CN103212077B - Erbin inhibitor is preparing the application in antitumor drug - Google Patents

Abstract

The invention provides Erbin inhibitor and prepare the application in antitumor drug.Described inhibitor specifically can be the interactional compound of expression or the interference Erbin-ErbB2 reducing Erbin.The interaction of Erbin and ErbB2 to the short cancer of ErbB2 and carcinogenesis very crucial, the PDZ structure fragment of process LAN Erbin, or the excessive C-terminal peptide fragment giving ErbB2 in cell, the growth of the dependent cancer cell multiplication of ErbB2 or tumor can be suppressed.The invention provides the approach of novel treatment ErbB2 positive tumor, namely by reducing the expression of Erbin, or the combination of interference Erbin and ErbB2 suppresses protein stability and the kinase activity of ErbB2, thus suppresses the effect of the carcinogenic and short cancer of ErbB2.

Description

Erbin inhibitor is preparing the application in antitumor drug

(1) technical field

The present invention relates to Erbin inhibitor and prepare the application in antitumor drug.

(2) background technology

The receptor of endothelial cell growth factor (ECGF) (Epidermal growth factor receptor) family, comprises EGFR(also known as HER1, ErbB1), ErbB2(HER2, neu), ErbB3(HER3) and ErbB4(HER4) all participate in the growth and development of tumor.Wherein ErbB2 especially causes concern, because surpass overexpression ErbB2 being detected in the breast cancer tissue of 25%, and the breast cancer patients of ErbB2 process LAN often has high recurrent, and easily shifts and prognosis mala.Except breast carcinoma, ErbB2 process LAN also involves ovarian cancer, gastric cancer and uterus carcinoma.(an oncogene carcinogen-inducedneuroblastoma, neu that the proto-oncogene that ErbB2 starts to cause conversion as one exactly is most found; Or an oncogene of avian erythoblastosis virus, ErbB2).As a member of endothelial growth factor receptor family, the not direct and any known ligand of ErbB2 combines, but is formed heterodimer with other by the endothelial cell growth factor (ECGF) family member of ligand activation and carry out transduction signal.Although be orphan receptor, ErbB2 is that other is inclined to by the ErbB receptor of ligand activation forms dimeric member most, and forms dimer with ErbB2 and significantly can promote the degree that other ErbB receptor is activated.The more important thing is, in breast cancer cell the ErbB2 of high expressed can not rely on part stimulation and directly form different aggressiveness or with aggressiveness thus activate downstream passages.This mechanism has increased the weight of the effect of the carcinogenic and short cancer of ErbB2.The downstream signaling pathway that ErbB2 can activate after activating comprises Ras, Src, PI-3K/Akt, 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 the erosion to hypothallus.

The ErbB2 of process LAN has the confirmation making the ability of cell cancerization obtain various in vitro and experiments in vivo.ErbB2 process LAN can make l cell NIH3T3 and NR6 transform, and normal human mammary epithelial cell MCF-10A can be made to form the solid polyadenous bubble structure of similar ductal carcinoma in situ (DCIS).In mammary gland of mouse epithelium, specificity process LAN ErbB2 can cause mice generate breast tumor and be transferred to pulmonary.All these results of study all show to suppress the protein level of ErbB2 to be the important channel for the treatment of breast carcinoma.The monoclonal antibody trastuzumab for ErbB2 developed now and pertuzumab, and the inhibitor lapatinib of anti-ErbB kinase activity has been applied to clinically.But these medicines often cause Drug resistance, the patient for the treatment of is also easy to recurrence.Thus, how to find the new therapy approach for ErbB2 to be the direction that scientific researcher is made great efforts always.

ErbB receptor can be degraded by endocytosis after by ligand stimulation.But the endocytosis comprising the receptor dimer of ErbB2 is but suppressed; Not only can stop the endocytosis inactivation of other ErbB receptor with ErbB2 dimerization, can also promote that they are transferred back to cell membrane.Suppress ErbB2 and be molecule in the born of the same parents that are combined with ErbB2 with the mechanism of the receptor endocytosis of ErbB2 dimerization.The C-end of ErbB2 is extremely important to its stability, remove C-end only 3 amino acid residues significantly will reduce the protein stability of ErbB2 and increase its degraded.These C-terminal amino acid residues form one in conjunction with PSD95-Dlg1-zo-1(PDZ) region of structure, imply that an ErbB2 associated proteins containing PDZ structure fragment is crucial especially to its stability.The present invention, using the C-terminal fragment of ErbB2 as bait (Bait), filters out the associated proteins Erbin(ErbB2-Interacting protein of ErbB2 in yeast two-hybrid library).Erbin is a LAP albumen, and N-end is containing 16 leucine enrichment repetitive sequences (Leucine-rich repeats, LRR), and its C-end is a PDZ structure fragment.Erbin is combined by PDZ structural specificity ground and ErbB2, and not in conjunction with other ErbB family receptors.Present inventor finds that the protein stability of Erbin to ErbB2 is extremely important, and Erbin can also promote the ErbB2 dimerization of process LAN thus activation HER2 activity and the carcinogenic and short cancer signal path in downstream.The present invention with cell and animal model prove Erbin to the carcinogenic of process LAN ErbB2 and tumor promotion very crucial; The effect suppressing the carcinogenic and short cancer of ErbB2 is all shown with the combination of multiple means specificity interference Erbin and ErbB2.

(3) summary of the invention

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

By the enlightenment that ErbB2 occurs in breast carcinoma and plays a crucial role in development, inventors have investigated its associated proteins Erbin to the impact of these processes.Erbin is specific expressed in breast ductal epithelial cells in result display, plays necessary effect to the dependent Cells Proliferation of Human Breast Cancer of ErbB2.Suddenly change Erbin in mammary gland of mouse epithelium, makes it can not interact with ErbB2, and the dependent breast cancer animal model MMTV-neu mice of ErbB2 can be suppressed not generate tumor.And the tumor of the breast cancer animal model MMTV-PyVT mice of ErbB2 dependent/non-dependent generates unaffected.The interaction of Erbin and ErbB2 to the short cancer of ErbB2 and carcinogenesis very crucial, the PDZ structure fragment of process LAN Erbin, or the excessive C-terminal peptide fragment giving ErbB2 in cell, the growth of the dependent cancer cell multiplication of ErbB2 or tumor can be suppressed.In tumor cell, strike the expression of low Erbin with specificity shRNA in the mode of Gene interfere, also have remarkable inhibitory action to the propagation of the dependent cancerous cell of ErbB2 and tumor growth.These results display Erbin generates the dependent tumor of ErbB2 and growth has crucial regulating action, also provides novel to press down cancer approach and medicament forms.In the research and analysis display cancerous tissue of clinical breast cancer specimen, the expression ratio cancer beside organism of Erbin significantly raises, and the pathological grading of the expression of Erbin and tumor and the expression of ErbB2 closely bound up, the expression providing to detect Erbin carries out the feasibility of clinical tumor diagnosis.

The technical solution used in the present invention is:

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

Concrete, described inhibitor is reduce the expression of Erbin or the interactional compound of interference Erbin-ErbB2.

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

SEQ ID No.1 sequence is as follows:

EIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLL

QPGDKIIQANGYSFINIEHGQAVSLLKTFQNTVELIIVREV

SEQ ID No.3 sequence is as follows:

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

Preferably, described inhibitor is the shRNA of Erbin.

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

Encode the gene order of this polypeptide, its nucleotide sequence is as 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 as shown in SEQ ID No.3.

Encode the gene order of this polypeptide, its nucleotide sequence is as shown in SEQ ID No.4:

CCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTG。

Due to the particularity of aminoacid sequence; the fragment of any polypeptide containing aminoacid sequence shown in SEQ ID NO.1 and 3 or its variant; 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 sequences homology are more than 90% and have identical function, all belong to the row of scope.Concrete, described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, the conservative for variant changes, and the aminoacid replaced has the structure similar to original acid or chemical property, and as replaced isoleucine with leucine, variant also can have non-conservation and change, as replaced glycine with tryptophan.

Due to the particularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ NO.2 and 4, as long as it has more than 90% homology with these polynucleotide and has identical function, all belongs to the row of scope.The variant of described polynucleotide refers to a kind of polynucleotide sequence having one or more nucleotide and change.The variant of these polynucleotide can make raw allelic variant or the variant of non-life, comprises and replaces variant, Deletion variants and insertion variant.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of a multiple nucleotide, disappearance or insertion, but can not from the amino acid whose function changing in fact its coding.

Beneficial effect of the present invention is mainly reflected in: Erbin inhibitor of the present invention is preparing the application in antitumor drug, for tumor diagnosis and therapy provides new approach.

(4) accompanying drawing explanation

Fig. 1 is the impact that Erbin breeds ErbB2 independent mammary tumor cells; A is ability and the degree that the shErbin in different breast cancer cell line detected with Western blotting reduces Erbin protein expression; B ~ D is with the proliferative conditions of the different stable cell lines of MTS method detection; N=3, * P<0.05, * * P<0.01;

Fig. 2 is the impact that at body generate and grow of Erbin on breast cancer tumour; D0 is injection day, and D10 is latter 10th day of injection;

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

Fig. 4 is the impact of Erbin-PDZ structural region on ErbB2 dependent tumors; A be Kaplan-Meier without tumor scattergram, Log-rank(Mantel-Cox) detect, P<0.0002; B is MMTV-neu; Erbin ^{c/ C}and MMTV-neu; Erbin ^{+ /+}mice exposes mammary gland through dissecting; C is fixing after mammary gland of mouse or lung are separated section, through hematoxylin-Yin Hong (H/E) stained photographs; D carries out overall brazilwood extract dyeing photo after the mammary gland of mouse of the non-bearing tumor be separated is fixed; E is that Kaplan-Meier is without tumor scattergram; F is the growth that the excision of PDZ structural region does not affect MMTV-PyVT mouse mammary tumor, n=8, P>0.05; G is at MMTV-PyVT; Erbin ^{+ /+}and MMTV-PyVT; Erbin ^{c/ C}the histologic characteristics of the mastadenoma generated in mice.

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

Fig. 6 is the ErbB2 protein stability that TAT-B2tail polypeptide fragment reduces breast cancer cell, kinase activity and cell proliferation; A is that the cell after process cleavedly carries out Western blot testing result; B is the quantitative statistics analysis of experimental result in A; C is that polypeptide treatment S KBR3 or BT474 cell are on the impact of the rate of increase;

Fig. 7 is the representative experimental results of TAT-B2tail polypeptide fragment in the growth of body Tumor suppression;

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

(5) detailed description of the invention

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

Embodiment 1: confirm that Erbin is of crucial importance to ErbB2 dependent breast carcinoma growth and development with cell model and animal model, provide and strike the expression of low Erbin to treat approach and the means of ErbB2 dependent tumors with specificity shRNA

In order to study the impact of Erbin on Cells Proliferation of Human Breast Cancer, have selected the dependent breast cancer cell line BT474(ATCC of ErbB2) and SKBR3(ATCC), and do not rely on the breast cancer cell line ZR751(Public Health England with ErbB2).When these cells are cultivated to dishful, the shRNA(shErbin with the specific recognition Erbin of slow virus packaging) (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) to invade and harass cell respectively (be 8x10 by titre ⁸the virion suspension 1 μ l of IU/ml adds in the cell dish containing the 100mm of 10ml culture fluid, changes fresh medium after 24 hours).Carrying Green Fluorescent Protein (GFP) on the package carrier of shErbin or shCtrl, thus with trypsinization and suspension cell, with selected by flow cytometry apoptosis go out by shRNA infect into cell and go down to posterity to set up stable cell line.

Western blot result shows, and contrast with shCtrl, the level of Erbin albumen in three cell lines can be reduced by 70 ~ 90%(Figure 1A by shErbin respectively).

The stable cell line that shErbin or shCtrl integrates grows at identical conditions with identical inoculum density, and respectively at 1, the MTS accepting cell proliferation after 2,3 days detects, and the results are shown in Figure 1B ~ D.Very have enlightening significance, in cell, Erbin is struck low BT474(Figure 1B by shRNA) and SKBR3(Fig. 1 C) propagation significantly slow down, and ZR751(Fig. 1 D of ErbB2 dependent/non-dependent) cell proliferation by the impact of shErbin.This cell model proves that Erbin promotes the propagation of the dependent breast cancer cell of ErbB2 specifically, and the protein level reducing Erbin can suppress the dependent Cells Proliferation of Human Breast Cancer of ErbB2.

In order to verify further Erbin to ErbB2 dependent tumors in body effect, the shErbin(of the integration right arrow by same quantity) or shCtrl(left arrow) BT474 cell (100 μ l, 3.0 × 10 ⁵individual cell suspension is in 50%Matri-gel) be injected into right side or the 4th pair, the left side mammary gland inside (D0 of nude mice respectively, injection day), construct the animal model that development occurs a people source breast carcinoma, within 10 days, observe into tumor size (D10) later, the results are shown in Figure 2.Left and right sides the 4th pair of mammary gland of same nude mice is being vaccinated with breast cancer cell, and meaningfully, the tumor of 10 days rear left side joint kind shErbin-BT474 significantly reduces, and the tumor that the shCtrl-BT474 on right side is formed enlarges markedly.This experiment confirmed under body environment, the generation development of expression to the tumor that ErbB2 relies on reducing Erbin in the mode of Gene interfere has inhibitory action.

Embodiment 2: confirm that the PDZ structure fragment of Erbin generates the dependent breast carcinoma of ErbB2 with cell model and animal model extremely crucial.The PDZ fragment of process LAN Erbin can be disturbed the interaction of Erbin-ErbB2 thus reduce protein level and its kinase activation of ErbB2, thus provides PDZ structure fragment for Erbin to treat therapy approach and the means of ErbB2 dependent tumors

The early stage work of inventor has been disclosed Erbin and ErbB2 and has been combined by PDZ structural region.The PDZ binding sequence of the PDZ structure fragment of the C-end of Erbin and the C-end of ErbB2 combines them and interacts very crucial.In order to study Erbin and ErbB2 combination whether to Erbin regulate the protein stability of ErbB2 and function very important, construct the plasmid of the PDZ structure fragment of only expressing Erbin

(ATGGAGATTCGAGTGAGGGTTGAAAAGGATCCAGAACTTGGATTTAGCATATCAGG TGGTGTCGGGGGTAGAGGAAACCCATTCAGACCTGATGATGATGGTATATTTGTAA CAAGGGTACAACCTGAAGGACCAGCATCAAAATTACTGCAGCCAGGTGATAAAATT ATTCAGGCTAATGGCTACAGTTTTATAAATATTGAACATGGACAAGCAGTGTCCTT GCTAAAAACTTTCCAGAATACAGTTGAACTCATCATTGTACGAGAAGTTGAACAAA AACTTATTTCTGAAGAAGATCTGTAG is built into pRK5 carrier with BamH1 and EcoR1 restriction enzyme site), whether the PDZ structure fragment then studying process LAN Erbin has the combination spending interference Erbin and ErbB2 of dominant negative (dominant-negative), thus affects protein level and the function of ErbB2.For this purpose, the Erbin-PDZ plasmid of varying number is entered Human fetal glomerular mesangial cell HEK293(ATCC from quantitative Erbin with ErbB2 transfection) in (in different rotaring redyeing systems, the plasmid total amount of transfection is supplied identical by empty vectors), then detect the protein level of ErbB2 and phosphorylation level (pErbB2) with Western blotting.As shown in Figure 3, the expression inhibiting of the Erbin-PDZ protein level of ErbB2, and inhibit the phosphorylation level of ErbB2 with larger degree, shows the impact larger on its kinase activity to result.More ironically, the suppression of Erbin-PDZ to ErbB2 protein level and phosphorylation level is dose dependent, and Erbin-PDZ plasmid transfection must be more, and ErbB2 albumen and phosphorylation decline more remarkable.With cell model, these experiments confirm that the expression of excessive Erbin-PDZ fragment can affect level and the kinase activation of ErbB2, provide approach and the means of being disturbed ErbB2 function by interference Erbin-PDZ.

In order to further with animal model checking, construct the mutant mice system erbin of Erbin ^{Δ C/ Δ C}.In this sudden change Mus, the Erbin of wild type by the mutain of the Erbin of a C-tip cut-off substitute (only surplus N-end 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 type Mus source Erbin ^1-693:

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

In order to verify this hypothesis, by erbin ^{Δ C/ Δ C}mus and the dependent breast cancer mouse model MMTV-neu of ErbB2 are hybridized, thus obtain holding MMTV-neu mice progeny that is that Erbin excises and wild type Erbin, called after MMTV-neu respectively containing C-; Erbin ^{Δ C/ Δ C}and MMTV-neu; Erbin ^{+ /+}mice.

Erbinerbin ^{Δ C/ Δ C}after mice and the white mouse backcross of wild type FVB 5 times, with the MMTV-neu mouse hybrid of FVB background, the female descendant MMTV-neu of generation; Erbin ^{Δ C/ Δ C}and MMTV-neu (n=20); Erbin ^{+ /+}(n=22) mice is raised under the condition of not copulation, and carry out in each week subcutaneous touch detect mastadenoma generation whether, the results are shown in Figure 4A ~ D.Note until about 20 monthly age above MMTV-neu; Erbin ^{Δ C/ Δ C}all do not detect in mice that tumor generates, and MMTV-neu; Erbin ^{+ /+}mice 100% has tumor to generate.Fig. 4 B is the MMTV-neu at representational a pair 12 monthly ages; Erbin ^{Δ C/ Δ C}and MMTV-neu; Erbin ^{+ /+}the dissection photo of sister mice.Display does not have MMTV-neu; Erbin ^{Δ C/ Δ C}the mammary gland of mice can detect visual tumors.Fixing section after mammary gland of mouse or lung are separated, through hematoxylin-Yin Hong (H/E) dyeing, does not detect MMTV-neu; Erbin ^{Δ C/ Δ C}tumor tissues is had in mammary gland of mouse, 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 mouse lung.Carry out overall brazilwood extract dyeing after the mammary gland of mouse of the non-bearing tumor be separated is fixing, Fig. 4 D is the local pictures after representational hematoxylin bulk dyeing, visible MMTV-neu; Erbin ^{+ /+}the mammary gland of the non-bearing tumor of mice has the phenomenon of obvious Epithelial hyperplasia, and MMTV-neu; Erbin ^{Δ C/ Δ C}the conduit form of mammary gland of mouse is normal.

Female mice MMTV-PyVT; Erbin ^{+ /+}and MMTV-PyVT (n=23); Erbin ^{Δ C/ Δ C}(n=15) raise under the condition of not copulation, subcutaneous touch per week generates with or without breast tumor to detect, and the results are shown in Figure 4E ~ G.The excision of result display PDZ structural region does not affect the growth of MMTV-PyVT mouse mammary tumor.MMTV-PyVT mouse mammary tumor is laid one's hand on and rear mistake two months, and tumor is stripped the growth rate of weighing with comparison of tumor, at MMTV-PyVT; Erbin ^{+ /+}and MMTV-PyVT; Erbin ^{Δ C/ Δ C}the mastadenoma generated in mice has identical histologic characteristics.

MMTV-neu breast cancer mouse model is specific model carcinogenic with MMTV promoter process LAN rat ErbB2 gene neu in galactophore epithelial cell.The excessive ErbB2 that MMTV-neu expresses stimulates cyclomastopathy and final canceration.About this carcinogenic mouse model breast carcinoma early start betides the 4-6 month.The breast carcinoma of major part Mus occurs more late, but the MMTV-neu mice of final 100% can produce breast carcinoma.And breast carcinoma occurs in latter two moon, the mice of 80% has Lung metastases.But the Erbin mutain of expression C-tip cut-off instead of the mice of wild type Erbin albumen all do not detect breast tumor when 20 monthly ages, also do not have the phenomenon (Fig. 4 A-D) of cyclomastopathy and Lung metastases.And ironically, by erbin ^{Δ C/ Δ C}in offspring after hybridizing with another breast cancer mouse model MMTV-PyVT, the Erbin mutain of expressing C-tip cut-off can not stop generation and the growth (Fig. 4 E-G) of breast tumor.From the experimental result that animal model comes, these support that Erbin-PDZ structural region, to ErbB2 dependent tumors, the importance of development occurs strongly, support to disturb Erbin-PDZ to treat novel approach and the means of tumor.

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

In order to develop be easy to apply for the interactional treatment means of Erbin-ErbB2, synthesized the polypeptide fragment B2tail(PTAENPEYLGLDVPV comprising ErbB2 end 15 amino acid residues).Myc-Erbin and Flag-ErbB2 plasmid is entered in HEK293 cell by transfection respectively, and wait protein expression after one day, cell is cleaved.The B2tail polypeptide of the cell pyrolysis liquid comprising Myc-Erbin and Flag-ErbB2 albumen of equivalent and 200 μ l50 μMs or irrelevant polypeptide (Ctrl) blending incubation one hour, then carry out immunoprecipitation with the antibody of anti-Myc.Immunoprecipitation product detects with Western blotting, the results are shown in Figure 5.As seen in Fig., B2tail decreases with the content of the Flag-ErbB2 under 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 mixing expressing Erbin and ErbB2 respectively, Erbin and ErbB2 can generate complex, finds expression in and the existence of Erbin can be detected in the product under the antibody mediated immunity of the label protein of anti-ErbB precipitation.

Enter in cell to allow polypeptide, add by activating transcription factor (the transactivator of transcription in HIV virus at the N-end of B2tail polypeptide, TAT) 12 derivative and next amino acid residue sequences, form the fused polypeptide (YGRKKRRQRRR-G-PTAENPEYLGLDVPV) of a TAT-B2tail.TAT fused polypeptide has strong cell membrane penetration capacity, thus can enter functionating in cell smoothly.With 20 μMs of TAT-B2tail or TAT-ctrl polypeptide treatment S KBR3 cells, after 1 hour, add the cycloheximide(CHX that 50 μMs stop albumen synthesis), and then process different time.It is cleaved that process terminates rear cell, then detects the ErbB2(pErbB2 of intracellular ErbB2 and tyrosine phosphorylation with Western blotting).Result as shown in fig. 6 a-b, after CHX stops albumen synthesis, can degrade by ErbB2 albumen.And TAT-B2tail accelerates the protein degradation speed of ErbB2 greatly, the protein half-life of prompting ErbB2 shortens greatly.More meaningfully, with in the cell of TAT-B2tail process, because endogenic Erbin can not be combined with each other with the ErbB2 of high expressed, the speed that the kinase activation of ErbB2 reduces is faster than the protein level of ErbB2, the protein level of the interaction of Erbin-ErbB2 except stable ErbB2 is described, also strengthens the kinase activator ability of ErbB2.TAT-B2tail suppresses protein level and the kinase activator of ErbB2, thus suppresses the dependent Cells Proliferation of Human Breast Cancer of ErbB2 (detection of MTS method).This is all verified on the dependent breast cancer cell SKBR3 of ErbB2 and BT474 cell, the two propagation TAT-B2tail process reduce respectively after 1 day 40% and 35%(Fig. 6 C).

In order to verify that TAT-B2tail Tumor suppression grows further in bulk effect, BT474 cell is inoculated in (100 μ l, 3.0 × 10 in the 4th pair of mammary gland of nude mice ⁵individual cell suspension is in 50%Matri-gel), TAT-B2tail is injected in the tumor of left side, and injection contrasts TAT-Ctrl(YGRKKRRQRRR-G in the right side tumor of same Mus), the tumor observing the equal size in the left and right sides after fortnight generates (Fig. 7 A, A ').Now rise and contrast TAT-Ctrl(25 μ l toward intra-tumoral injection TAT-B2tail or its twice weekly, 5mM, is dissolved in PBS).After one month, the tumor of TAT-B2tail injection side obviously reduces, and the lasting increase of the tumor of TAT-Ctrl injection side (Fig. 7 B, B ').This experiment demonstrates the useful effect of TAT-B2tail Tumor suppression growth further with animal model.

Embodiment 4: the detection of clinical breast cancer specimen is analyzed and confirmed 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 pathological grading of the expression of Erbin and tumor and cancerous tissue, confirm the novel markings thing being expressed as clinical tumor diagnosis of Erbin

In order to seek the probability of Erbin in clinical practice and importance, utilizing the antibody of the high specific Erbin of affinity purification purification, have detected the Erbin expression in 171 routine human breast carcinoma specimen with Immunohistochemical Method.As shown in Figure 8 A, the immunostaining of Erbin clearly, and almost to can't detect the immunohistochemical staining result of representational Erbin in the section of clinical breast cancer specimen in cancerous tissue in Carcinoma side normal tissue.The more important thing is, the immunostaining intensity of Erbin, is also the protein level of Erbin in cancerous tissue, with the pathological grading close association of tumor.Tumor grade is higher, and the immunostaining intensity of Erbin is darker, and the expression representing Erbin is higher.

The ImmunohistochemistryResults Results of Erbin is according to international German sxemiquantitative Quantitative marking standard (German semi-quantitative scoring), being provided the score value of staining power and stained area by three different staff when not knowing patient and pathological information, after being multiplied, obtaining the scoring of each group of change result.The statistics of appraisal result show that the high expressed of Erbin to distribute (Fig. 8 B at the height of cancerous tissue, the SABC score value of Erbin is 0-12, in case figure, horizontal line represents mean scores, the edge up and down of chest represents the case place score value of 25% and 75% respectively, the scope of vertical line representative data, the fragmentary case exceeding this scope represents with ringlet.N=171 example specimen, P<0.001, Kruskal-Wallis detect add 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 adds Games-Howell ' s through unidirectional ANOVA detection and revises).

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 be dependency, and in the contiguous slices that namely ErbB2 dyes specimen section strong, the dyeing of Erbin also comparatively by force (Fig. 8 D).The SABC score value of Erbin is divided into low (low, 0-4 divide) by quantitative statistics analysis, in (medium, 5-8) and high (high, 9-12) three groups, find that the case of ErbB2 high expressed (high groupization score value) concentrates on and neutralize high Erbin group, especially high Erbin group (Fig. 8 E).With Western blotting, detect Erbin and the ErbB2 content in homogenate cancerous tissue and Carcinoma side normal tissue, find the expression of Erbin equally more than high in Carcinoma side normal tissue in cancerous tissue, and the protein level of Erbin and ErbB2 is dependency (Fig. 8 F).These experimental results confirm that Erbin is the molecular marked compound that a novel cancer pathology detects, and can analyze generation and the pathological grading of tumor by its expression.And the expression of Erbin and ErbB2 in cancerous tissue is correlated with, further affirm clinically with the feasibility of Erbin targeted therapy ErbB2 positive tumor the most.

Conclusion:

ErbB2 is clinical tumor diagnosis, an especially important conventional index of breast cancer diagnosis.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 for the treatment of ErbB2 positive tumor.Clinical used medicine to comprise anti-ErbB monoclonal antibody trastuzumab and pertuzumab, and suppress the micromolecular compound lapatinib of ErbB2 and EGFR kinase activity.But these medicines usually cause Drug resistance to cause failure in treatment.Trastuzumab and pertuzumab is incorporated into the born of the same parents outer end of ErbB2.Cancerous cell can by cutting off the born of the same parents outer end of ErbB2 or fleeing from the process of these medicines with the born of the same parents outer end of the outer glycoprotein of born of the same parents such as mucin-4 covering ErbB2.The therapeutic effect playing longer-term with the novel therapeutic means of and tumor promotion carcinogenic to ErbB2 for intracellular protein Erbin by overcoming these defects of our invention.

The invention provides the approach of novel treatment ErbB2 positive tumor, namely by reducing the expression of Erbin, or the combination of interference Erbin and ErbB2 suppresses protein stability and the kinase activity of ErbB2, thus suppresses the effect of the carcinogenic and short cancer of ErbB2.The invention provides and reduce Erbin with Gene interfere method and express, process LAN Erbin-PDZ structure fragment or disturb the interaction of Erbin-ErbB2 with TAT-B2tail polypeptide thus suppress the generation of ErbB2 dependent tumors and the concrete means of development.But protection scope of the present invention is not limited in this.The adjustment of Erbin protein level, and the interaction of Erbin-ErbB2 can also by alternate manner, such as micromolecular compound, antibody, polypeptide derivative, realizes etc. multiple means.All for reducing the expression of Erbin or disturbing the interactional technological means of Erbin-ErbB2 all in protection scope of the present invention.

ErbB2 positive tumor is also not limited only to breast carcinoma, ovarian cancer, gastric cancer, and uterus carcinoma all finds the tissue of the ErbB2 positive in the cell carcinomas such as head and neck cancer.Also can be applied in the treatment except other ErbB2 positive tumors of breast carcinoma for regulating the treatment means of ErbB2 protein level.Consider that the existing treatment means for ErbB2 positive tumor often causes Drug resistance, therefore propose the mode of multiple treatment means compound use.For the interactional treatment means of Erbin or Erbin-ErbB2 can with for the medicine or the means that regulate ErbB2 protein level or kinase activity, such as trastuzumab, 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 Erbin to be also found to be present in the tissue of other system and to regulate signal transduction and the function of ErbB2, such as nervous system.Therefore, the present invention also can be applicable to the Diagnosis and Treat of the disease to other system.Allly to diagnose for the expression detecting Erbin, and treat, all in protection scope of the present invention with the expression reducing Erbin or the interactional technological means of interference Erbin-ErbB2.

Claims (3)

1.Erbin inhibitor is preparing the application in antitumor drug, it is characterized in that the shRNA that described inhibitor is the polypeptide shown in SEQ ID No.3 or Erbin, and described tumor is the tumor that ErbB2 relies on.

2. disturb the interactional polypeptide of Erbin-ErbB2, its aminoacid sequence is as shown in SEQ ID No.3.

3. the gene order of polypeptide described in coding claim 2, its nucleotide sequence is as shown in SEQ ID No.4.