| Breast cancer is the most common malignancy in women, with increasing incidence. Therapeutic options for this type of cancer range from primary surgery to adjuvant chemotherapy, radiotherapy, hormonal therapy or targeted therapy. Breast cancer is a heterogeneous disease, and therefore, no golden standard therapy exists suitable for all tumors of the mammary gland.Currently, tumor immunotherapy has become the fourth therapeutic pattern of tumors following surgery, chemotherapy and radiotherapy, especially for those tumors with poor surgerical therapeutic efficiency. Identification of appropriate target antigens is the first and most crucial step in the successful development of antigenspecific cancer immunotherapy. For tumor antigens to be potential immunotherapeutic targets, the antigens must have no or highly restricted expression in normal tissues so that the autoimmunity can be prevented.In recent years, cancer/testis antigens (CTA) possess several features of ideal targets for cancer immunotherapy. They are expressed in a wide variety of malignant tumors, but their expression in normal tissue is mostly restricted to germ cells of the testis and the placenta cells, which are immunoprivileged because of their lack of human leukocyte antigen (HLA) molecules. Therefore, the discovery and characterization of CTA has provided the first group of target antigens for cancer immunotherapy that can be used in various common cancers. Melanoma-associated antigens (MAGE) are a group of well-characterized members of the CTA family, which was firstly separated from melanoma cells. MAGE gene encodes tumor-sepicific antigenic peptides which were presented to CD8+T lymphocytes by HLA-I class Ⅰ molecules, and therefore inducing tumor sepicific killing.In recent years, our group mainly focused on the biological functions and their effects on tumor immonology of MAGE family. At least55MAGE closely related proteins were found. Based on their tissue-specific gene expression, the MAGEs have been subdivided into two categories:MAGE-Ⅰ and MAGE-Ⅱ type Ⅰ cancer-testis antigens and type Ⅱ ubiquitous MAGEs. Most of MAGE-Ⅰ antigens are relevant cancer/testis antigens and therefore are rarely expressed in normal adult tissues except for testis, but highly expressed in various forms of cancer. Unlike the type Ⅰ MAGE cancer-testis antigens, MAGE Ⅱ antigens are not restricted to germline expression and are expressed in a variety of normal adult tissues. The MAGE-Ⅰ family includes MAGE-A, MAGE-B and MAGE-C subfamilies. The MAGE-A gene family is comprised of12family members called MAGE-A1-MAGE-A12. The MAGE-A antigens are of particular interest for cancer immunotherapy because they are strictly tumor specific and are shared by many kinds of tumors. However, to identify which MAGE-A antigen should be the target of a breast cancer vaccine, the expression of each individual member of MAGE-A family still has to be defined.A few studies have reported on the expression of MAGE-A1and MAGE-A3in breast cancers. In the present study, we collected the formalin-fixed and paraffin-embedded tissues and the clinicopathological parameters from75primary breast cancer patients, immunohistochemically detected the expressions of MAGE-A10and MAGE-A11proteins, and analyzed their association with the clinicopathological parameters and the survival of breast cancer patients. The MAGE-A11expression plasmid was successfully constructed. Gene transfection, RT-PCR, western-blot analysis and immunoprecipitation were adopted to detect the the exogenous MAGE-A11on estrogen-dependent cell proliferation.The main research contents and results were shown as follows:Part Ⅰ The expression of MAGE-A10and MAGE-A11in breast cancer tissues and their correlation with the clinicopathological parameters and the prognosisObjective:To investigate the expression of MAGE-A10and MAGE-A11 in breast cancer tissues and the normal breast tissues, and explore their correlation with the clinicopathological parameters and the prognosisMethods:Immunohistochemical staining was used to detect the protein expression of MAGE-A10and MAGE-A11in75formalin-fixed and paraffin-embedded breast cancer and normal breast tissues. Retrospective analysis was used to analyze the correlation between MAGE-A10and MAGE-A11protein expression and the clinicopathological parameters of breast cancer patients, including age of the patients, pathological types, histology grades, clinical stages, tumor size, metastasis of axillary lymph nodes, estrogen receptor (ER), progestrogen receptor (PR) and HER-2status. The correlation between MAGE-A10and MAGE-A11protein expression and the prognosis of breast cancer was also analyzed.Results:1Expression of MAGE-A10and MAGE-A11proteins in normal testis tissuesNormal testis tissue was used as the positive control for MAGE-A10and MAGE-A11protein expression. Both MAGE-A10and MAGE-A11expressions were mainly observed on spermatogonia and primary spermatocytes and both the cytoplasm and the nucleus were stained.2Expression of MAGE-A10and MAGE-A11proteins in breast cancer tissues and normal breast tissuesThe immunohistochemical staining results showed that MAGE-A10expression was mainly observed on spermatogonia and primary spermatocytes and both the cytoplasm and the nucleus were stained. No MAGE-A10expression was found in75tumor-free breast specimens.55out of75(73.3%) breast cancer specimens were found positive with MAGE-A10antibody, which was significantly higher that the normal breast tissues (P<0.05), suggesting that MAGE-A10is a tumor specific antigen.The immunohistochemical staining results showed that MAGE-A11expression was mainly observed on spermatogonia and primary spermatocytes and both the cytoplasm and the nucleus were stained. No MAGE-A11 expression was found in75tumor-free breast specimens.39out of75(52.0%) breast cancer specimens were found positive with MAGE-A11antibody, which was significantly higher that the normal breast tissues (P<0.05), suggesting that MAGE-A11is a tumor specific antigen.3Correlation between MAGE-A10and MAGE-A11expressions and the clinicopathological features of breast cancer patientsThe correlation between MAGE-A10and MAGE-A11expressions and the clinicopathological features was statistically evaluated. The result showed that no association at all was observed for MAGE-A10expression and the clinicopathological features of breast cancer patients, such as the age of the patients (χ2=1.003, P=0.606), pathological type (χ2=2.327, P=0.127), histological grade (χ2=0.645, P=0.724), clinical stage (χ2=1.364, P=0.506), tumor size (χ2=0.642, P=0.725), lymphatic metastasis (χ2=0.052, P=0.820), ER (χ2=1.847, P=0.174) and PR (χ2=0.487, P=0.485) status.No association was observed for MAGE-A11expression and the age of the patients (χ2=0.542, P=0.763), pathological type (χ2=0.033, P=0.855), histological grade (χ2=0.739, P=0.691), clinical stage (χ2=0.666, P=0.717), tumor size (χ2=2.350, P=0.309), lymphatic metastasis (χ2=2.376, P=0.123), and PR (χ2=0.153, P=0.696) status. MAGE-A11expression was more frequent in estrogen-receptor (ER) positive breast carcinomas (25/36,69.4%) compared with ER negative breast carcinomas (14/39,35.9%)(χ2=8.441, P=0.004).4Correlation between MAGE-A10and MAGE-A11expressions and the HER-2status of breast cancer patientsBecause HER-2has been reported to be overexpressed in25-30%breast cancers, and is correlated with the poor prognosis of breast cancer we immunohistochemically examined the expression of HER-2in breast cancer specimens and analyzed the association between MAGE-A10and MAGE-A11expressions and HER-2expression, in order to explore the association between MAGE-A10and MAGE-A11expressions and the prognosis of breast cancer patients. The immunohistochemical staining results showed that HER-2 expression was mainly observed on cell membrane. The statistical analysis showed that7out of75(9.3%) was HER-2(-),29out of75(38.7%) was HER-2(+),18out of75(24%) was HER-2(++),21out of75(28%) was HER-2(+++). No association at all was observed between MAGE-A10expression and HER-2expression. MAGE-A11expression was positively associated with HER-2expression(χ2=14.029, P=0.003).5Correlation between MAGE-A10and MAGE-All expressions and the survival of breast cancer patientsAll75patients were followed up for16-60months, of those,26patients were lost. The results showed that overall survival of patients with MAGE-A11negative expression was significantly longer than those patients with positive MAGE-A11expression (χ2=4.697, P=0.030), but no difference of overall survival was found between patients with MAGE-A10negative expression and positive expression,(χ2=0.022, P=0.881), suggesting that MAGE-A11protein expression may be one of the poor prognosis factor of breast cancer patients.Conclusions:1Normal breast tissues do not express MAGE-A10and MAGE-A11protein. The expression rate of MAGE-A10and MAGE-A11in breast cancer was73.3%and52.0%, respectively, suggesting that MAGE-A10and MAGE-A11were tumor specific antigens.2No association at all was observed for MAGE-A10expression and the clinicopathological features of breast cancer patients, such as the age of the patients, pathological type, histological grade, clinical stage, tumor size, lymphatic metastasis, ER and PR status.3No association at all was observed for MAGE-A11expression and the clinicopathological features of breast cancer patients, such as the age of the patients, pathological type, histological grade, clinical stage, tumor size, lymphatic metastasis, and PR status. MAGE-A11expression was more frequent in ER positive breast carcinomas compared with ER negative breast carcinomas. 4No association at all was observed between MAGE-A10expression and HER-2expression. MAGE-A11expression was positively associated with HER-2expression, suggesting that MAGE-A11protein expression may be one of the poor prognosis factors of breast cancer patients.5No difference of overall survival was found between patients with MAGE-A10negative expression and positive expression. The overall survival of patients with MAGE-A11negative expression was significantly longer than those patients with positive MAGE-A11expression, suggesting that MAGE-A11protein expression may be one of the poor prognosis factor of breast cancer patients.Part Ⅱ The construction of MAGE-A11expression plasmid and its effect on the proliferation of breast cancer cellsObjective:To construct the MAGE-A11expression plasmid with GFP tag, and investigate its effects on the proliferation of breast cancer MCF-7cells under overexpression condition.Methods:Gene recombination technique was used to construct the pCMV6-AC-MAGE-A11-GFP expression plasmid. MTT assay and colony formation assay were used to investigate the effects of exogenous MAGE-A11on the proliferation of breast cancer MCF-7cells.Results:1pCMV6-AC-MAGE-A11-GFP expression plasmid was successfully constructed.2Westem-blot results showed that MAGE-A11protein could be detected after transfection, suggesting that that this expression plasmid could be used in the next experiments.3MTT assay showed that after pCMV6-AC-MAGE-All-GFP transfection for48h, the cell survival rate of MCF-7cells was significantly increased than the control (p<0.05).4The result of colony formation assay showed that after pCMV6-AC-MAGE-A11-GFP transfection and G418selection for two weeks, the number of colonies of MCF-7cells was118±6, which was significant lower than the control group55±2(p<0.05), suggesting that exogenous MAGE-A11promoted the proliferation of MCF-7cells.Conclusions:1pCMV6-AC-MAGE-All-GFP expression plasmid was successfully constructed.2Exogenous MAGE-A11promoted the proliferation of MCF-7cells.Part Ⅲ The effect and molecular mechanism of MAGE-A11on proliferation of estrogen-dependent breast cancer cellsObjective:To investigate the effect and the molecular mechanism of MAGE-A11on proliferation of estrogen-dependent breast cancer cells.Methods:RT-PCR and western-blot were used to detect the expression of ER in MCF-7and MDA-MB-231cells. Gene transfection, RT-PCR and western-blot were used to detect the expression of ER-downstream genes at the mRNA and protein levels after17β-estradiol treatment. Gene transfection and immunoprecipitation were used to detect the physical interaction between MAGE-A11and ER at the presence of17β-estradiol. MTT and colony formation assay were used to detect the effect of MAGE-A11on proliferation of estrogen-dependent breast cancer MCF-7cells.Results:1To detect ER expression in breast cancer cellsRT-PCR and western-blot results showed that ER was positive in MCF-7cells and negative in MDA-MB-231cells, suggesting that MCF-7was ER positive cells, and MDA-MB-231was ER-negative cells.2Effect of MAGE-A11on estrogen-induced ER downstream Efp expressionRT-PCR and western-blot results showed that in ER positive breast cancer MCF-7cells, the expression of ER-downstream gene Efp was significantly increased at mRNA and protein levels after10nM17β-estradiol treatment for24h. MAGE-A11transfection alone can not increase the expression of Efp at mRNA and protein levels. However, exogenous MAGE-A11increased estrogen-dependent Efp mRNA and protein expressions.3Physical interaction between MAGE-A11and ERGene transfection and immunoprecipitation were used to detect the physical interaction between MAGE-A11and ER at the presence of17β-estradiol. The immunoprecipitation experiment showed that after transfected with MAGE-A11and treatment of17β-estradiol, the immunoprecipitates of MAGE-A11contained ER protein and the immunoprecipitates of ER contained MAGE-A11protein, suggesting that there exists physical interaction between MAGE-A11and ER, and MAGE-A11may increases ER-induced Efp expression through physical interaction with ER.4Effect of MAGE-A11on the proliferation of estrogen-dependent breast cancer cellsMTT assay result showed that the proliferation of MCF-7cells was significantly increased after17β-estradiol treatment. MAGE-A11could increase17β-estradiol-induced proliferation of MCF-7cells. Colony formation assay results showed that exogenous MAGE-A11could increase the colony numbers of MCF-7cells. After17β-estradiol treatment, the colony numbers of MCF-7cells were significantly increased. MAGE-A11could increase the proliferation of17β-estradiol-induced breast cancer MCF-7cells. These results suggested that MAGE-A11could increase the proliferation of estrogen-dependent breast cancer cells.Conclusions:1MCF-7was ER positive cells, and MDA-MB-231was ER-negative cells.2In ER positive breast cancer MCF-7cells, as a co-activator, MAGE-A11may increase ER-induced Efp expression through physical interaction with ER at the presence of17β-estradiol, thereby promoting the proliferation of estrogen-dependent breast cancer cells. |
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