≤2.3ng/ml of the serum DKK1 of detection object (subject) is (preferably, 0.05-2.3ng/
Ml or 0.5-2.0ng/ml), then prompt the risk that breast cancer Lung metastases occur for the detection object high, and Bone of Breast Cancer occurs and turns
The risk of shifting is low；
(ii) when detecting the concentration > 2.3ng/ml and≤3.0ng/ml of serum DKK1 of object (preferably, 2.3-
It 3.0ng/ml) then prompts the risk that breast cancer Lung metastases occur for the detection object relatively low (risk≤20% in 3 years), and breast occurs
The risk of gland cancer Bone tumour is higher (risk >=20% in 3 years)；
(iii) as the concentration > 3ng/ml (such as 3.0-5.5ng/ml) of the serum DKK1 of detection object, then the detection is prompted
The risk that Bone tumour occurs for object is high (risk >=30% in 3 years), the low (risk in 3 years of the probability for occurring Lung metastases<10%).
In a preferred embodiment, the present invention also provides the diagnostic kits of DKK1, including：DKK1mRNA diagnosis examinations
Agent box or DKK1 enzyme linked immunologicals (ELISA) detection kit.
Detection method and kit
The present invention relates to quantitative and detection and localization people DKK1 protein levels or mRNA level in-site diagnostic testing process.These examinations
It is known in the art to test.The people's DKK1 protein levels detected in experiment can be used for diagnosing (including auxiliary diagnosis) breast
The position whether gland cancer shifts or shift.
A kind of method in detection sample with the presence or absence of DKK1 albumen is examined using the specific antibody of DKK1 albumen
It surveys, it includes：Sample is contacted with DKK1 protein specific antibodies；It sees whether to form antibody complex, it is multiple to form antibody
It closes object and means that there are DKK1 albumen in sample.
DKK1 albumen or its polynucleotides can be used for the diagnosing and treating of DKK1 protein related diseases.The multinuclear glycosides of the present invention
Part or all of acid can be used as probe and be fixed in microarray or DNA chip, the difference table for analyzing gene in tissue
Up to analysis and gene diagnosis.The antibody of anti-DKK1 can be fixed on protein-chip, for detecting the DKK1 albumen in sample.
Main advantages of the present invention include：
(1) present invention firstly discovers that, joint targeted inhibition is carried out to two signal path JNK and the TGF β in the downstreams DKK1,
It can inhibit breast cancer to the transfer of lung and bone simultaneously, thus provide a kind of combination therapy side for the transfer of breast cancer multiple organ
Case.
(2) present invention firstly discovers that, the serum DKK1 protein levels of the patient with breast cancer of Bone tumour group are significantly higher than nothing and turn
The control group of shifting, and the DKK1 of Lung metastases group patient is then substantially less than control group.Also, the apneumia of high serum DKK1 group patients turns
It moves survival rate and is significantly higher than low DKK1 groups, and on the contrary, this shows that DKK1 is a new class of while pre- in no Bone tumour survival rate
The biological marker for surveying Bone of Breast Cancer transfer and Lung metastases fully assesses the more target organs of patient and shifts risk simultaneously to play
The effect of guiding treatment.
(3) present invention firstly discovers that, the Bone tumour and lung of patient with breast cancer can be assessed according to the DKK1 protein levels of serum
It shifts risk, can be used as more accurate Prognosis and more targeted therapeutic strategy offer reference frame is provided.
(4) present invention firstly discovers that, the correlation of DKK1 and bone, Lung metastases have specificity in its protein family.
(5) present invention firstly discovers that, JNK and TGF beta inhibitors are used in combination can reduce breast cancer to bone and to lung simultaneously
Transfer ability, the patient for the even multiple target organ transfer of clinical treatment mammary gland carcinogenesis bone or lung's far-end transfer carries
New therapeutic scheme is supplied.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
Unless stated otherwise, otherwise material therefor and reagent are commercial product in the embodiment of the present invention.
Universal method
(1) detection of patients serum's sample：After obtaining patients serum's sample, frozen immediately at -80 DEG C.Before detection, by blood
Final proof is originally melted on ice, is centrifuged 5 minutes with 3000 revs/min at 4 DEG C, draws supernatant and according to the (purchase of DKK1ELISA kits
From R&D Systems), DKK3 kits (be purchased from Abcam) and (the being purchased from Abcam) dilution of DKK4 kits and detect patients serum
DKK1, DKK3 and DKK4 concentration of sample.
(2) THP-1 double smalls room migration experiment (24 orifice plate)：First by inhibited dose of stimulation or the tumour cell item of control group
Part culture solution presses 1 with 1640 culture mediums of RPMI (being purchased from Thermo)：1 ratio mixes, and takes 500 μ l mixed liquors that lower section is added small
Room.By 2 × 105THP-1 cells (being purchased from Chinese Academy of Sciences's cell bank) are resuspended in 300 μ lRPMI1640 culture mediums, are carefully added into
Square cell.At 37 DEG C, 5%CO2Collect liquid in lower chamber after 16 hours of culture, and to the cell count in liquid,
Obtain the number of migrating cell.
(3) MDSC double smalls room migration experiment (24 orifice plate)：1. vitro differentiation MDSC：It sacrifices BALB/c mouse and (is purchased from Shanghai
This Lake experimental animal Co., Ltd), the long bone of its lower limb is taken out, bone marrow cell is gone out, in differential medium (RPMI
1640,10% fetal calf serum, 1% penicillin/streptomycin is dual anti-, and 40ng/ml GM-CSF (are purchased from Peprotech), 10ng/
MlIL-6 (be purchased from SinoBiological)) in culture 7 days, the cell centrifuged i.e. MDSC.2. double small room migrates：Basic step
Suddenly consistent with the migration of THP-1 double smalls room, but 1 × 10 need to be planted in the cell of upper layer6Cell, and bottom chamber is collected after 3 hours
In liquid and counting.
(4) osteoclast vitro differentiation：Mouse is sacrificed, the long bone of its lower limb is taken out, bone marrow cell is gone out and in α-MEM
Overnight incubation in culture medium (being purchased from Thermo).Draw upper layer culture medium within second day, 1500 revs/min are collected by centrifugation cell.It will
The cell of collection is with 2 × 106The concentration of/ml is resuspended in osteoclast differential medium (α-MEM, 20% inactivated fetal bovine serum)：
Tumour cell CMC model 3：In 1 mixed liquor, and be added final concentration of 25ng/ml recombination RANKL (being purchased from Peprotech) and
50ng/ml rhM-CSFs (are purchased from Peprotech), cultivate 6 days.With TRAP staining kits (being purchased from Sigma) dyeing after 6 days
And count the mature osteoclast of the TRAP positives of check figure >=3.
Mouse experiment：1.DKK1 functional verifications：The breast cancer cell for being overexpressed DKK1 or control group is digested and is counted, respectively
By 2 × 105With 1 × 105Cell by left ventricle and tail vein injection enter nude mice (purchased from Shanghai Si Laike experimental animals it is limited
Company) in vivo, and regular injections luciferase substrate is injected into mouse vivo detection transfer stove growing state.
(5) JNK and TGF beta inhibitor combination therapies：By 4T1.2 mouse tumor cells (be obtained from the laboratories Miller) with 1 ×
105It is injected into the mammary fat pad of mouse.Injection started to measure mouse tumor volume after a week twice a week, in injection two weeks
Mouse is randomly divided into four groups afterwards, keeps the average of every group of in situ tumor volume close and without significant difference.Injection is in situ swollen
Tumor starts that inhibitor (SP600125 (20mg/kg), SB431542 (10mg/kg)) or control solvent are injected intraperitoneally daily after two weeks
Control.Injection starts irradiation X-ray photograph after two weeks and observes Bone tumour, and the lung for taking out mouse checks Pulmonary metastasis focuses quantity.
The inhibitor used in this patent：SP600125,JNK-IN-8,SB431542,SD-208,LY2109761,
SB525334 and Galunisertib is purchased from Selleckchem, and BI 78D3 and TCS JNK 6o are purchased from Tocris.This patent
The middle antibody used：JUN (9165), p-JUN (9164), SMAD2 (5339) and pSMAD2 (3108) is purchased from and Cell
Signaling Technologies.GAPDH (G9545) is purchased from Sigma.TGF β 1 (18978-1-AP) are purchased from
Proteintech。
Embodiment 1
Expression patterns of the DKK1 in breast cancer sample
It is found by the enzyme linked immunosorbent assay (ELISA) (ELISA) to patients serum, the blood of the patient with breast cancer of Bone tumour group
Clear DKK1 protein levels are significantly higher than the control group of no transfer, and the DKK1 of Lung metastases group patient is then substantially less than control group (figure
1a).Also, high serum DKK1 group patients' is significantly higher than low DKK1 groups without Lung metastases survival rate, and in no Bone tumour survival rate
In opposite (Fig. 1 b).Research before this almost never reported it is similar with DKK1 in different tissues transspecific have phase
The biological marker of anti-expression pattern, showing that DKK1 has becomes a new class of while predicting Bone of Breast Cancer transfer and Lung metastases
The possibility of biological marker, the shifting risk for more target organs to play the role of fully assessing patient and guide treatment.Such as
Fruit is using DKK1 contents height is grouped patient in patients serum, it can be seen that the Bone tumour and lung of difference group patient
Shifting risk has very big difference (table 1), can be used as more accurate Prognosis and formulates more targeted therapeutic strategy
Reference frame is provided.
Table 1：The bone of the patient of difference DKK1 contents, Lung metastases risk in serum
Embodiment 2
Functional studies of the DKK1 in the transfer of breast cancer group-specific
In order to detect whether DKK1 has function in breast cancer tissue's transspecific, what is marked with luciferase
It has been overexpressed DKK1 in MDA-MB-231 breast cancer cell lines (being obtained from the laboratories Joan Massague), has then passed through cell
Tail vein and atrium sinistrum are injected into immunodeficient mouse (being purchased from Shanghai Slac Experimental Animal Co., Ltd.), pass through luciferase
The injection of substrate is able to observe that optical signal corresponding with transfer load.It is consistent with clinical correlation, the light of tumour cell
The DKK1 that signal display is overexpressed can significantly inhibit the Lung metastases of breast cancer and significantly increase Bone tumour (Fig. 1 c, d, e).
Embodiment 3
The mechanism of action of DKK1
DKK1 is the inhibition albumen of classical WNT signal paths.The present invention the study found that DKK1 passes through non-classical WNT
Signal path-JNK and NF- κ B accesses adjust microenvironment cell (macrophage and the immunosupress of medullary system source in Pulmonary metastasis focuses
Cell, MDSC) recruitment and important cytokine TGF β secretion influence Pulmonary metastasis focuses growth.DKK1 can inhibit JNK logical
The phosphorylation level of road downstream transcription regulatory factor JUN causes downstream gene expression to decline to reduce its transcriptional activity, suppression
Tumour cell processed generates the ability (Fig. 2 a) for attracting macrophage/MDSC factors；DKK1 can also inhibit NF- κ B access weights simultaneously
The phosphorylation level of transcription regulaton factor RELA is wanted to reduce the secretion (Fig. 2 b) of downstream TGF β, common inhibition breast cancer lung turns
Move the growth of stove.
Embodiment 4
The inhibitor SP600125 and SB431542 that JNK and TGF β are used in combination can significantly inhibit breast cancer Lung metastases and bone
Transfer
The inhibitor SP600125 (Sellckchem) and SB431542 of JNK and TGF β are used in combination in Mice Body
(Selleckchem) detect whether they can significantly reduce breast cancer cell from original position to its hetero-organization (including lung tissue
And bone tissue) transfer ability.Through treatment after a period of time, although the growth effect to primary tumor is little (Fig. 2 c), connection
The tumor nodule number for sharing medicine group mouse lung surface substantially reduces (Fig. 2 d).
In addition, being also further observed that breast cancer also has significant decrease to the ability of Bone tumour in the case of drug combination
(Fig. 2 e).In Bone of Breast Cancer transfer, the osteoclast in the cancer cell meeting excessive activation bone tissue of bone tissue is reached, it is ripe
Osteoclast can degrade the bone matrix rich in growth factor, further nourish tumour cell and simultaneously provide growing space.It is osteoclastic thin
The excessive activation of born of the same parents shows as the destruction and dissolving of bone tissue, and observed after SP600125 and SB431542 is used in combination by
The osteoclastic area that tumor cell induction corrodes is reduced significantly.
To sum up, result of study of the invention is found, be used in combination JNK and TGF beta inhibitors can reduce simultaneously breast cancer to
Bone and transfer ability to lung are the even multiple target organ transfer of clinical treatment mammary gland carcinogenesis bone or lung's far-end transfer
Patient provide new therapeutic scheme.
Embodiment 5
Verify reliabilities of the JNK with TGF beta inhibitors in treatment Lung metastases and Bone tumour
Present invention employs a variety of inhibitor and combinations thereof for JNK and TGF β, verify its to macrophage recruit and
Whether osteoclast cell maturation has effect.First, can verify JNK the and TGF beta inhibitors of use effectively inhibit JNK and TGF β to believe
Number access.By the phosphorylation level for detecting downstream JUN and SMAD2 albumen, it is thus identified that the inhibitor used can effectively inhibit
Corresponding signal path (Fig. 3 a).Using tumour cell conditioned medium as attractant, using the migration experiment of double small room come mould
Quasi- tumour cell recruits the precursor monocyte (Fig. 3 b) of cycle macrophage in lung.
Experimental result find, other used jnk inhibitors (BI 78D3, TCS JNK 6o and JNK-IN-8) and
SP600125 can significantly reduce the monocyte emigration of tumour cell conditioned medium induction equally effectively, this display is a variety of
Jnk inhibitor may all inhibit migration of the monocyte into Pulmonary metastasis focuses in vivo.It is opposite, except Galunisertib have compared with
Other than weak effect, TGF beta inhibitors (SB525334, SD-208, SB431542 and LY2109761) are to breast cancer cell condition
The monocyte emigration of culture solution induction is almost without effect.The above results are consistent with the Mechanism Study of the present invention, i.e. under JNK-JUN
Trip Gene on Tumor Cells attracts monocyte to play a major role.In addition, the combination of JNK and TGF beta inhibitors also show with
JNK signal pathway inhibitors are similar as a result, the antagonism that can't influence each other is used in combination in two kinds of inhibitor of display, still have
Effect.
Meanwhile to detect effect of these inhibitor to Bone tumour, mouse primary Bone Marrow Cells In Vitro osteoclast is utilized
Differentiation technique.Mouse primary bone marrow cell includes the various kinds of cell ingredient including osteoclast precursor cells, can be more
The interaction of true simulation tumour cell and bone marrow cell component.Identical, the present invention also has detected JNK and TGF β and inhibits
Whether agent influences the osteoclast cell maturation (Fig. 3 c) of breast cancer cell induction.
Experimental result shows have part jnk inhibitor to have the osteoclast cell maturation differentiation that breast cancer cell induces certain
Inhibiting effect, and TGF beta inhibitors then all show the stronger inhibiting effect to osteoclast cell maturation.With the present invention it is small
Mouse result is consistent, the inhibiting effect of two kinds of inhibitor being used in combination to osteoclast cell maturation.For mature osteoclast
Anti- tartaric acid alkaline phosphatase (TRAP) coloration result show, the present invention tested part jnk inhibitor, all TGF β suppression
Preparation and combination thereof can effectively reduce TRAP under the visual field+Multinuclear mature osteoclast quantity (Fig. 3 d).
It discusses
In conclusion the present invention the study found that clinically, DKK1 is one new can predict that breast cancer is suffered from simultaneously
The biological marker gene of person's Bone tumour and Lung metastases risk, for patient shift risk prediction and Index for diagnosis provides new side
Method and foundation.In addition, the combination for the inhibitor of downstream signaling pathway JNK and the TGF β of its regulation and control can effectively reduce lung
Lung metastases are alleviated in the infiltration of macrophage and MDSC in transfer stove；Meanwhile combination can inhibit osteoclast in Bone tumour stove
Maturation differentiation, slows down Bone tumour, and new treatment means are provided for treatment Bone tumour, Lung metastases or the patient of the multiple transfer of bone lung
(Fig. 4).
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Bibliography
1.Bos,P.D.,et al.Genes that mediate breast cancer metastasis to the
brain.Nature 459,1005-1009(2009).
2.Minn,A.J.,et al.Lung metastasis genes couple breast tumor size and
metastatic spread.P Natl Acad Sci USA 104,6740-6745(2007).
3.Minn,A.J.,et al.Genes that mediate breast cancer metastasis to
lung.Nature 436,518-524(2005).
Claims (10)
1. the purposes of a kind of DKK1 genes, mRNA, cDNA or albumen or its detection reagent, which is characterized in that (i) is used as detection
The marker of metastatic breast cancer or Metastasis in Breast Cancer risk；And/or (ii) is used to prepare detection metastatic breast cancer or mammary gland
The diagnostic reagent or kit of metastasis of cancer risk.
2. purposes as described in claim 1, which is characterized in that the Metastasis in Breast Cancer is selected from the group：Bone tumour, Lung metastases,
Brain metastes, hepatic metastases, or combinations thereof.
3. a kind of for detecting metastatic breast cancer or the diagnostic kit of Metastasis in Breast Cancer risk, which is characterized in that described
Kit contains a container, contains the detection reagent for detecting DKK1 genes, mRNA, cDNA or albumen in the container；And mark
Label or specification, the label or specification indicate the kit for detecting metastatic breast cancer or Metastasis in Breast Cancer wind
Danger.
4. diagnostic kit as claimed in claim 3, which is characterized in that the detection metastatic breast cancer or breast cancer turn
It moves risk and refers to the metastasis site whether detection Metastasis in Breast Cancer has occurred, occurred, and/or
Judge the possibility size of generation Metastasis in Breast Cancer.
5. diagnostic kit as claimed in claim 3, which is characterized in that the detection DKK1 genes, mRNA, cDNA or
The detection reagent of albumen includes：
(a) specific antibody of the anti-DKK1 albumen of；And/or
(b) specific primer of the mRNA or cDNA of specific amplifications DKK1.
6. diagnostic kit as claimed in claim 3, which is characterized in that indicated in following in the label or specification
Hold：
(i) when concentration≤2.3ng/ml of the serum DKK1 of detection object (subject) (preferably, 0.05-2.3ng/ml, or
0.5-2.0ng/ml), then it prompts the risk that breast cancer Lung metastases occur for the detection object high (risk >=30% in 3 years), and sends out
The risk of lactogenesis gland cancer Bone tumour is low (risk≤10% in 3 years)；
(ii) when detecting the concentration > 2.3ng/ml and≤3.0ng/ml of serum DKK1 of object (preferably, 2.3-3.0ng/
Ml it) then prompts the risk that breast cancer Lung metastases occur for the detection object relatively low (risk≤20% in 3 years), and Bone of Breast Cancer occurs
The risk of transfer is higher (risk >=20% in 3 years)；
(iii) as the concentration > 3ng/ml (such as 3.0-5.5ng/ml) of the serum DKK1 of detection object, then the detection object is prompted
The risk that Bone tumour occurs is high (risk >=30% in 3 years), the low (risk in 3 years of the probability for occurring Lung metastases<10%).
7. diagnostic kit as claimed in claim 3, which is characterized in that the kit is additionally operable to prediction metastatic mammary gland
The life span of cancer patient or prognosis.
8. a kind of method of detection metastatic breast cancer or Metastasis in Breast Cancer risk, which is characterized in that the method includes：
A) test sample from subject is provided；
B) concentration of DKK1 albumen in test sample is detected；With
C) concentration of the DKK1 albumen measured in step b) is compared with control,
Wherein compared with the control, the concentration of DKK1 albumen is less than reference value in the sample, shows that subject occurs lung and turns
The probability of shifting is higher than general patient with breast cancer crowd；And/or the concentration of DKK1 albumen is higher than reference value, shows that bone occurs for subject
The probability of transfer is higher than general patient with breast cancer crowd.
9. method as claimed in claim 8, which is characterized in that the test sample includes the blood sample of patient with breast cancer
And/or serum sample.
10. a kind of method of determining therapeutic scheme, which is characterized in that including：
A) test sample from subject is provided；
B) concentration of DKK1 albumen in test sample is detected；With
C) therapeutic scheme is determined based on the concentration of the DKK1 albumen in the sample.
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