| Breast cancer is one of the most common malignancies of women worldwide. The newly diagnosed global breast cancer cases were 1,671,000 accounting for 25% of the whole cancer patients according to GLOBOCAN 2012. Though patients with breast cancer have taken advantage in prognosis with the advance in diagnosis and treatment, some cases will be dead due to distant metastasis. Based on the molecular phenotype, breast cancer is mainly classified into Luminal A, Luminal B, HER-2(+) and basal-like subtype which is mainly composed by triple negative breast cancer(TNBC) characteristic with ER(-), PR(-) and HER-2(-). Owing to no effective therapeutic target and heterogeneity of the disease, in spite of sensitivity of chemotherapy, most of the TNBC patients are likely to die within the first three years after initial treatment because of visceral metastasis. In consequence, it is pivotal to research on the mechanism in metastasis of breast cancer in depth and explore new treatment target for the whole prognosis improvement.Metastasis is the leading cause of cancer related death. It has been proved that epithelial mesenchymal transition(EMT) is a crucial step for cancer metastasis. EMT not only plays a role in embryogenesis but also works in inflammation, fibrosis and cancer progress. In the early stage of carcinogenesis, some detached tumor cells infiltrate into blood and vascular system and spread to the whole body via EMT, forming the second neoplastic foci in the distance. This process includes morphology changes and cytoskeleton rearrangement of the cells, the loss of epithelial markers and gain of mesenchymal traits, enhanced migration. It is also reported that EMT is responsible for chemotherapy and radiotherapy resistance. Hence, EMT is associated with aggressiveness and poor outcomes of cancer. It has been reported that EMT occurs in TNBC cells which has mesenchymal traits. Hence, exploring underlying mechanisms of EMT in TNBC cells is helpful for discovering new target and therapeutic ideas.Melanoma associated antigen-MAGEs, a member of tumor-associated antigen, was initially discovered in melanoma but was found in tissues of various origins. According to the locations in chromosome and distribution in tissues, MAGEs are grouped into type I and type II. MAGE-I is composed of MAGE-A,-B,-C families. MAGE-As, the widely studied family, is aberrantly expressed in various cancers derived from different tissues, such as breast cancer, liver cancer, lung cancer, whereas exclusively expressed in testicular germ cells and placenta. MAGE-As antigens can elicit CD8+ T lymphocytes directed anti-tumor immunity. Consequently, MAGE-As is crucial for disease assessment and target therapy of cancer patients. Accumulating evidence suggested that MAGE-As expression is linked to development and metastasis of breast cancer cells. Moreover, it is highly expressed in aggressive cancer types, such as TNBC.Thus, we speculate that MAGE-As may be associated with aggressiveness and EMT of TNBC. To address this issue, we firstly performed immune-histochemical technique to detect the expression profile of MAGE-As, E-cadherin, β-catenin and vimentin in normal breast tissue and breast cancer tissues. Further, MAGE-As expression was knocked down in TNBC cell lines to explore its impact on proliferation, migration and invasion of the cancer cells and the potential mechanism. The main research contents and results are as follows: Part I Research on expression of MAGE-As and EMT markers in breast cancer tissuesObjective: To investigate expression profile of MAGE-As in normal breast tissues and breast cancer tissues and detect E-cadherin,vimentin and β-catenin expression in breast cancer tissues. The association between their expression and clinical pathological indexes was also analyzed. Correlation between MAGE-As and EMT markers was also determined.Methods:The subjected breast cancer tissue was classified into Luminal, HER-2(+) and TNBC subtype according to the newest classification criteria firstly. Then, IHC was used to analyze the contents as follows:1 The expression profile of MAGE-As was detected in normal breast tissue as well as breast cancer tissue, and its positive rate was also compared in different subtypes.2 The relevance between MAGE-As expression and clinical pathological indicators of breast cancer was explored.3 The aberrant expression of E-cadherin and β-catenin and positivity of vimentin was tested in the whole breast cancer tissue and different subtypes.4 The association between E-cadherin, β-catenin, vimentin and clinical pathological indicators of breast cancer was studied.5 The correlation between MAGE-As and E-cadherin, β-catenin, vimentin was analyzed.Results:1 The proportion of Luminal set, HER-2(+) set and TNBC set was 75%,10.83% and 14.17%, respectively.2 MAGE-As was absent in normal breast tissues but expressed in breast cancer tissues with the positive rate of 49.17%(59/120)in the whole cohort, its prevalence is higher in breast cancer tissue than normal breast tissue(P<0.05). MAGE-As was more frequently expressed in TNBC(76.47%,13/17)than HER-2(+)(53.85%,7/13)and Luminal subtype(43.33%,39/90)(P<0.05). Furthermore, MAGE-As was more frequent in TNBC compared to non-TNBC(P<0.05).3 MAGE-As was associated with ER(-), PR(-), HER-2(-), high histological grade and metastatic axillary lymph nodes(P<0.05). No relevance was established between distribution of MAGE-As in cells and breast cancer pathological indexes(P>0.05).4 In the whole subjected breast cancer tissue, the normal expression rate of E-cadherin and β-catenin was 30% and 25%, the aberrant expression rate was 70% and 75%, respectively. Normal expression of E-cadherin and β-catenin was mostly observed in non-TNBC cases(P<0.05), however, TNBC cases were always with aberrant expression of E-cadherin and β-catenin(P<0.05). The prevalence of vimentin was 34.16%(41/120), and it was more frequent in TNBC compared to non-TNBC(P<0.05).5 Abnormal E-cadherin expression was associated with metastatic axillary lymph nodes, vascular invasion, ER(-), PR(-) and HER-2(-)(P<0.05); aberrant β-catenin was mainly connected with larger breast tumors(>2cm), metastatic axillary lymph nodes, ER(-), and higher Ki-67(P<0.05); positive vimentin was correlated with ER(-), PR(-) and metastatic axillary lymph nodes(P<0.05).6 In breast cancer tissues, MAGE-As positivity was correlated with aberrant expression of E-cadherin and positive vimentin. There was no relationship between MAGE-As and β-catenin.Conclusions:1. MAGE-As is expressed in breast cancer tissues with desperate positive rates in different subtypes, which make it a new marker for classification in breast cancer.2. MAGE-As expression is associated with poor prognosis and metastasis of breast cancer.3. EMT occurs in TNBC, and MAGE-As may be responsible for EMT of TNBC. Part II Research on the association between expression of MAGE-As and EMT of TNBC cellsObjective: To explore the potential correlation between MAGE-As expression and EMT of TNBC cells.Methods: Highly metastatic human triple negative breast cancer cell lines MDA-MB-231 and BT549 with mesenchymal characters were chosen for study. Because of the high conservation in structures, members of MAGE-As share similar functions and have complementary functions, hence, MAGE-A si RNA was used to knock down all the MAGE-A members. Non-transfected group, control group(scrambled MAGE-A si RNA transfection) and experiment group were set for study as follows.1 RT-PCR and WB were performed to confirm the down-regulation of MAGE-As.2 Wounding healing assay and transwell migration and invasion assay were applied to determine migratory and invasive potential of the cells when MAGE-As were down-regulated.3 MTS was used to test the proliferation of the cells.4 The morphology changes of the cells were observed after MAGE-A s expression was decreased.5 RT-PCR and WB were carried out to examine the expression profile of EMT markers(E-cadherin, β-catenin, vimentin, MMP9) and EMT related transcriptional factors(snail, slug, ZEB1/2, twist)6 Immunofluorescence(IF) assay was performed to detect the expression of E-cadherin, β-catenin and vimentin.Results:1 MAGE-As expression was knocked down successfully in the two types of TNBC cell lines.2 Wound healing assay showed that the horizontal migratory potential was inhibited in the TNBC cell lines when MAGE-As expression was decreased. In MDA-MB-231 and BT549, the relative migration(%) at 24 h of the experimental group was lower than nontransfected and control group(P<0.05).3 Transwell assay displayed that vertical migration and invasion of the cells were declined. The migration assay uncovered that the invaded cells of the experimental group in MDA-MB-231 and BT549 was less than nontransfected and control group(P<0.05). In invasion assay of MDA-MB-231 and BT549, the number of the invaded cells was less in experimental group compared to nontransfected and control group(P<0.05)。4 MTS showed that the proliferative ability of the cells with MAGE-As down-regulation was significantly repressed. At 24 h and 48 h, OD value in the experimental group of MDA-MB-231 and BT549 was lower than nontransfected and control group(P<0.05). The expression of proliferation associated genes cyclin-D1 and c-myc were also decreased in the two kinds of TNBC cells with MAGE-As declination at both m RNA and protein level.5 When MAGE-As was down-regulated, morphology change was not observed in MDA-MB-231, but in BT 549, the cells became small and round with disappearance of protrusion.6 In realtime-PCR assay of MDA-MB-231, E-cadherin was elevated(P<0.05), but vimentin and MMP9 were decreased with MAGE-As downregulation(P<0.05). No significant changes of β-catenin was observed(P>0.05). In BT549, E-cadherin and β-catenin did not significantly changed(P>0.05), while vimentin and MMP9 were markedly declined(P<0.05). According to WB, no obvious alteration of E-cadherin was observed(P>0.05), but vimentin, β-catenin, MMP9 were evidently down-regulated in both MDA-MB-231 and BT549.7 The IF assay showed that, with the diminution of MAGE-As, E-cadherin was not evidently changed, but vimentin and β-catenin were significantly down-regulated in the two kinds of TNBC cell lines.8. In realtime-PCR assay of MDA-MB-231, slug and twist were not changed significantly, but snail and ZEB1/2 were decreased with MAGE-As downregulation(P<0.05). No significant changes of snail was observed(P>0.05)in BT549, but slug, ZEB1/2 and twist were decreased(P<0.05). According to WB, no obvious alteration of twist was observed(P>0.05), but snail and ZEB1/2 were evidently down-regulated in MDA-MB-231. Slug was elevated(P<0.05). In BT549, snail and twist were not changed markedly( P>0.05), but slug and ZEB1/2 were declined with MAGE-As down-regulation(P<0.05)Concluisions1 The proliferation, migration and invasion protential are inhibited when MAGE-As expression are knocked down in TNBC cell lines.2 EMT of TNBC is partly reversed when MAGE-As is down-regulated.3 EMT of TNBC may be regulated by MAGE-As via transcriptional factors ZEB1/2. Part III Research on mechanism in regulation of EMT via MAGE-As in TNBC cellsObjective: To explore the mechanim behind regulation of EMT in TNBC by MAGE-As.Methods:1 Real-time PCR and WB were performed to investigate the expression profile of the downstream target genes(cyclin-D1, c-myc, MMP7) in the Wnt/β-catenin/GSK-3β signal pathy when MAGE-As was down-regulated.2 WB assay was carried out to detect the expression of GSK-3β/pGSK-3β of the cell lines when MAGE-As were down-regulated. IF was utilized to examine the GSK-3β expression changes.Inhibitor of GSK-3β SB216763 which can active Wnt/β-catenin pathy was administrated to the cell lines of control and experiment groups in the following studies.3. Wound healing and transwell assay were applied to analyze the migration and invasion changes of the control and experiment groups after SB216763 treatment.4 MTS was employed to test the proliferation of the cells in control and experiment groups after SB216763 treatment.5 Morphology changes of the cells were observed after SB216763 treatment.6 WB was adopted to determine the expression profile of EMT markers(E-cadherin, β-catenin, vimentin, MMP9) and EMT related transcriptional factors(Snail, Slug, ZEB1/2, Twist) of the control and experiment groups after SB216763 treatment.7 WB was used to investigate the expression of cyclin-D1, c-myc, MMP7 in the control and experiment groups after SB216763 treatment.Results:1 Cyclin-D1, c-myc and MMP7 were all significantly down-regulated after MAGE-A si RNA transfection in the cell lines(P<0.05).2 WB assay showed that GSK-3β expression was significantly elevated, while p-GSK-3β was obviously decreased with MAGE-As depletion(P<0.05)..3 The inhibited migration and invasion were reversed after SB216763 administration in the MAGE-As down-regulated cells. Wound healing and transwell assay showed that migratory and invasive ability of the cells in control group were significantly increased after SB216763 application and were reversed in the experiment group(P<0.05).4 The proliferation of the cells with MAGE-As declination was significantly reversed after SB216763 application. MTS results uncovered that the OD value of the cells in control group was significantly increased after SB216763 application and was significantly reversed in the experiment group(P<0.05).5 The inhibition of EMT was relieved after SB216763 treatment. The mesenchymal morphology of control and experimental group of MDA-MB-231 became more obvious. This change was also observed in the control group of BT549, and the round shape of the cells in BT549 experimental group turned into spindle shape again. WB results showed that E-cadherin was obviously decreased in MDA-MB-231 control group after SB216763 treatment(P<0.05), while no significant change was seen in BT549(P>0.05). Vimentin、β-catenin and MMP9 were increased in the control groups of both MDA-MB-231 and BT549 cell lines(P<0.05). In the experiment group, E-cadherin was not changed in both of the two cells(P<0.05), but the down-regulated vimentin、β-catenin and MMP9 were up-regulated again(P<0.05). ZEB1 and ZEB2 were increased in control groups, and reversed in experiment groups(P<0.05).6 The blocked Wnt/β-catenin signal was reactivated again after SB216763 treatment. WB results showed that cyclin-D1, c-myc and MMP7 were elevated in control groups(P<0.05), those declined in the experiment groups reversed after SB216763 treatment(P<0.05).Conclusions:1 Wnt/β-catenin signal is blocked when MAGE-As was knocked down.2 The inhibited EMT by MAGE-As down-regulation is reversed by GSK-3β inhibitor SB216763.3 EMT of TNBC is regulated by MAGE-As via GSK-3β mediated Wnt/β-catenin signal in part. |
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