The Regulation Of Epidermal Growth Factor Like Domain Protein-6(EGFL6)on Malignant Biological Behavior Of Breast Cancer Cells And The Targeting Intervention Of EGFL6 Antibody

Breast cancer is a malignant tumor that produced by the carcinogenesis of the mammary epithelial cells.Breast cancer is also one of the most common malignancy tumors and the main cause diseases which threaten the women's health in the world,with the changing of the social,environmental and many other factors,the incidence of the breast cancer is increasing rapidly.Although targeted therapy based on new tumor targets played an important role in the treatment of the breast cancer,due to the characteristics of breast cancer distal recurrence and migration,the prognosis of some breast cancer patients still remains very poor.Therefore,finding the novel target and prognostic markers of the treatment of breast cancer,exploring the regulatory mechanism of key molecules on the pathogenesis of breast cancer and developing specific targeted therapy preparations are still the key and the difficult point in the field of breast cancer research in recent years.The epidermal growth factor(EGF)is consisting by a single polypeptide chain,which has 53 amino acid residues.It is characterized by three disulfide bonds composed of cysteine interlaced in space.These disulfide bonds are important structure to retain the biological activity of the EGF.EGFL6 belong to the EGF like superfamily.In 1999,Yeung first found and identified the EGFL6 mapped to the human Xp22 chromosome.EGFL6 is also named MAEG,because it has both the EGF domain and the MAM domain.EGF family members involved in regulating various cellular and physiological processes,such as cell cycle,cell adhesion,cell proliferation,cell migration and the neural development.As reported that in oral squamous cell carcinoma,the high level of plasma EGFL6 is associated with the clinic pathological characteristics.EGFL6 is highly expressed in some tumor cells,such as in melanoma,lung cancer and other tumor tissues,especially in the endothelial cells of ovarian cancer.A recent report from Cancer Research revealed the involvements and mechanism of EGFL6 in helping ovarian cancer cells spread and multiplies.The blocking antibody could decrease the growth of the cancer cells and stop the metastasis.This finding has potential meaning for the treatment of the high-risk ovarian cancer in women.Several studies have shown that the activation of EMT can promote EGF induced cancer cell migration and invasion.It is still necessary to further explore whether EGFL6 can mediate the malignant biological behavior of breast cancer cells by inducing EMT.Epithelial-mesenchymal Transition(EMT),in which the epithelial cells are transformed to the mesenchymal cells,which is an important processes that triggers the cancer occur and the migration of the cancer cells.The carcinogenesis of epithelial cells occupies more than 90%of all the incidence of the malignant tumor,and the metastasis is the main cause of the death on the cancer patients.Common metastases include metastases to the liver,lung and bone.Metastasis of tumor cells seriously affects the function of the organs,and further threatens the life of the patients.Meanwhile,the unlimited prolifration of the cancer cells,destroying the normal cell tissues and inhibiting the apoptosis have also brought many difficulties to the treatment of cancer.During the initiation of invasion and metastasis of tumor cells in EMT,the tumor cells obtained the self-renewal characteristics similar to the tumor stem cells.Sternness cells also play an important role in the formation and development of tumors.A group of tumor cell subgroups existed as CD44high/CD24low antigen phenotype in human breast tumors.Overexpression of Snail,Twist,or TGF-beta will increase the production of tumor spheres and the production of stem cell markers,CD44high/CD24low.This evidence suggests that EMT may have a direct link with the increasing of tumor stem cells,which may be a prerequisite for cancer cell metastasis.A better understanding of the molecular mechanism of the pathogenesis and progression of breast cancer and the identification of the key molecules in the process of breast cancer growth and metastasis are particularly important for developing antibodies against metastatic breast cancer patients.Antibody therapy has been developed to be an important option for the treatment of cancer.The main mechanism of antibody killing tumor cells is to block the proliferation of the tumor cells,activate the signal transduction of the apoptotic pathway,and activate the immune system to mediate the killing of the cancer cells.Recent years,with further understanding of the pathogenesis of cancer,targeting use of antibodies for treatment has achieved significant clinical efficacy.The antibody has high target specificity,therefore studying the molecular biological mechanism of cancer pathogenesis,discovering the key molecules,which induce cancer,and developing the specific molecular therapeutic antibodies will open up a new way for the immunotherapy of breast cancer.Because the highly expression of EGFL6 in tumor cells may be related to the occurrence and the development of some cancers,EGFL6 is likely to become a new target for cancer diagnosis and treatment.Therefore,the in-depth study of the relationship between EGFL6 and tumors and the discovery of relevant molecular mechanisms is greatly significance for the treatment of certain tumors.Meanwhile,a new approach for the treatment of breast cancer by monoclonal antibodies targeting EGFL6 is developed.This study focused on the growth and metastasis of breast carcinoma,which used variety features of breast cancer cell model and tumor model system after EGFL6 gene silencing or overexpression.Multiple levels from in vitro and in vivo study were used to reveal the regulation of the breast cancer cell growth and molecular mechanism of metastasis.In addition,the specific anti human EGFL6 monoclonal antibody was developed,and used to intervene the migration,invasion and proliferation of breast cancer cells in vitro.Then the mouse tumor-bearing model was used to investigate the effect of these two antibodies on tumor growth,to further dev elop the clinical application value of EGFL6 target intervention for breast tumor.Part I Regulatory effect of epidermal growth factor like domain protein-6 on malignant biological behavior of breast cancer cellsObjective:To detect the expression of membrane or soluble EGFL6 in breast cancer cell,increase or silent expression of EGFL6 in breast cancer cells was used to analysis of the effect and mechanism of EGFL6 on the migration and invasion of breast cancer cells.Methods:(1)qPCR and Western blot were performed to detect the expression of EGFL6 in six breast cancer cells at mRNA and protein level.ELISA assay was used to detect the protein expression level from the serum of the breast cancer cell lines.(2)Lentiviral system was used in the establishment of the up-regulating EGFL6 plasmid pLVX-Puro-EGFL6 and the control plasmid pLVX-Puro-Control.The stable EGFL6 overexpressed clone MCF-7/EGFL6 and EGFL6 down-regulated clones T47D/shRNA and MDA-MB-231/shRNA were produced.(3)Cell migration and invasion were detected by scratches wound healing and transwell assays.The effects of the recombinant human protein EGFL6 and the conditioned medium of overexpressing EGFL6 on the migration of breast cancer cells were verified.(4)qPCR,Western blot and Immunofluorescence staining were used to detect the epithelial mesenchymal transition(EMT)related markers E-cadherin,N-cadherin,Vimentin and fibronectionin gene and protein level with EGFL6 up-regulated or down regulated in breast cancer cells and detect the expression of the relative transcription factors.(5)Alamar blue,colony formation and 3D culture assays were performed to detect the effects of EGFL6 modulation on cell proliferation.The expression of apoptosis markers Bcl-2,Fas and Caspase3 were detected by qPCR.The effect of EGFL6 on the apoptosis of breast cancer cells was also detected by Annexin V flow cytometry.(6)Flow cytometry assay and qPCR were used to reveal the population of CD24-/CD44+and cancer stem cell markers in breast cancer cells under EGFL6 up or down regulated.(7)Breast cancer cells MCF-7/EGFL6 with stable overexpressing or control cell MCF-7/NC were injected subcutaniously into nude mice to explore the effect of EGFL6 regulation to the breast cancer growth.Regular detect the growth of tumors in vivo.Frozen slices of tumor tissue were stained by immunohistochemical staining to explore the expression of CD31 and EGFL6.The phosphorylation expression of AKT and ERK were observed by Western Blot in breast cancer cell lines.Results:(1)qPCR and Western blot assay suggested that the difference expression of EGFL6 in six breast cancer cells which significantly higher in T47D but lower in MCF-7.EGFL6 binding assay also indicated that EGFL6 can bind to the breast cancer cells and played a potential role for tumor growth.Elisa detected the soluble EGFL6 expression in breast cancer cell lines T47D and human endothelial cell RF24.It was found that soluble EGFL6 was highly expressed in human endothelial cells,and the expression of T47D cells in breast cancer cells was the highest.(2)EGFL6 was successfully upregulated or silenced by Lentiviral-mediated overexpression or Lentiviral-mediated shRNA,respectively.The stable overexpression EGFL6 cells MCF-7/EGFL6 and the stable knockdown EGFL6 cells T47D/shEGFL6,MDA-MB-231/shEGFL6 were successfully constructed.(3)After the expression of EGFL6 was up regulated,the number of migration and invasion of breast cancer cells and the wound healing rate of cell scratch were significantly higher than those of the control group.Exogenous recombinant human EGFL6 protein and EGFL6 conditioned medium could also enhance the migration and invasion of breast cancer cells and the rate of cell scratch healing coverage.(4)When EGFL6 expression was up-regulation,the MCF-7 cells has the EMT change.The expression of E-cadherin was inhibited,while the expression of N-cadherin and Vimentin was upregulated.On the contrary,downregulation of EGFL6 resulted in the opposite effects.The expression of E-cadherin was up-regulation,while the expression of N-cadherin and Vimentin was inhibited.(5)Cell function experiment results showed that EGFL6 upregulation resulted in enhanced cell viability and colony formation efficiency in breast caner cells and the diameter of 3D culture is greater than that of the control group.EGFL6 downregulation significanted inhibited the above mentioned biological behaviors.The Fas and Caspase3 expressions were evidently up regulated while Bcl-2 mRNA expression was downregulated.(6)Flow cytometry assay revealed that EGFL6 knockdown decreased the CD24-/CD44+population.qPCR assay showed that in EGFL6 downregulated,ITGB1,BMI1,ITAG6,ALCAM,CD44,FGFR2,DNMT1,KLF4,MYC which as the cancer stem cell markers or involved in self-renewal had been down regulated.The phosphorylation level of downstream key signaling molecules AKT and ERK was affected.(7)In vivo studies suggested that EGFL6 overexpression significantly increased the tumor volume and quality in MCF-7 cell xenografts compared with the control.The results of immunohistochemical frozen slices showed that compared with the control cells,EGFL6 obviously enhanced the CD31 level in the EGFL6 overexpress tumor sample.The phosphorylation level of downstream key signaling molecules AKT and ERK was affected.Conclusion:Up-regulation EGFL6 in promoting migration and invasion,inducing the epithelial mesenchymal transition,maintaining cancer stem cells in breast cancer.Up regulation of EGFL6 can promote the proliferation and tumorigenesis in breast cancer cells and inhibit cell apoptosis and promote angiogenesis in tumor tissue.These data indicate that EGFL6 played an important role in the progression and metastasis of breast cancer,and provided a strong basis for the preparation of EGFL6 monoclonal antibody to inhibit the proliferation and metastasis of breast cancer.Part II The inhibition role of anti-EGFL6 monoclonal antibody to the biological properties of human breast cancer cell lines and tumors and the exploration of the anti-tumor mechanismsObjective:To develop the antibodies and test their suppressive effects on the migration,invasion and proliferation of human breast cancer cell lines and the effect of EGFL6 on the growth of breast cancer tumors,to investigate the role and mechanism of EGFL6 monoclonal antibody in the treatment of breast cancer.Methods:(1)To identify the monoclonal antibodies which targeting EGFL6,we used the PBMCs of immunized rabbit with human EGFL6,isolated the single memory B cells,and used the ELISA assay to screen EGFL6 antibodies.(2)Transwell and wound healing assay were used to detect the inhibition effect of the cell migration and invasion ability after the antibody treatment.(3)Alamar blue assay was used to detect the inhibition effect of the proliferation after the antibody treatment.Mouse IgG was used as a parallel control.(4)Western blot method was used to detect the changes of the downstream signal pathway with the EGFL6 antibody treatment.(5)Furthermore,we validated the EGFL6 potential therapeutic application in vivo using xenograft mouse model.The breast cancer cell lines T47D,MCF-7/NC and MCF-7/EGFL6,which overexpress EGFL6,were inoculated in the nude mice.The mice were treated with EGFL6 monoclonal antibody on the seventh day after the inoculation,and tumor volume was detected regularly in nude mice bearing tumor.IgG was used as parallel control in nude mice.(6)Western blot was used to detect the changes of the expression of key signaling pathways in the tumor tissues after the treatment with EGFL6 mAbs in human breast cancer mice.(7)The correlation between the expression of EGFL6 and CD31 in tumor tissues was detected by freezing immuno histochemical method.Results:(1)Two functional mAbs(#93 and#135)were produced which used for the following study in in vitro and in vivo treatment.The two antibodies were identified at different sites of EGFL6.In which#93 identified the EGF repeat regions and#135 identified the RGD and MAM regions.(2)EGFL6 enhanced cell migration and invasion were significantly abrogated by EGFL6 antibody treatment.Meanwhile,conditional medium from RF24/EGFL6 stimulated migration of T47D/shEGFL6 and MDA-MB-231 cells can be reversed by anti-EGFL6 antibody treatment.Scratch wound healing assay gave consistent results in which antibody treatment reduced the scratch coverage of T47D and MDA-MB-231 cells induced either by ectopic expression of EGFL6 or conditional media culture.(3)The increased number of cells caused by overexpression of EGFL6 in T47D and MCF-7 cells can be inhibited by EGFL6 antibody.Increased viability of T47D/shEGFL6 and MCF-7 cells by condition medium from RF24/EGFL6 can also be blocked by anti-EGFL6 antibody treatment.(4)Cell growth signaling phosphorated AKT and ERK were blocked by anti-EGFL6 antibody treatment.(5)The EMT makers like E-cadherin and Vimentin also been down regulated by the antibody treatment.(6)Anti-EGFL6 antibody treatment could block the tumor growth in MCF-7/EGFL6 overexpressed EGFL6 cells,but not the MCF-7 parental cells with vector control MCF-7/NC.Also the antibody could reduce the T47D xenograft tumor growth similar as the inhibition of tumor growth of T47D cells when the EGFL6 had been knocked down.(7)In vivo tumor samples indicated that AKT and ERK signal had been blocked by the antibody treatment resulting the inhibition of tumor growth.(8)Compared with the IgG parallel control group,the immune-histochemical results of frozen slices showed that the expression of CD31 was decreased with the treatment of EGFL6 antibody.Conclusion:Our study suggested that EGFL6 play a critical role for the metastasis and growth of cancer cells.We developed two therapeutic antibodies which binding to different sites to against EGFL6 in previous study.In vitro experiments showed that anti-EGFL6 monoclonal antibody can binding to the cell membrane,changing the biological properties of human breast cancer cell lines,such as reducing the proliferation,decreasing the migration and invasion ability,reducing the colony formation,the tumor volume and quality were also significantly lower than the control group.These phenomena not only further confirmed the characteristics of EGFL6,and also showed that the anti-EGFL6 monoclonal antibody have anti-tumor activity,these data are provided for further experiments in vivo.After treatment with the mAbs,the expressions of related signaling pathways pAKT and pERK were down regulated in tumor tissues.It is preliminarily proved that the anti-tumor activity of EGFL6 is related to the inactivation of PI3K/AKT and MAPK/ERK.Meanwhile,EGFL6 monoclonal antibody inhibits the angiogenesis of tumor tissue.Therefore,EGFL6 could be a new potential gene therapy targets for the breast cancer.Therapeutic antibodies showed the inhibition of metastasis and tumor growth,which provides valuable theoretical parameters for EGFL6 therapeutic antibodies and a powerful basis for targeted clinical treatment.In summary,we verified that the EGFL6 was closely related to the growth and migration of breast cancer through the in vivo and in vitro experiments.We have found that EGFL6 accelerated the mesenchymal transition by EMT mechanism in breast cancer cells,promote tumor progression of breast cancer;the increased expression of EGFL6 can promote the pAKT and pERK signals and the corresponding biological function;EGFL6 antibodies can effectively block the proliferation and migration of breast cancer cells and tumor growth in breast cancer.Furthermore,the mechanisms of EGFL6 regulation of breast cancer and the mechanism of promoting tumor growth and metastasis are elucidated,which provides a new target for immune intervention of breast cancer.