Detection And Significance Of MUC1 In Breast Tissues And Antibodies Against MUC1 VNTR Core Peptides In Sera Of Patients With Benign And Malignant Breast Tumors

MUC1, a transmembrane glycoprotein, is less expressedin many normal epithelial cells and overexpresses in cancercells such as breast cancer, ovarian cancer and so on. Itsextracellular domain is composed of a polypeptide corecontaining variable number tandem repeats of a 20-amino acidsequence. When the cell cancerates, its extracellular domaincovered numerous carbohydrate side chains under normalcircumstances exposes itself due to deficient glycosylation.Then, the exposure evokes humoral and cellular immuneresponse against MUC1 VNTR core peptides in vivo. Althoughthe immunity to MUC1 VNTR core peptides in vivo is notintensive enough to control the extension of tumors, theantibodies or CTLs against MUC1 VNTR core peptides caninhibit growth of tumors. At present, MUC1 VNTR core peptidesantigen has become a target for specific tumor immunotherapy.Usually, MUC1 is mainly expressed on the apical surface ofepithelial cells of various tissues and organs. That is, it showstop expression or polar contribution and no expression atendochylema. However, MUC1 in breast cancer tissue is ofoverexpression, reduced glycosylation and aberrant top location,and it is expressed at both cell membrane and cytoplasm andits expression intensity positively correlates with the malignancygrade.Studies have showed that antibodies against MUC1 maybe effective in eradicating these circulating tumor cells and itsmechanism probably involves antibody-dependent cell-mediatedcytotoxicity and complement-mediated lysis. In addition,antibodies to MUC1 may be involved in restoring cell adhesionand limiting cancer invasion, uncovering cell surface receptorsinvolved in immune recognition, and neutralizing theimmunosuppressive effect of soluble MUC1 and so on. Ingeneral, the antibodies may check cancer spread in patientswith breast cancer possibly by destroying circulating, seeded,isolated or disseminated tumor cells. The stronger immuneresponse and the immunologic memory that are associatedwith active immunotherapy would provide a constantsurveillance mechanism to protect cancer patients fromrecurrence of disease.Investigates have paid close attention to the natural andinitial state antibodies to MUC1 VNTR core peptides in serumas well as CTLs since earlier times in order to provide evidenceof tumor biotherapy targeting MUC1 VNTR core peptides. Thefree auto-antibodies agaist MUC1 VNTR core peptides havebeen found in healthy subjects, in patients with benign breastdisease and in carcer patients, but there has not been the reportabout detecting domestic women's sera. Several studies haveshowed that serum levels of antibodies against MUC1 VNTRcore peptides were closely correlated with survival time ofpatients with malignant tumors, and higher levels of anti-MUC1VNTR core peptides antibodies may be a favorable prognosticfactor for cancers. Approach to more effective, specific andsensitive method detecting anti-MUC1 antibodies in serum canprovide tumor vaccine targeting MUC1 VNTR core peptides withpotent monitoring tool. Current detection to anti-MUC1 VNTRcore peptides antibodies in serum mainly involves indirectELISA with synthetic peptides antigen of MUC1 VNTR corepeptides, but synthetic peptides with higher cost and tediouscouple procedure are not easily being coated. By literatureretrieval, indirect ELISA method detecting natural MUC1 VNTRcore peptides antibodies in serum with recombinant proteinantigen has not been found to report.In our study, MUC1 expression was detected in normal,benign breast tumor and breast cancer tissues. We obtainedrecombinant protein of MUC1 VNTR core peptides by generecombination technique and established an indirect ELISAmethod detecting MUC1 VNTR core peptides antibodies inserum with recombinant protein antigen, and the detected serasamples from 130 patients with breast tumors and 56 healthywomen, and probed into the mechanism of role of MUC1 VNTRcore peptides antibodies in serum in carcinogenesis andextension of breast cancer.The major parts of this paper is as follows:1. The construction and expression and purification of8R-MUCPTWith pET28a-HSP65-MUC1 plasmid from our lab astemplete, we isolated the MUCPS cDNA by PCR with specificprimers. The specific cDNA with molecular size of 164 bp andtwo repeats sequence was cloned into the pMD-18-T vector.The recombinant pMD-18-T-MUCPS plasmid was respectivelydigested with PstⅠ, BglⅡand PstⅠ, BamHⅠ, and thereleased insert was subcloned into pMD-18-T-MUCPS digestedwith PstⅠ, BglⅡ. The recombinant pMD-18-T-MUCPD wasidentified with EcoRⅠ, and then was digested with PstⅠ,BamHⅠ. The released insert with the molecular size of 331bpwas subcloned into pMD-18-T-MUCPS. The MUCPT insertreleased from the recombinant pMD-18-T-MUC-PT plasmidwas cloned into pET 26b+ vector with NcoⅠand XhoⅠ. Theinsert released from pET26b+-8R-MUCPT, with the molecularsize of 477bp, was confirmed by DNA sequencing. It encodes apeptide consisting of 159 amino acids. ThepET26b+-8R-MUCPT plasmid was transformed into BL21.Incubated cells were harvested after being induced by IPTG for3h. The lysate of the harvested cells was analyzed usingSDS-PAGE. A major species with the molecular weight 23KDwas revealed in induced cells but not in uninduced cells. Thisdemonstrated that 8R-MUCPT cDNA was expressed in E.Coli.Through a metal ion chelate affinity chromatography, theprotein was purified to 95.5% purity. .2. Preparation and identification of polyclonal antibodyagainst MUC1 VNTR core peptidesIn order to identify whether 8R-MUCPT protein hasantigenic capacity or not and intensity of antigenic capacity, theprotein was used to immunize a rabbit to prepare polyclonalantibody against MUC1 VNTR core peptides. With completeadjuvant and subcutaneous multipoint injection being used, thehigher titer of 1:256000 of the polyclonal antibody was inducedwithin 6 weeks. HSP65-MUC1 protein was used as antigen todetermine the titer of antibody against MUC1 VNTR corepeptides, and the titer was 1:16000. Both western blotting andblocker of ELISA had identified the specific antibody againstMUC1 VNTR core peptides which demonstrated the polyclonalantibody contained the specific antibody against MUC1 VNTRcore peptides, and 8R-MUCPT protein had antigenic capacityand was prone to become antigen in indirect ELISA detectingMUC1 VNTR core peptides antibodies in serum. At the sametime, the polyclonal antibody provides a better experimentalmaterial for tumor immunotherapy targeting MUC1 VNTR corepeptides.3. Detection of expression of MUC1 in human breasttissuesIn order to elucidate expression of MUC1 VNTR corepeptides in benign and malignant breast tumor tissues andfurther identify the relationship between MUC1 expression inbreast tissues and serum levels of antibodies against MUC1VNTR core peptides, the immunohistochemical withanti-MUC1VNTR mAb was used to examine the MUC1expression in 15 cases of breast cancer, 8 cases of benignbreast tumor and 5 cases of normal tissues beside breastcancer. The results showed that MUC1 expressed in normal,benign breast tumor and breast cancer tissues respectively.MUC1 VNTR core peptides in normal tissues beside breastcancer was low expression (most of them showed +), whichwas mostly located at the apical side of epithelial cells. MUC1VNTR core peptides in benign breast tumor was lowexpression (most of them showed +~++), which was mostlylocated at the apical side of epithelial cells, and a few waslocated at the external surface of luman and still a few locatedat the cell membranes and cytoplasms. There was a highexpression in breast cancer tissues (most of them showed+++), which was mostly located at the cell membranes andcytoplasms.The polyclonal antibody against MUC1 VNTR corepeptides prepared by 8R-MUCPT was used to detect MUC1expression in breast cancer tissues by immunohistochemical,and positive expression of MUC1 VNTR core peptides in breastcancer tissues had been found by it. The expression intensityand location were similar to those detected by anti-MUCVNTRmonoclonal antibody, which verified that MUC1 VNTR corepeptides in 8R-MUCPT were consistent with MUC1 in breastcancer tissues.4. Establishment of an indirect ELISA for detecting MUC1VNTR core peptides antibodies in human serum4.1. Specificity testsA dot blot and an ELISA blocker were performed toinvestigate the specificity of detecting circulating MUC1 VNTRcore peptides antibodies in the indirect ELISA. In addition, acompetitive indirect ELISA (see 4.2) also verified the specificityof this indirect ELISA. The result of the dot blot demonstratedthat there existed specific MUC1 VNTR core peptides antibodyin serum because it had competed with anti-MUC1VNTR mAb.Same molar of poly-arginine (polyR), poly-histidine (polyH)peptides and 8R-MUCPT were respectively dropped into sera ofsame patient, which could respectively neutralize or block theserum antibodies against 8R, 6H and 8R-MUCPT. This resultshowed that antibodies to MUC1 VNTR core peptides ratherthan antibodies to 8R and 6H had been detected by the indirectELISA, and it simultaneously verified that the recombinant8R-MUCPT with six tandem repeat sequence would be asuitable antigen for detecting circulating MUC1 VNTR corepeptides antibodies.4.2. Establishment of a competitive indirect ELISAThe established method could qualitatively detected specificMUC1 VNTR core peptides antibodies in serum, which willprovide the indirect ELISA detecting circulating MUC1 VNTRcore peptides antibodies with a reference test. A competitiveindirect ELISA ELISA is usually used to test specificity. Its meritlies in higher specificity;that is, it can find "true" antibody.4.3. Establishment of an indirect ELISA detecting MUC1VNTR core peptides antibody in human serum4.3.1. Definition of cut off value for positivity31 cases of control sera were from the samples whoseoptical density (OD) values were less than mean value of 45serum samples from healthy women which could meet withrequirement of negative control and would make the resultsmore reliable. X±3SD was considered as standard of the cut-offvalue of positivity which further improved reliability of the results.Under the above standard, the detected positive rates weresimilar to those of previous reports which demonstrated thedefinition was correct.4.3.2. Validity analysisValidity analysis showed the indirect ELISA had asensitivity of 91.3% and specificity of 94.1% when taking thecompetitive indirect ELISA as a reference test. The coincidencewas 92.5%.4.3.3. Results of the indirect ELISAThe indirect ELISA was used to detect circulatingantibodies against MUC1 VNTR core peptides in 186 serumsamples. The IgG was respectively detected in 13 of 56 (23.3%)healthy subjects, 14 of 22(63.6%) patients with benign breasttumors, 34 of 104 (31.5%) patients with malignant breast tumors;furthermore, 5 of 13 (38.5%) stage Ⅰ , 19of60 (31.7%) stage Ⅱ,1of 7(14.3%) stage Ⅲ and 6 of17 (35.3) stage Ⅳ . The IgM wasrespectively detected in 13 of 45(28.9%) healthy subjects, 3 of20(15.0%) patients with benign breast tumors, 15 of 98(15.3%)patients with malignant breast tumors;furthermore, 0 of 11(0)stage Ⅰ , 9 of 57(15.8%) stage Ⅱ , 0 of 6(0) stage Ⅲ and 4 of16(25%) stage Ⅳ .Our group found that the incidence of positivity ofanti-MUC1 VNTR core peptides IgG increased significantly inbreast benign patients than that in breast cancer patients(before or after primary treatment) or in the healthy women(P<0.01), which was different from the overseas result. It isconsidered that the reasons for it are probably due to increasein shedding of MUC1 VNTR core peptides after treatment andrelatively fewer sample cases in our study compared with that ofprevious study. Considering that breast benign tumor patientshas higher levels of circulating IgG against MUC1 VNTR corepeptides and decrease in positive rates of anti-MUC1 VNTRcore peptides IgG from benign tumor to stage Ⅲ of breastcancer, we presumed circulating anti-MUC1 VNTR corepeptides IgG probably played a role of neutralizing MUC1 VNTRcore peptides antigen in breast cancer.In our study, it was found that mean value of OD ofanti-MUC1 VNTR core peptides IgG increased significantly inCA15-3 antigen-negative sera than in CA15-3 antigen-positivesera(p=0.041). There is less anti-MUC1 VNTR core peptidesIgG in CA15-3 antigen-positive serum and more anti-MUC1VNTR core peptides IgG in CA15-3 antigen-negative serum(p=0.037). What mentioned above implied that there was anegative correlation of serum levels between anti-MUC1 VNTRcore peptides antibodies and MUC1 VNTR core peptidesantigens in serum. Because the detected sera above mainlycame from patients with advanced malignant tumors, it could beinfered that anti-MUC1 VNTR core peptides IgG in serum alsoprobably played a role of neutralizing MUC1 VNTR corepeptides antigen in stageⅣ of breast cancer. It was also inferedthat diversion of malignant tumors in stageⅣ of breast cancerwas not only restricted to the tumor with expression of MUC1VNTR core peptides. Higher levels of anti-MUC1 VNTR corepeptides antibodies and low levels of MUC1 VNTR corepeptides antigens in serum of stageⅣ demonstrated that forsome patients, no or less MUC1 VNTR core peptides werelocated in the extending or transferring tumors tissues, andMUC1 with abnormal glycosylation or other epitopes probablybecame predominant. There were still the other patients withexpression of MUC1 VNTR core peptides in the extending ortransferring tumors because higher levels of MUC1 VNTR corepeptides antigens and low levels of anti-MUC1 VNTR corepeptides antibodies in some serum samples of stageⅣ could befound.To sum up, MUC1 was respectively expressed in breastcancer, benign breast tumor and normal tissue beside breastcancer in different extent and at different locations. Theestablished indirect ELISA in detecting circulating MUC1 VNTRcore peptides antibodies with 8R-MUCPT recombinant proteinas antigen was reliable. The incidence of positivity of anti-MUC1VNTR core peptides IgG increased significantly in breast benignpatients than that in breast cancer patients (before or afterprimary treatment) or in the healthy women (Both areP<0.01).Through analyzing its mechanism in breast cancer, weinfered that the antibody probably played a role of neutralizingMUC1 VNTR core peptides antigen in genesis and growth ofbreast cancer. With the established indirect ELISA of detectinganti-MUC1 VNTR core peptides antibodies in human serumbeing further consummated, it is expected that it would takeplace of the current indirect ELISA with synthetic peptideantigen in order to appraise validity of MUC1-drived vaccines orpredict prognosis of cancer patients. Our group also providedsome evidence for the mechanism of serum anti-MUC1 VNTRcore peptides antibodies in breast cancer.