| In the incidence of female malignancy,breast cancer has been ranked the first,and in recent years it has been a growing trend that more and more young women suffered from that in China.Invasive Breast Cancer,as a type of subtype,has poor effect in present clinical treatment and the main reason is distant metastasis at its early stage,which makes it of high recurrence and metastasis rate of the tumor.Epithelial-Mesenchymal Transformation(EMT)is an important factor in the invasion and metastasis of tumor cells in breast cancer patients.Previous studies have shown that abnormal expression of multiple transcription factors also promotes EMT in breast cancer.In this paper,we made a preliminary research on the mechanism of Brachyury in the process of breast cancer metastasis as a member of the T-box transcription factor.The Brachyury gene is originally found specifically expressed in the embryonic chord and tail buds.Overexpression of Brachyury in malignant tumors has been found in recent years.We collected 573 cases of primary breast cancer and 29 cases of adjacent normal tissues around the cancer.A total of 317 specimens were selected and included in the statistics excluding those failed to get immunohistochemical results because of dye stripping in the chip and those with incomplete medical records.Among them,288 cases were of cancer tissue and 29 cases were of the adjacent normal tissue around cancer,28 out of which were matching tissue.We successfully constructed seven tissue chips,six of which were cancer tissue chips and one chip was a normal tissue chip.Immunohistochemical methods were used to get the qualitative position of Brachyury expressed in breast cancer and its adjacent tissue.93 cases out of 288 breast cancer specimens showed positive Brachyury expression and its rate was 32.29%.3 cases out of 29 adjacent cancer tissue specimens showed positive Brachyury expression and its rate was 10.34%.It was suggested when conducting Pearson X~2,X~2=5.0162,P=0.02511,that Brachyury protein expression had significant difference in cancer and its adjacent tissue,which indicated that Brachyury might have great importance in the development of breast cancer.In addition,we also used the Mann-Whitney U nonparametric test to check the correlation that the positive expression of Brachyury protein have with pathological parameters on the age of the patients,pathological type,histological grade,tumor size,lymph node metastasis,AJCC staging,and menopause status using Pearson X~2 or Fisher exact probability for statistical analysis.The results showed that the difference in Brachyury protein at different ages was not statistically significant(P=0.1950).The results of Pearson X~2 test showed that the difference of Brachyury protein expression in difference menopausal status was of no statistical significance(P=0.1131).Pearson X~2 test results suggested that the difference of Brachyury protein expression in different pathological types was of no statistical significance(P=0.6157).Pearson X~2 test results suggested that the difference of Brachyury protein expression in different histological differences was of no statistical significance(P=0.9549).Pearson X~2 test results showed that the expression of Brachyury protein in different tumor sizes was not statistically significant(P=0.1986).Pearson X~2 test results suggested that the difference of Brachyury protein expression in the presence and absence of lymph node metastasis was of no statistical significance(P=0.9136).Pearson X~2 test results suggested that the difference of Brachyury protein expression in AJCC staging was of no statistical significance(P=0.3459).There were 46 cases of Luminal A type taking up 41.44%,23 cases of Luminal B type taking up 29.87%,5cases of HER2 overexpression type,taking up 14.71%and 19 cases of three-negative type taking up 28.79%in those with positive Brachyury protein expression,while the corresponding number in negative Brachyury protein expression are:65 cases of Luminal A type taking up 58.56%,54 cases of Luminal B type taking up 70.13%,29cases of HER2 overexpression taking up 85.29%and 47 cases of three-negative type taking up 71.21%.Pearson X~2 test results showed that the expression of Brachyury protein was statistically significant in different molecular typing of breast cancer(P=0.0219).Among the patients with positive Brachyury protein expression,there were28 cases with positive expression of HER2 taking up 25.23%and 65 cases with negative expression of HER2 taking up 36.72%,while for those with negative Brachyury protein expression,there are 83 cases with positive HER2 expression taking up 74.77%and 112 cases with negative expression of HER2 taking up 63.28%.Pearson X~2 test results suggested that the difference of Brachyury protein expression in different HER2 expression was of no statistical significance(P=0.0572).Among the patients with positive expression of Brachyury protein,there were 69 cases with positive ER expression taking up 36.7%and 24 cases with negative expression of ER taking up 24%,while for those with negative Brachyury protein expression,there are119 cases with positive ER expression taking up 63.3%and 76 cases with negative ER expression taking up 76%.Pearson X~2 test results suggested that the difference of Brachyury protein expression in different ER expression was statistically significant(P=0.0392).Among the patients with positive Brachyury protein expression,there were 55 cases with positive PR expression taking up 36.42%and 38 cases with negative PR expression taking up 27.74%,while for those with negative Brachyury protein expression,there were 96 cases with positive PR expression taking up 63.58%and 99 cases with negative PR expression taking up 72.26%.Pearson X~2 test results suggested that the difference of Brachyury protein expression in different PR expression was not statistically significant(P=0.1475).By analyzing these results,we found surprisingly that there was a significant difference in the expression of Brachyury protein among different molecular subtypes of breast cancer,especially in ER positive breast cancer.This is consistent with the previous reports that Brachyury is associated with the resistance to tamoxifen which is an endocrine therapy drug for ER-positive breast cancer.Along this clue,we explored the potential role of Brachyury in tamoxifen resistance.In ER positive breast cancer cells,by small interfering RNA plasmid transfection,knockdown of Brachyury protein expression can increase the sensitivity of breast cancer cells to tamoxifen,and up-regulation of Brachyury expression enhanced tamoxifen resistance.At the same time,we found that Sirtuin-1(SIRT1)was a downstream effector of Brachyury protein,and Brachyury influenced tamoxifen resistance by regulating the expression of SIRT1 in breast cancer cells.Overexpression of SIRT1 can also overcome tamoxifen resistance in breast cancer cells.Meanwhile,we also confirmed the above conclusions in the mouse tumor model.In addition,we also identified the relationship between Brachyury protein and EMT phenotype in three-negative breast cancer cells.The results showed that overexpression of Brachyury increased EMT phenotype,while restrained expression of Brachyury reduced EMT phenotype in triple-negative breast cancer cells.In addition,we also confirmed that the expression of Brachyury is regulated by chromosome structural maintenance protein 1(SMC1)in three-negative breast cancer.When the expression of SMC1 increased,the expression of Brachyury increased and EMT enhanced accordingly.When the expression of SMC1 was decreased,the expression of Brachyury decreased and EMT weakened.In summary,we analyzed the samples of the patients by tissue chip technique and suggested a hypothesis that the Brachyury protein may be associated with tamoxifen resistance in breast cancer,which was verified in subsequent cells and animal experiments.At the same time,we also propose that its specific mechanism of action is that Brachyury can affect the expression of SIRT1.In addition,we found that Brachyury protein is a key factor in the process of EMT in three-negative breast cancer,and its expression is regulated by SMC1.Therefore,Brachyury may be a potential therapeutic target for reversing the resistance to endocrine therapy in ER positive breast cancer and inhibiting invasion and metastasis in three-negative breast cancer. |
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