CN103212077A

CN103212077A - Application of Erbin inhibitor in preparation of antitumor drug

Info

Publication number: CN103212077A
Application number: CN2013101212756A
Authority: CN
Inventors: 梅林�; 陶艳梅; 沈承勇; 熊文诚; 罗时文
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-09
Filing date: 2013-04-09
Publication date: 2013-07-24
Anticipated expiration: 2033-04-09
Also published as: CN103212077B

Abstract

The invention provides application of an Erbin inhibitor in preparation of an antitumor drug. The inhibitor is specifically a compound capable of lowering expression of Erbin or interfering Erbin-ErbB2 interaction. The Erbin-Erbin interaction is crucial to tumor-prompting action and carcinogenic action of ErbB2; the ErbB2 dependent cancer cell proliferation or tumor growth can be inhibited by over-expressing a PDZ structural segment of Erbin or excessively giving tail-end polypeptide segments to the ErB2 in the cell. The invention provides a novel way of treating ErbB2 positive tumors, i.e., a way of inhibiting protein stability and kinase activity of the ErbB2 by reducing the expression of Erbin or interfering with the combination of ErBin and ErbB2, so that the tumor-prompting action and carcinogenic action of the ErbB2 are inhibited.

Description

The application of Erbin inhibitor in the preparation antitumor drug

(1) technical field

The present invention relates to the application of Erbin inhibitor in the preparation antitumor drug.

(2) background technology

The receptor of endothelial cell growth factor (ECGF) (Epidermal growth factor receptor) family comprises that EGFR(claims HER1 again, ErbB1), ErbB2(HER2, neu), ErbB3(HER3) and ErbB4(HER4) all participate in the generation and the development of tumor.Wherein ErbB2 especially causes concern, because surpass the overexpression that detects ErbB2 in 25% the breast cancer tissue, and ErbB2 crosses the breast carcinoma patient of expression and often has high recurrent, and shifts easily and prognosis mala.Except that breast carcinoma, ErbB2 crosses expression and also involves ovarian cancer, gastric cancer and uterus carcinoma.ErbB2 begins exactly as proto-oncogene found (an oncogene carcinogen-induced neuroblastoma, a neu who causes conversion most; Perhaps an oncogene of avian erythoblastosis virus, ErbB2).As a member of endothelial growth factor receptor family, the not direct and any known ligand combination of ErbB2 comes transduction signal but formed heterodimer with other by the activated endothelial cell growth factor (ECGF) family member of part.Though be orphan receptor, ErbB2 is that other is inclined to by the activated ErbB receptor of part forms dimeric member most, and forms dimer with ErbB2 and can significantly promote the degree that other ErbB receptor is activated.The more important thing is, thereby the ErbB2 of high expressed can not rely on the stimulation of part and directly forms different aggressiveness or activate downstream passages with aggressiveness in breast cancer cell.This mechanism has increased the weight of the effect of the carcinogenic and short cancer of ErbB2.ErbB2 can comprise Ras, Src, PI-3K/Akt by activated downstream signal path after activating, GAB2, and small G-protein, the activation of these paths finally causes propagation, the survival of cancerous cell, and epithelial cell is to the conversion of Interstitial cell, migration and to the erosion of hypothallus.

Crossing the ErbB2 that expresses has the ability that makes the cell cancerization and has obtained various exsomatize and in the confirmation of body experiment.ErbB2 crosses expression can make l cell NIH3T3 and NR6 transform, and can make normal people's galactophore epithelial cell MCF-10A form the solid polyadenous bubble structure of similar ductal carcinoma in situ (DCIS).Specificity is crossed expression ErbB2 and can be caused mice to generate breast tumor and be transferred to pulmonary in the mice breast epithelium.All these results of study show that all the protein level that suppresses ErbB2 is the important channel of treatment breast carcinoma.Monoclonal antibody trastuzumab and the pertuzumab that have now developed at ErbB2, and the inhibitor lapatinib of anti-ErbB kinase activity is applied to clinically.But these medicines tend to cause Drug resistance, and the patient of treatment also is easy to recurrence.Thereby how to find the new treatment approach at ErbB2 is the direction that scientific research person makes great efforts always.

The ErbB receptor can be degraded by endocytosis after being subjected to ligand stimulation.But the endocytosis that comprises the receptor dimer of ErbB2 but is suppressed; Not only can stop the endocytosis inactivation of other ErbB receptor with the ErbB2 dimerization, can also promote them to shift back cell membrane.Suppress ErbB2 and with the mechanism of the receptor endocytosis of ErbB2 dimerization be with the bonded born of the same parents of ErbB2 in molecule.The C-end of ErbB2 is extremely important to its stability, remove the C-end only 3 amino acid residues will significantly reduce the protein stability of ErbB2 and increase its degraded.These several C-terminal amino acid residues constitute one in conjunction with PSD95-Dlg1-zo-1(PDZ) zone of structure, hint that an ErbB2 who contains the PDZ structure fragment is conjugated protein to its stability key especially.The present invention as bait (Bait), filters out the conjugated protein Erbin(ErbB2-Interacting protein of ErbB2 with the C-terminal fragment of ErbB2 in the yeast two-hybrid library).Erbin is a LAP albumen, and the N-end contains 16 leucine enrichment repetitive sequences, and (Leucine-rich repeats, LRR), and its C-end is a PDZ structure fragment.Erbin by the PDZ structure specifically with the ErbB2 combination, and not in conjunction with other ErbB family receptors.The present application people finds that Erbin is extremely important to the protein stability of ErbB2, thereby and Erbin can also promote the ErbB2 dimerization activation ErbB2 kinase activity and the carcinogenic and short cancer signal path in downstream of expressing.The present invention is very crucial to crossing the carcinogenic and short cancer effect of expressing ErbB2 with cell and animal model proof Erbin; Disturb Erbin and combining of ErbB2 all to show the effect of the carcinogenic and short cancer of inhibition ErbB2 with the multiple means specificity.

(3) summary of the invention

The invention provides a kind of novel cancer detection label---albumen or the mRNA level of Erbin, and the application of Erbin inhibitor in the preparation antitumor drug is provided, for new tumour medicine research and development provide the foundation.

The enlightenment that played a crucial role in breast carcinoma takes place and develops by ErbB2, the inventor has studied the influence of its conjugated protein Erbin to these processes.The result shows that Erbin is specific expressed in the breast duct epithelial cell, and the dependent breast cancer cell of ErbB2 has been bred necessary effect.The Erbin that suddenlys change in the mice breast epithelium can not interact it with ErbB2, can suppress the dependent breast cancer animal model MMTV-neu mice of ErbB2 and can not generate tumor.And the tumor of the breast cancer animal model MMTV-PyVT mice of ErbB2 dependent/non-dependent generation is unaffected.The short cancer of the interaction partners ErbB2 of Erbin and ErbB2 and carcinogenesis are very crucial, cross the PDZ structure fragment of expressing Erbin, or the excessive C-terminal polypeptide fragment that in cell, gives ErbB2, can suppress dependent cancer cell multiplication of ErbB2 or growth of tumor.The expression of striking low Erbin with specificity shRNA in the interferential mode of gene in tumor cell, also propagation and the tumor growth to the dependent cancerous cell of ErbB2 has remarkable inhibitory action.These results show that Erbin generates the dependent tumor of ErbB2 and growth has crucial regulating action, and novel cancer approach and the medicament forms of pressing down also is provided.The research and analysis of clinical breast carcinoma specimen show that the expression ratio cancer beside organism of Erbin in the cancerous tissue significantly raises, and the expression of the expression of Erbin and the pathological grading of tumor and ErbB2 is closely bound up, and the feasibility of carrying out the clinical tumor diagnosis with the expression that detects Erbin is provided.

The technical solution used in the present invention is:

The application of Erbin inhibitor in the preparation antitumor drug.Described tumor comprises breast carcinoma, ovarian cancer, gastric cancer and uterus carcinoma etc.

Concrete, described inhibitor is expression that reduces Erbin or the interactional chemical compound that disturbs Erbin-ErbB2.

Preferably, described inhibitor is the polypeptide shown in SEQ ID No.1 or the SEQ ID No.3.

SEQ ID No.1 sequence is as follows:

EIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLL

QPGDKIIQANGYSFINIEHGQAVSLLKTFQNTVELIIVREV

SEQ ID No.3 sequence is as follows:

PTAENPEYLGLDVPV(annotates: last four amino acids DVPV are the most key for the peptide chain C-terminal, and the peptide Duan Jun that it is DVPV that all C hold last four amino acids is in protection scope of the present invention)

Preferably, described inhibitor is the shRNA of Erbin.

The invention still further relates to the PDZ structure fragment of a kind of Erbin of comprising, play the polypeptide of dominant negative effect, its aminoacid sequence is shown in SEQ ID No.1.

The encode gene order of this polypeptide, its nucleotide sequence is shown in SEQ ID No.2:

GAGATTCGAGTGAGGGTTGAAAAGGATCCAGAACTTGGATTTAGCATATCAGGTGGTGTCGGGGGTAGAGGAAACCCATTCAGACCTGATGATGATGGTATATTTGTAACAAGGGTACAACCTGAAGGACCAGCATCAAAATTACTGCAGCCAGGTGATAAAATTATTCAGGCTAATGGCTACAGTTTTATAAATATTGAACATGGACAAGCAGTGTCCTTGCTAAAAACTTTCCAGAATACAGTTGAACTCATCATTGTACGAGAAGTT

The invention still further relates to the interactional polypeptide of a kind of interference Erbin-ErbB2, its aminoacid sequence is shown in SEQ ID No.3.

The encode gene order of this polypeptide, its nucleotide sequence is shown in SEQ ID No.4:

CCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTG。

Because the particularity of aminoacid sequence; any fragment or its variant that contains the polypeptide of aminoacid sequence shown in SEQ ID NO.1 and 3; as its examples of conservative variations, bioactive fragment or derivant; as long as the fragment of this polypeptide or polypeptide variants and aforementioned amino acid sequence homology all belong to the row of protection domain of the present invention more than 90% and have identical functions.Concrete, described change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative change of variant, the aminoacid of being replaced has structure similar to original acid or chemical property, and as replacing isoleucine with leucine, variant also can have non-conservation and change, as replacing glycine with tryptophan.

Because the particularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ NO.2 and 4 as long as itself and this polynucleotide have 90% above homology and have identical functions, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide is meant a kind of polynucleotide sequence that one or more nucleotide change that has.The variant of these polynucleotide can make the living allelic variant or the variant of non-life, comprises replacing variant, deletion mutation body and inserting variant.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of a plurality of nucleotide, but can be from not changing the function of its amino acids coding in fact.

Beneficial effect of the present invention is mainly reflected in: the application of Erbin inhibitor of the present invention in the preparation antitumor drug, and for diagnosing tumor and treatment provide new approach.

(4) description of drawings

Fig. 1 is the influence of Erbin to ErbB2 independent mammary tumor cells propagation; A be with Western blotting detected in different breast cancer cell lines shErbin reduce the ability and the degree of Erbin protein expression; B～D is the propagation situation with the different stable cell lines of MTS method detection; N=3, * P＜0.05, * * P＜0.01;

Fig. 2 is the influence in body generation and growth of Erbin to breast cancer tumour; D0 is injection day, and D10 is for injecting the back the 10th;

Fig. 3 is protein stability and the kinase activity that the PDZ structure fragment dominant negative of Erbin is destroyed ErbB2 in the breast cancer cell; A detects the protein level of ErbB2 and represents the tyrosine phosphorylation level (pErbB2) of kinase activity with Western blotting; B-C is the quantitative statistics analysis of the result among the experiment A; N=3, * * P＜0.01;

Fig. 4 is the influence of Erbin-PDZ structural region to the ErbB2 dependent tumors; A is that Kaplan-Meier does not have the tumor scattergram, Log-rank(Mantel-Cox) detects P＜0.0002; B is MMTV-neu; Erbin ^{C/ C}And MMTV-neu; Erbin ^{+ /+}Mice exposes mammary gland through dissecting; C be mice mammary gland or lung separate the fixing section in back, through hematoxylin-Yin Hong (H/E) dyeing photo; D carries out whole brazilwood extract dyeing photo after the mice mammary gland of isolating not bearing tumor is fixed; E is that Kaplan-Meier does not have the tumor scattergram; F excises the growth that does not influence the MMTV-PyVT mouse mammary tumor, n=8, P for the PDZ structural region〉0.05; G is at MMTV-PyVT; Erbin ^{+ /+}And MMTV-PyVT; Erbin ^{C/ C}The histologic characteristics of the mastadenoma that generates in the mice.

Fig. 5 is the interaction that the C-terminal polypeptide sequence B 2tail of ErbB2 disturbs Erbin and ErbB2; A is that the immunoprecipitation product is with the Western blotting testing result; B is the quantitative statistics analysis of experimental result among the A, n=3, * P＜0.05;

Fig. 6 is the ErbB2 protein stability of TAT-B2tail polypeptide fragment reduction breast cancer cell, kinase activity and cell proliferation; A is the cleaved Western blot testing result of carrying out of cell after handling; B is the quantitative statistics analysis of experimental result among the A; C is the influence to the rate of increase of polypeptide treatment S KBR3 or BT474 cell;

Fig. 7 is the TAT-B2tail polypeptide fragment suppresses growth of tumor at body a representative experimental results;

Fig. 8 is the clinical case result of study; A is the immunohistochemical staining result of representational Erbin in clinical breast carcinoma specimen section; B is the case figure that the SABC result of quantized Erbin distributes in cancerous tissue and cancer beside organism; C is the case figure of the distribution of the painted quantized result of Erbin groupization in different tumor pathology ranks; D is representational Erbin and the immunohistochemical staining result of ErbB2 in adjacent clinical breast carcinoma specimen section; E is the case figure that the SABC quantized result of ErbB2 distributes in the different case group of Erbin expression; F be fresh cancerous tissue and cancer beside organism after the tissue homogenate cracking, detect the expression of Erbin and ErbB2 by Western blotting.

(5) specific embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: confirm that with cell model and animal model Erbin generates the dependent breast carcinoma of ErbB2 and develops of crucial importancely, provides the expression of striking low Erbin with specificity shRNA to treat the approach and the means of ErbB2 dependent tumors

In order to study the influence of Erbin, selected the dependent breast cancer cell line BT474(ATCC of ErbB2 to breast cancer cell propagation) and SKBR3(ATCC), and do not rely on breast cancer cell line ZR751(Public Health England with ErbB2).When these cells are cultivated dishful, with the shRNA(shErbin of the specific recognition Erbin of slow virus packing) (normal chain: 5 ' TGCAT CCCTC TAGAG AACAA CTTTC AAGAG AAGTT GTTCTCTAGA GGGAT GCTTT TTTC; Anti-chain: 5 ' TCGAG AAAAA AGCAT CCCTCTAGAG AACAA CTTCT CTTGA AAGTT GTTCT CTAGA GGGAT GCA.) and another contrast shRNA(shCtrl) (pLL3.7 empty carrier) invade and harass cell respectively (with titre be 8x10 ⁸The virion suspension 1 μ l of IU/ml adds in the cell dish of the 100mm that contains the 10ml culture fluid, changes fresh medium after 24 hours).Carry green fluorescent protein (GFP) on the package carrier of shErbin or shCtrl, thereby with trypsinization and suspension cell, go out to be infected cell into by shRNA and go down to posterity to set up stable cell line with selected by flow cytometry apoptosis.

The Western blot result shows that contrast with shCtrl, shErbin can reduce by 70～90%(Figure 1A respectively with the proteic level of Erbin in three cell lines).

The stable cell line that shErbin or shCtrl integrate is grown under identical condition with identical inoculum density, and respectively at 1,2, the MTS that accepts cell proliferation after 3 days detects, and the results are shown in Figure 1B～D.What the enlightenment meaning was arranged very much is that Erbin is struck low BT474(Figure 1B by shRNA in the cell) and SKBR3(Fig. 1 C) propagation significantly slow down, and ZR751(Fig. 1 D of ErbB2 dependent/non-dependent) cell proliferation be not subjected to the influence of shErbin.This cell model proof Erbin promotes the propagation of the dependent breast cancer cell of ErbB2 specifically, and the protein level that reduces Erbin can suppress the dependent breast cancer cell propagation of ErbB2.

For further checking Erbin to the ErbB2 dependent tumors in the body effect, with the shErbin(of the integration right arrow of same quantity) or the shCtrl(left arrow) BT474 cell (100 μ l, 3.0 * 10 ⁵Individual cell is suspended among the 50%Matri-gel) be injected into right side or the 4th couple of mammary gland inside (D0 in left side of nude mice respectively, injection day), made up an animal model that people source breast carcinoma takes place to develop, observed into later tumor size (D10), and the results are shown in Figure 2 in 10 days.The 4th pair of mammary gland of the left and right sides of same nude mice is being inoculated breast cancer cell, and meaningfully, the tumor of left side inoculation shErbin-BT474 significantly reduces after 10 days, and the formed tumor of the shCtrl-BT474 on right side enlarges markedly.This experiment confirm under the body environment, the expression that reduces Erbin in the interferential mode of gene has inhibitory action to the generation development of the tumor that ErbB2 relies on.

Embodiment 2: the PDZ structure fragment that confirms Erbin with cell model and animal model generates extremely crucial to the dependent breast carcinoma of ErbB2.Can disturb the interaction of Erbin-ErbB2 to reduce protein level and its kinase activation of ErbB2 thereby cross the PDZ fragment of expressing Erbin, thereby the treatment approach and the means for the treatment of the ErbB2 dependent tumors at the PDZ structure fragment of Erbin are provided

The early stage work of inventor has disclosed Erbin and has combined by the PDZ structural region with ErbB2.The PDZ binding sequence of the PDZ structure fragment of the C-end of Erbin and the C-end of ErbB2 is to their combinations and interact very crucial.That Erbin is regulated protein stability and the function of ErbB2 is very important in order to study whether combining of Erbin and ErbB2, has made up the plasmid of the PDZ structure fragment of only expressing Erbin

ATGGAGATTCGAGTGAGGGTTGAAAAGGATCCAGAACTTGGATTTAGCATATCAGGTGGTGTCGGGGGTAGAGGAAACCCATTCAGACCTGATGATGATGGTATATTTGTAACAAGGGTACAACCTGAAGGACCAGCATCAAAATTACTGCAGCCAGGTGATAAAATTATTCAGGCTAATGGCTACAGTTTTATAAATATTGAACATGGACAAGCAGTGTCCTTGCTAAAAACTTTCCAGAATACAGTTGAACTCATCATTGTACGAGAAGTTGAACAAAAACTTATTTCTGAAGAAGATCTGTAG with BamH1 and EcoR1 restriction sites built into pRK5 vector), and then studied the expression of the PDZ Erbin fragments have dominant negative (dominant-negative) role to interfere with the binding Erbin and ErbB2, thus affecting the ErbB2 protein levels and functions.For this purpose, the Erbin-PDZ plasmid of varying number is advanced people's tire nephrocyte HEK293(ATCC with quantitative Erbin and ErbB2 transfection) in (the plasmid total amount of transfection in the different rotaring redyeing systems is supplied identical by blank carrier), detect protein level and the phosphorylation level (pErbB2) of ErbB2 then with Western blotting.The result as shown in Figure 3, the expression inhibiting of Erbin-PDZ the protein level of ErbB2, and suppressed the phosphorylation level of ErbB2 with bigger degree, shown the influence bigger to its kinase activity.More ironically, Erbin-PDZ is a dose dependent to the inhibition of ErbB2 protein level and phosphorylation level, and the Erbin-PDZ plasmid transfection must be many more, and ErbB2 albumen and phosphorylation descend just significantly more.These experiments have confirmed that with cell model the segmental expression of excessive Erbin-PDZ can influence level and the kinase activation of ErbB2, provide by disturbing Erbin-PDZ to disturb the approach and the means of ErbB2 function.

With the animal model checking, the mutant mice that has made up Erbin is erbin for further ^{Δ C/ Δ C}In this sudden change Mus, the Erbin of wild type substituted by the mutain of the Erbin of a C-tip cut-off (only surplus N-holds 1-693 amino acid residue, wild-type mice source Erbin:

MTTKRSLFVRLVPCRCLRGEEETVTTLDYSHCSLEQVPKEIFTFEKTLE

ELYLDANQIEELPKQLFNCQSLHKLSLPDNDLTTLPASIANLINLRELD

VSKNGIQEFPENIKNCKVLTIVEASVNPISKLPDGFSQLLNLTQLYLND

AFLEFLPANFGRLTKLQILELRENQLKMLPKTMNRLTQLERLDLGSNE

FTEVPEVLEQLSGLREFWMDGNRLTFIPGFIGSLRQLTYLDVSKNNIEM

VEEGISTCENLQDFLLSSNSLQQLPETIGSLKNVTTLKIDENQLMYLPD

SIGGLRSIEELDCSFNEIEALPSSIGQLTNMRTFAADHNYLQQLPPEIGN

WKNITVLFLHCNKLETLPEEMGDMQKLKVINLSDNRLKNLPFSFTKLQ

QLTAMWLSDNQSKPLIPLQKETDTETQKMVLTNYMFPQQPRTEDVMF

ISDNESFNPALWEEQRKQRAQVAFECDEDKDEREAPPREGNLKRYPTP

YPDELKNMVKTVQTIVHRLKDEETNEESGRDLKQHEDQQVVNKDKC

VKTSESTTTKSKLDEREKYMNSVQKMSEPEAETNGGNLPVTASMKLS

GNLKHIVNHDDVFEESEELSSDEEMKMAEMRPPLIESSINQPKVVALS

NNKKDDAKDADSLSDEVTHNSNQNNSNCSSPSRMSDSVSLNTDSSQD

TSLCSPVKQTPVDSNSKVRQEDENFNSLLQNGVNLNNSPEEKFKINDK

KDFKLPEYDLNIEEQLVLIEKDIDSKATSDDSRQLDHINMNINKLVTNN

IFQPEVMERSKMQDIVLGTGFLSIHPKNEAEHIENGAKFPNLESINKVN

GLCEDTAPSPGRVEPQKASSSADVGISKSTEDLSPQRSGPTGAVVKSHS

ITNMETGGLKIYDILGDDGPQPPSAAVKIASAVDGKNIVRSKSATLLYD

QPLQVFTAASSSSELLSGTKAVFKFDSNHNPEEPDIIRAATVSGPQSTP

HLYGPPQYNVQYSGSATVKDTLWHPKQNPQIDPVSFPPQRLPRSESAE

NHSYAKHSANMNFSNHNNVRANTGYHLQQRLAPARHGEMWAISPN

DRLVPAVTRTTIQRQSSVSSTASVNLGDPTRRTEGDYLSYRELHSMGR

TPVMSGSQRPLSARAYSIDGPNTSRPQSARPSINEIPERTMSVSDFNYSR

TSPSKRPNTRVGSEHSLLDPPGKSKVPHDWREQVLRHIEAKKLEKSML

SRSFNSNLTAVSSSHYGSSRDLHGSQGSLALSVADGRGSGGHIFRHPQ

TSSPGECCQDDRFMSEEQNHPSGALSHRGLPDSLMKMPLSNGQMGQP

LRPQAHYSQTHHPPQASVARHPSREQLIDYLMLKVAHQPPYTHPHCSP

RQGHELAKQEIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRV

QPEGPASKLLQPGDKIIQANGYSFINIEHGQAVSLLKTFHNAVDLIIVRE

VSS

Saltant Mus source Erbin ^1-693:

MTTKRSLFVRLVPCRCLRGEEETVTTLDYSHCSLEQVPKEIFTFEKTLEELYLDANQIEELPKQLFNCQSLHKLSLPDNDLTTLPASIANLINLRELDVSKNGIQEFPENIKNCKVLTIVEASVNPISKLPDGFSQLLNLTQLYLNDAFLEFLPANFGRLTKLQILELRENQLKMLPKTMNRLTQLERLDLGSNEFTEVPEVLEQLSGLREFWMDGNRLTFIPGFIGSLRQLTYLDVSKNNIEMVEEGISTCENLQDFLLSSNSLQQLPETIGSLKNVTTLKIDENQLMYLPDSIGGLRSIEELDCSFNEIEALPSSIGQLTNMRTFAADHNYLQQLPPEIGNWKNITVLFLHCNKLETLPEEMGDMQKLKVINLSDNRLKNLPFSFTKLQQLTAMWLSDNQSKPLIPLQKETDTETQKMVLTNYMFPQQPRTEDVMFISDNESFNPALWEEQRKQRAQVAFECDEDKDEREAPPREGNLKRYPTPYPDELKNMVKTVQTIVHRLKDEETNEESGRDLKQHEDQQVVNKDKCVKTSESTTTKSKLDEREKYMNSVQKMSEPEAETNGGNLPVTASMKLSGNLKHIVNHDDVFEESEELSSDEEMKMAEMRPPLIESSINQPKVVALSNNKKDDAKDADSLSDEVTHNSNQNNSNCSSPSRMSDSVSLNTDSSQDTSLCSPVKQTPVDSNSKVR）。The sudden change Erbin of excision C-end can not combine with ErbB2 again, thereby should lose the effect of regulating the ErbB2 function.

In order to verify this hypothesis, with erbin ^{Δ C/ Δ C}The dependent breast carcinoma mouse model of Mus and ErbB2 MMTV-neu hybridization, thus obtain containing C-end Erbin MMTV-neu mice offspring excision and wild type Erbin, called after MMTV-neu respectively; Erbin ^{Δ C/ Δ C}And MMTV-neu; Erbin ^{+ /+}Mice.

Erbinerbin ^{Δ C/ Δ C}Mice and the white mice of wild type FVB are backcrossed after 5 times, with the MMTV-neu mice hybridization of FVB background, the female descendant MMTV-neu of generation; Erbin ^{Δ C/ Δ C}(n=20) and MMTV-neu; Erbin ^{+ /+}(n=22) mice is raised under the condition of not copulation, and whether carries out generation that subcutaneous touch detects mastadenoma each week, the results are shown in Figure 4A～D.Note until about 20 above MMTV-neu of monthly age; Erbin ^{Δ C/ Δ C}All do not detect tumor in the mice and generate, and MMTV-neu; Erbin ^{+ /+}Mice 100% has tumor to generate.Fig. 4 B is the MMTV-neu at representational a pair of 12 monthly ages; Erbin ^{Δ C/ Δ C}And MMTV-neu; Erbin ^{+ /+}The dissection photo of sister mice.Showing does not have MMTV-neu; Erbin ^{Δ C/ Δ C}The mammary gland of mice can detect visible tumor.Mice mammary gland or lung separate the fixing section in back, through hematoxylin-Yin Hong (H/E) dyeing, do not detect MMTV-neu; Erbin ^{Δ C/ Δ C}In the mice mammary gland tumor tissues is arranged, and MMTV-neu; Erbin ^{+ /+}The breast tumor of mice presents the tissue signature (Fig. 4 C) of typical adenoma, MMTV-neu; Erbin ^{Δ C/ Δ C}Mouse lung also not detection place as MMTV-neu; Erbin ^{+ /+}Metastatic tumour tissue in the mouse lung.Carry out whole brazilwood extract dyeing after the mice mammary gland of isolating not bearing tumor is fixing, Fig. 4 D is the local pictures behind the representational hematoxylin bulk dyeing, visible MMTV-neu; Erbin ^{+ /+}The mice not mammary gland of bearing tumor has the outgrowth phenomenon of tangible ductal epithelium, and MMTV-neu; Erbin ^{Δ C/ Δ C}The conduit form of mice mammary gland is normal.

Female mice MMTV-PyVT; Erbin ^{+ /+}(n=23) and MMTV-PyVT; Erbin ^{Δ C/ Δ C}(n=15) raise under the condition of not copulation, the subcutaneous touch of jede Woche has or not breast tumor to generate with detection, the results are shown in Figure 4E～G.The result shows that the excision of PDZ structural region does not influence the growth of MMTV-PyVT mouse mammary tumor.The MMTV-PyVT mouse mammary tumor is laid one's hand on and it's two months is past the back, and tumor is stripped from the growth rate of weighing with comparison of tumor, at MMTV-PyVT; Erbin ^{+ /+}And MMTV-PyVT; Erbin ^{Δ C/ Δ C}The mastadenoma that generates in the mice has identical histologic characteristics.

MMTV-neu breast carcinoma mouse model is that specific the mistake with the MMTV promoter in galactophore epithelial cell expressed rat ErbB2 gene neu and come carcinogenic model.The excessive ErbB2 that MMTV-neu expresses stimulates cyclomastopathy and final canceration.This carcinogenic mouse model breast carcinoma early start betides 4-6 about the month.The breast carcinoma of most of Mus takes place later, but final 100% MMTV-neu mice can produce breast carcinoma.And breast carcinoma takes place in latter two month, and 80% mice has lung and shifts.Yet the Erbin mutain rather than the proteic mice of wild type Erbin of expressing the C-tip cut-off all do not detect breast tumor, the phenomenon (Fig. 4 A-D) that does not yet have cyclomastopathy and lung to shift when 20 monthly ages.And ironically, with erbin ^{Δ C/ Δ C}In offspring after another breast carcinoma mouse model MMTV-PyVT hybridization, the Erbin mutain of expressing the C-tip cut-off can not stop the generation and the growth (Fig. 4 E-G) of breast tumor.These experimental results of coming from animal model are supported the importance of Erbin-PDZ structural region to ErbB2 dependent tumors generation development strongly, support to disturb Erbin-PDZ to treat the novel approach and the means of tumor.

Embodiment 3: confirm that with cell model and animal model the C-terminal polypeptide fragment of ErbB2 can disturb the interaction of Erbin-ErbB2 and reduce protein level and the kinase activity of ErbB2 in the cancerous cell, provide the C-terminal polypeptide B2tail that gives excessive ErbB2 with external source to treat the approach and the means of tumor

For develop be easy to use at the interactional treatment means of Erbin-ErbB2, synthesized the polypeptide fragment B2tail(PTAENPEYLGLDVPV that comprises terminal 15 amino acid residues of ErbB2).Myc-Erbin and Flag-ErbB2 plasmid quilt transfection respectively advance in the HEK293 cell, wait protein expression after one day, and cell is cleaved.The B2tail polypeptide or irrelevant polypeptide (Ctrl) mixing that comprise Myc-Erbin and proteic cell pyrolysis liquid of Flag-ErbB2 and 200 μ l50 μ M of equivalent were hatched one hour, and the antibody with anti-Myc carries out immunoprecipitation then.The immunoprecipitation product detects with Western blotting, the results are shown in Figure 5.As seen in Fig., B2tail has reduced the content with the Flag-ErbB2 under the immuno-precipitation co-precipitation, and the interaction of prompting Erbin-ErbB2 is affected, and B2tail can disturb the interaction of Erbin and ErbB2.After the cell pyrolysis liquid of expressing Erbin and ErbB2 was respectively mixed, Erbin and ErbB2 can generate complex, found expression in to detect existing of Erbin in the product under the antibody mediated immunity precipitation of the label protein of anti-ErbB.

In order to allow polypeptide enter in the cell, add by (the transactivator of transcription of the activating transcription factor in the HIV virus at the N-of B2tail polypeptide end, TAT) derive and 12 next amino acid residue sequences, form the fused polypeptide (YGRKKRRQRRR-G-PTAENPEYLGLDVPV) of a TAT-B2tail.The TAT fused polypeptide has intensive cell membrane penetration capacity, thereby can enter functionating in the cell smoothly.With 20 μ MTAT-B2tail or TAT-ctrl polypeptide treatment S KBR3 cell, add 50 μ M after 1 hour and stop the synthetic cycloheximide(CHX of albumen), and then handle different time.It is cleaved that processing finishes the back cell, detects the ErbB2(pErbB2 of intracellular ErbB2 and tyrosine phosphorylation then with Western blotting).The result is shown in Fig. 6 A-B, and after CHX stoped albumen synthetic, ErbB2 albumen can be degraded.And TAT-B2tail has accelerated the protein degradation speed of ErbB2 greatly, and the albumen half-life of prompting ErbB2 shortens greatly.More meaningfully, in the cell with the TAT-B2tail processing, because endogenic Erbin can not mutually combine with the ErbB2 of high expressed, the speed that the kinase activation of ErbB2 reduces is faster than the protein level of ErbB2, the interaction that Erbin-ErbB2 is described is also strengthened the kinase activator ability of ErbB2 except stablizing the protein level of ErbB2.TAT-B2tail suppresses protein level and the kinase activator of ErbB2, thereby suppresses the dependent breast cancer cell propagation of ErbB2 (detection of MTS method).This all is verified on dependent breast cancer cell SKBR3 of ErbB2 and BT474 cell, the propagation of the two after TAT-B2tail handled 1 day, reduce respectively 40% and 35%(Fig. 6 C).

For further checking TAT-B2tail suppress tumor growth in bulk effect, with BT474 cell inoculation (100 μ l, 3.0 * 10 in the 4th pair of mammary gland of nude mice ⁵Individual cell is suspended among the 50%Matri-gel), in the tumor of left side, inject TAT-B2tail, and injection contrasts TAT-Ctrl(YGRKKRRQRRR-G in the right side tumor of same Mus), observe the equal size tumor in the left and right sides after fortnight and generate (Fig. 7 A, A ').Rise this moment weekly and inject TAT-B2tail or its contrast TAT-Ctrl(25 μ l twice in tumor, 5mM is dissolved in PBS).After one month, the tumor of TAT-B2tail injection side is obviously dwindled, and the tumor of TAT-Ctrl injection side continues to increase (Fig. 7 B, B ').This experiment has verified further that with animal model TAT-B2tail suppresses the useful effect of tumor growth.

Embodiment 4: the check and analysis of clinical breast carcinoma specimen confirm that the expression of Erbin in cancerous tissue is significantly higher than cancer beside organism, and the expression of ErbB2 is closely related in the expression of Erbin and the pathological grading of tumor and the cancerous tissue, confirms the novel markings thing that is expressed as the clinical tumor diagnosis of Erbin

In order to seek probability and the importance of Erbin in clinical practice, utilize the antibody of the high specific Erbin of affinity purification purification, detected Erbin expression in the 171 routine human breast carcinoma specimen with the SABC method.The immunohistochemical staining result of representational Erbin in the section of clinical breast carcinoma specimen is shown in Fig. 8 A, and the immunostaining of Erbin is very obvious in cancerous tissue, and in the other normal structure of cancer, almost detect less than.The more important thing is that the immunostaining intensity of Erbin also is the protein level of Erbin in cancerous tissue, with the pathological grading close association of tumor.Tumor grade is high more, and the immunostaining intensity of Erbin is dark more, represents the expression of Erbin high more.

The SABC result of Erbin quantizes standards of grading (German semi-quantitative scoring) according to international German sxemiquantitative, under the situation of not knowing patient and pathological information, provide staining power and the score value of the area that dyes by three different staff, obtain each groupization result's scoring after multiplying each other.The statistics of appraisal result draws height distribution (Fig. 8 B of the high expressed of Erbin at cancerous tissue, the SABC score value of Erbin is 0-12, horizontal line is represented mean scores among the case figure, the last lower edge of chest is represented 25% and 75% case place score value respectively, the scope of vertical line representative data, the fragmentary case that exceeds this scope is represented with ringlet.N=171 example specimen, P＜0.001, Kruskal-Wallis detects and adds that Pearson revises), and the high correlation of Erbin expression and pathologic grading of cancer (Fig. 8 C, G1, G2, G3 represent the 1-3 level of pathologic grading of cancer.Data statistics detects through unidirectional ANOVA and adds Games-Howell ' s correction).

Further, all breast carcinoma specimen SABC of having carried out ErbB2 of also cutting into slices detects, and found that the expression of Erbin and ErbB2 is dependency, i.e. in the contiguous slices of the specimen section that ErbB2 dyeing is strong, the dyeing of Erbin is strong (Fig. 8 D) also.The quantitative statistics analysis is divided into low (low, 0-4 branch) with the SABC score value of Erbin, in (medium, 5-8) and high (high, 9-12) three groups, find that the case of ErbB2 high expressed (high group is divided value) concentrates on the high Erbin group that neutralizes, especially high Erbin group (Fig. 8 E).With Western blotting, detect Erbin and ErbB2 content in the other normal structure of homogenate cancerous tissue and cancer, find being expressed in the cancerous tissue of Erbin equally, and the protein level of Erbin and ErbB2 is dependency (Fig. 8 F) more than high in the other normal structure of cancer.These experimental results confirm that Erbin is the molecular marked compound of a novel tumor pathology detection, can its expression analyze the generation and the pathological grading of tumor.And Erbin is relevant with the expression of ErbB2 in cancerous tissue, has further affirmed clinically the feasibility of targeted therapy ErbB2 positive tumor the most with Erbin.

Conclusion:

ErbB2 is clinical tumor diagnosis, especially breast cancer diagnosis important conventional index.Taking into consideration of the expression of ErbB2 and other molecule indication is the standard of important judgement patient's survival rate and medication.The protein level and the kinase activity that reduce ErbB2 are the first-selected approach of treatment ErbB2 positive tumor.Clinical monoclonal antibody trastuzumab and the pertuzumab that has used medicine to comprise anti-ErbB, and the micromolecular compound lapatinib that suppresses ErbB2 and EGFR kinase activity.Yet these medicines usually cause that Drug resistance causes treatment to be lost efficacy.Trastuzumab and pertuzumab are incorporated into the born of the same parents outer end of ErbB2.Cancerous cell can be by born of the same parents outer end that cuts off ErbB2 or the processing of fleeing from these medicines with the born of the same parents outer end that outer glycoprotein such as the mucin-4 of born of the same parents covers ErbB2.We will overcome these defectives and bring into play more long periods of treatment effect the novel therapeutic means with at intracellular protein Erbin carcinogenic and short cancer effect to ErbB2 of invention.

The invention provides the approach of novel treatment ErbB2 positive tumor, promptly pass through to reduce the expression of Erbin, or disturb Erbin and combining of ErbB2 to suppress protein stability and the kinase activity of ErbB2, thereby suppress the effect of the carcinogenic and short cancer of ErbB2.The invention provides with the gene interference method and reduce the Erbin expression, express the Erbin-PDZ structure fragment or disturb the interaction of Erbin-ErbB2 to suppress the generation of ErbB2 dependent tumors and the concrete means of development with the TAT-B2tail polypeptide thereby cross.But protection scope of the present invention is not limited in this.The adjusting of Erbin protein level, and the interaction of Erbin-ErbB2 can also be by alternate manner, such as micromolecular compound, antibody, polypeptide derivative is realized etc. multiple means.All at the expression that reduces Erbin or disturb the interactional technological means of Erbin-ErbB2 all in protection scope of the present invention.

The ErbB2 positive tumor also is not limited only to breast carcinoma, ovarian cancer, and gastric cancer, uterus carcinoma is all found the male tissue of ErbB2 in the cell carcinomas such as head and neck cancer.Also can be applied in the treatment except that other ErbB2 positive tumors of breast carcinoma at the treatment means of regulating the ErbB2 protein level.Consider that existing treatment means at the ErbB2 positive tumor often causes Drug resistance, therefore propose the mode of the compound use of multiple treatment means.At Erbin or the interactional treatment means of Erbin-ErbB2 can with at the medicine or the means of regulating ErbB2 protein level or kinase activity, trastuzumab for example, pertuzumab, or lapatinib etc., in conjunction with or exchange use, to reach best anticancer effect.

ErbB2 also plays a very important role in other system of human body, and signal transduction and function that Erbin also is found in the tissue that is present in other system and regulates ErbB2, for example nervous system.Therefore, the present invention also can be applicable to diagnosis and the treatment to the disease of other system.All at diagnosing with the expression that detects Erbin, and with the expression that reduces Erbin or disturb the interactional technological means of Erbin-ErbB2 to treat, all in protection scope of the present invention.

Claims

1.Erbin the application of inhibitor in the preparation antitumor drug.

2. application as claimed in claim 1 is characterized in that described inhibitor is expression that reduces Erbin or the interactional chemical compound that disturbs Erbin-ErbB2.

3. application as claimed in claim 2 is characterized in that described inhibitor is the polypeptide shown in SEQ ID No.1 or the SEQ ID No.3.

4. application as claimed in claim 2 is characterized in that described inhibitor is the shRNA of Erbin.

A PDZ structure fragment that comprises Erbin, play the polypeptide of dominant negative effect, its aminoacid sequence is shown in SEQ ID No.1.

6. the gene order of coding claim 5 described polypeptide, its nucleotide sequence is shown in SEQ ID No.2.

7. one kind is disturbed the interactional polypeptide of Erbin-ErbB2, and its aminoacid sequence is shown in SEQ ID No.3.

8. the gene order of coding claim 7 described polypeptide, its nucleotide sequence is shown in SEQ ID No.4.