Role of AP1S1 in inducing chemotherapy resistance in breast cancer
Objective To explore the correlation between the expression level of adaptor related protein complex 1 subunit sigma 1(AP1S1)gene and the prognosis of breast cancer patients undergoing chemotherapy,and analyze the role of AP1S1 in chemotherapy resistance of breast cancer.Methods The expression of AP1S1 between breast cancer patients and breast normal tissues in The Cancer Genome Atlas(TCGA)database was analyzed by bioinformatics.Kaplan-Meier survival analysis was used to explore the association between AP1S1 expression level and relapse-free survival prognosis of breast cancer patients undergoing chemotherapy.Through cell culture,RNA extraction,real-time fluorescent quantitative PCR,Western blot analysis and drug sensitivity test,and other experimental methods,based on the breast cancer cell model with high expression of AP1S1,the effect of high expression of AP1S1 gene on chemotherapy resistance of breast cancer cells was revealed.The effect of overexpression of AP1S1 on the expression of stem and epithelial-mesenchymal transformation(EMT)markers in tumor cells was investigated.Results The expression level of AP1S1 gene in breast cancer tissue was significantly higher than that in normal tissue;the difference was statistically significant(P＜0.05).At different tumor stages,the expression level of AP1S1 gene was significantly higher than that of normal tissue,with statistical significance(all P＜0.05).Among them,the overexpression level of AP1S1 gene of stage 4 tumors was higher,and the difference was statistically significant(P＜0.05).Luminal,human epidermal growth factor receptor 2(HER2)-positive and triple-negative breast cancer tissues showed high expression level of AP1S1 gene compared with normal tissues,with statistical significance(all P＜0.05).The expression levels of AP1S1 were higher in HER2-positive and triple-negative breast cancer tissues,and the differences were statistically significant(both P＜0.05).Kaplan-Meier survival analysis showed that the relapse-free survival of breast cancer patients with high AP1S1 expression after chemotherapy was significantly shorter than that of patients with low AP1S1 expression,the difference was statistically significant(P=0.038).High AP1S1 expression was significantly associated with poor prognosis after chemotherapy in triple-negative and HER2-positive breast cancer patients(P=0.037,0.017).In Luminal B patients,high AP1S1 expression was significantly associated with longer relapse-free survival(P=0.007).Real-time fluorescence quantitative PCR showed that compared with the control group,the transcription level of AP1S1 in the overexpressed cell lines CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 was significantly up-regulated,with statistical significance(t=23.51,6.22;both P＜0.05).Western blot results showed that compared with the control group,the AP1S1 protein levels in the AP1S1 overexpression cell lines CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 were significantly up-regulated,with statistical significance(all P＜0.05).The results of The Genomis of Drug Sensitivity in Cancer Project(GDSC)database analysis showed that the inhibitory concentration 50％(IC50)of CAL-51 and Hs-578T were 0.008 398 and 0.000 935 μmol/L,respectively.Drug toxicity test results showed that overexpression of AP1S1 significantly enhanced the tolerance of breast cancer cells CAL-51 and Hs-578T to the chemotherapy drug docetaxel,with statistical significance(t=8.74,9.90;both P＜0.05).The results of three-dimensional culture experiment showed that CAL-51 cells overexpressing AP1S1 formed more spherical cell clusters,while the structure of the control group was significantly deteriorated under the action of drugs(t=11.54,P＜0.05).Compared with the control group,in CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 breast cancer cell lines,overexpression of AP1S1 significantly upregulated stem cell marker gene Nanog homeobox(NANOG),octamer-binding transcription factor 4(OCT4),SRY associated HMG box gene 2(SOX2),BMI1 polycomb ring finger oncogene(BMI1),and multi-drug resistance associated gene ATP-binding cassette subfamily G member 2(ABCG2)expression at the transcription level;the differences were statistically significant(t=20.75,39.05,28.55,29.76,94.18;24.78,17.79,19.60,14.10,16.79;all P＜0.05).Meanwhile,the mRNA expression of epithelial cadherin(E-cadherin)(CDH1)and tight junction protein 1(TJP1)was significantly decreased;The expression of EMT-related markers neural cadherin(N-cadherin)(CDH2)and vimentin(VIM)were increased,and the differences were statistically significant(t=6.26,4.99,9.79,5.23;13.25,19.87,13.83,11.42;all P＜0.05).In CAL51 cell line,Western blot results showed that overexpression of AP1S1 led to increased expression level of stem cell marker gene SOX2 and multi-drug resistance gene ABCG2,and significantly inhibited the expression of cell adhesion protein E-cadherin,it also promoted the overexpression of N-cadherin,and the differences were statistically significant(all P＜0.05).Conclusion The high expression of AP1S1 in breast cancer not only could promote the resistance of chemotherapy drug docetaxel,but also promote the dry enhancement of breast cancer cells and induces EMT process.
AP1S1Breast cancerDocetaxelResistance to chemotherapy
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